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Sample records for primary astrocytes exposed

  1. Mitochondrial dysfunction and loss of glutamate uptake in primary astrocytes exposed to titanium dioxide nanoparticles

    NASA Astrophysics Data System (ADS)

    Wilson, Christina L.; Natarajan, Vaishaali; Hayward, Stephen L.; Khalimonchuk, Oleh; Kidambi, Srivatsan

    2015-11-01

    Titanium dioxide (TiO2) nanoparticles are currently the second most produced engineered nanomaterial in the world with vast usage in consumer products leading to recurrent human exposure. Animal studies indicate significant nanoparticle accumulation in the brain while cellular toxicity studies demonstrate negative effects on neuronal cell viability and function. However, the toxicological effects of nanoparticles on astrocytes, the most abundant cells in the brain, have not been extensively investigated. Therefore, we determined the sub-toxic effect of three different TiO2 nanoparticles (rutile, anatase and commercially available P25 TiO2 nanoparticles) on primary rat cortical astrocytes. We evaluated some events related to astrocyte functions and mitochondrial dysregulation: (1) glutamate uptake; (2) redox signaling mechanisms by measuring ROS production; (3) the expression patterns of dynamin-related proteins (DRPs) and mitofusins 1 and 2, whose expression is central to mitochondrial dynamics; and (4) mitochondrial morphology by MitoTracker® Red CMXRos staining. Anatase, rutile and P25 were found to have LC50 values of 88.22 +/- 10.56 ppm, 136.0 +/- 31.73 ppm and 62.37 +/- 9.06 ppm respectively indicating nanoparticle specific toxicity. All three TiO2 nanoparticles induced a significant loss in glutamate uptake indicative of a loss in vital astrocyte function. TiO2 nanoparticles also induced an increase in reactive oxygen species generation, and a decrease in mitochondrial membrane potential, suggesting mitochondrial damage. TiO2 nanoparticle exposure altered expression patterns of DRPs at low concentrations (25 ppm) and apoptotic fission at high concentrations (100 ppm). TiO2 nanoparticle exposure also resulted in changes to mitochondrial morphology confirmed by mitochondrial staining. Collectively, our data provide compelling evidence that TiO2 nanoparticle exposure has potential implications in astrocyte-mediated neurological dysfunction.Titanium dioxide (Ti

  2. Mitochondrial dysfunction and loss of glutamate uptake in primary astrocytes exposed to titanium dioxide nanoparticles.

    PubMed

    Wilson, Christina L; Natarajan, Vaishaali; Hayward, Stephen L; Khalimonchuk, Oleh; Kidambi, Srivatsan

    2015-11-28

    Titanium dioxide (TiO2) nanoparticles are currently the second most produced engineered nanomaterial in the world with vast usage in consumer products leading to recurrent human exposure. Animal studies indicate significant nanoparticle accumulation in the brain while cellular toxicity studies demonstrate negative effects on neuronal cell viability and function. However, the toxicological effects of nanoparticles on astrocytes, the most abundant cells in the brain, have not been extensively investigated. Therefore, we determined the sub-toxic effect of three different TiO2 nanoparticles (rutile, anatase and commercially available P25 TiO2 nanoparticles) on primary rat cortical astrocytes. We evaluated some events related to astrocyte functions and mitochondrial dysregulation: (1) glutamate uptake; (2) redox signaling mechanisms by measuring ROS production; (3) the expression patterns of dynamin-related proteins (DRPs) and mitofusins 1 and 2, whose expression is central to mitochondrial dynamics; and (4) mitochondrial morphology by MitoTracker® Red CMXRos staining. Anatase, rutile and P25 were found to have LC50 values of 88.22 ± 10.56 ppm, 136.0 ± 31.73 ppm and 62.37 ± 9.06 ppm respectively indicating nanoparticle specific toxicity. All three TiO2 nanoparticles induced a significant loss in glutamate uptake indicative of a loss in vital astrocyte function. TiO2 nanoparticles also induced an increase in reactive oxygen species generation, and a decrease in mitochondrial membrane potential, suggesting mitochondrial damage. TiO2 nanoparticle exposure altered expression patterns of DRPs at low concentrations (25 ppm) and apoptotic fission at high concentrations (100 ppm). TiO2 nanoparticle exposure also resulted in changes to mitochondrial morphology confirmed by mitochondrial staining. Collectively, our data provide compelling evidence that TiO2 nanoparticle exposure has potential implications in astrocyte-mediated neurological dysfunction.

  3. Accumulation of silver nanoparticles by cultured primary brain astrocytes

    NASA Astrophysics Data System (ADS)

    Luther, Eva M.; Koehler, Yvonne; Diendorf, Joerg; Epple, Matthias; Dringen, Ralf

    2011-09-01

    Silver nanoparticles (AgNP) are components of various food industry products and are frequently used for medical equipment and materials. Although such particles enter the vertebrate brain, little is known on their biocompatibility for brain cells. To study the consequences of an AgNP exposure of brain cells we have treated astrocyte-rich primary cultures with polyvinylpyrrolidone (PVP)-coated AgNP. The incubation of cultured astrocytes with micromolar concentrations of AgNP for up to 24 h resulted in a time- and concentration-dependent accumulation of silver, but did not compromise the cell viability nor lower the cellular glutathione content. In contrast, the incubation of astrocytes for 4 h with identical amounts of silver as AgNO3 already severely compromised the cell viability and completely deprived the cells of glutathione. The accumulation of AgNP by astrocytes was proportional to the concentration of AgNP applied and significantly lowered by about 30% in the presence of the endocytosis inhibitors chloroquine or amiloride. Incubation at 4 °C reduced the accumulation of AgNP by 80% compared to the values obtained for cells that had been exposed to AgNP at 37 °C. These data demonstrate that viable cultured brain astrocytes efficiently accumulate PVP-coated AgNP in a temperature-dependent process that most likely involves endocytotic pathways.

  4. Transcriptomic analyses of primary astrocytes under TNFα treatment.

    PubMed

    Birck, Cindy; Koncina, Eric; Heurtaux, Tony; Glaab, Enrico; Michelucci, Alessandro; Heuschling, Paul; Grandbarbe, Luc

    2016-03-01

    Astrocytes, the most abundant glial cell population in the central nervous system, have important functional roles in the brain as blood brain barrier maintenance, synaptic transmission or intercellular communications [1], [2]. Numerous studies suggested that astrocytes exhibit a functional and morphological high degree of plasticity. For example, following any brain injury, astrocytes become reactive and hypertrophic. This phenomenon, also called reactive gliosis, is characterized by a set of progressive gene expression and cellular changes [3]. Interestingly, in this context, astrocytes can re-acquire neurogenic properties. It has been shown that astrocytes can undergo dedifferentiation upon injury and inflammation, and may re-acquire the potentiality of neural progenitors [4], [5], [6], [7]. To assess the effect of inflammation on astrocytes, primary mouse astrocytes were treated with tumor necrosis factor α (TNFα), one of the main pro-inflammatory cytokines. The strength of this study is that pure primary astrocytes were used. As microglia are highly reactive immune cells, we used a magnetic cell sorting separation (MACS) method to further obtain highly pure astrocyte cultures devoid of microglia. Here, we provide details of the microarray data, which have been deposited in the Gene Expression Omnibus (GEO) under the series accession number GSE73022. The analysis and interpretation of these data are included in Gabel et al. (2015). Analysis of gene expression indicated that the NFκB pathway-associated genes were induced after a TNFα treatment. We have shown that primary astrocytes devoid of microglia can respond to a TNFα treatment with the re-expression of genes implicated in the glial cell development. PMID:26981349

  5. Gintonin stimulates gliotransmitter release in cortical primary astrocytes.

    PubMed

    Kim, Hyunsook; Lee, Byung-Hwan; Choi, Sun-Hye; Kim, Hyeon-Joong; Jung, Suk-Won; Hwang, Sung-Hee; Rhim, Hyewon; Kim, Hyung-Chun; Cho, Ik-Hyun; Nah, Seung-Yeol

    2015-08-31

    Lysophosphatidic acid (LPA) is a simple and minor phospholipid, but serves as a lipid-derived neurotransmitter via activation of G protein-coupled LPA receptors. Astrocytes abundantly express LPA receptors and contain gliotransmitters that modulate astrocyte-neuron interactions. Gintonin is a novel ginseng-derived G protein-coupled LPA receptor ligand. Gintonin induces [Ca(2+)]i transients in neuronal and non-neuronal cells via activation of LPA receptors, which regulate calcium-dependent ion channels and receptors. A line of evidence shows that neurotransmitter-mediated [Ca(2+)]i elevations in astrocytes are coupled with gliotransmitter release. However, little is known about whether gintonin-mediated [Ca(2+)]i transients are coupled to gliotransmitter release in astrocytes. In the present study, we examined the effects of gintonin on adenosine triphosphate (ATP) and glutamate release in mouse cortical primary astrocytes. Application of gintonin to astrocytes induced [Ca(2+)]i transients in a concentration-dependent and reversible manner. However, ginsenosides, other active ingredients in ginseng, had no effect on [Ca(2+)]i transients. The induction of gintonin-mediated [Ca(2+)]i transients was attenuated/blocked by the LPA1/3 receptor antagonist Ki16425, a phospholipase C inhibitor, an inositol 1,4,5-triphosphate receptor antagonist, and an intracellular Ca(2+) chelator. Gintonin treatment on astrocytes increased ATP and glutamate release in a concentration- and time-dependent manner. BAPTA and Ki16425 attenuated gintonin-mediated ATP and glutamate release in astrocytes. The present study shows that gintonin-mediated [Ca(2+)]i transients are coupled to gliotransmitter release via LPA receptor activation. Finally, gintonin-mediated [Ca(2+)]i transients and gliotransmitter release from astrocytes via LPA receptor activation might explain one mechanism of gintonin-mediated neuromodulation in the central nervous system. PMID:26191656

  6. Laminin is produced by early rat astrocytes in primary culture

    PubMed Central

    1983-01-01

    The production of laminin by early rat astrocytes in primary culture was investigated by double immunofluorescence staining for laminin and the glial fibrillary acidic protein (GFAP), a defined astrocyte marker. In early cultures (3 d in vitro; 3 DIV) cytoplasmic laminin was detected in all the GFAP-positive cells which formed the major population (80%) of the nonneuronal cells present in cultures from 20- 21-d embryonic, newborn, or 5-d-old rat brains. Monensin treatment (10 microM, 4 h) resulted in accumulation of laminin in the Golgi region, located using labeled wheat germ agglutinin. Laminin started gradually to disappear from the cells with the time in culture, was absent in star-shaped, apparently mature astrocytes, but remained as pericellular matrix deposits. The disappearance of cellular laminin was dependent on the age of the animal and the time in culture so that it started earlier in cultures from 5-d-old rat brains (5 DIV) and approximately following the in vivo age difference in cultures from newborn (12 DIV) and embryonic (14 DIV) rat brains. Our results indicate that laminin is a protein of early astrocytes and also deposited by them in primary culture, thus suggesting a role for this glycoprotein in the development of the central nervous system. PMID:6339524

  7. Effects of Ranolazine on Astrocytes and Neurons in Primary Culture

    PubMed Central

    Aldasoro, Martin; Guerra-Ojeda, Sol; Aguirre-Rueda, Diana; Mauricio, Mª Dolores; Vila, Jose Mª; Marchio, Patricia; Iradi, Antonio; Aldasoro, Constanza; Jorda, Adrian; Obrador, Elena; Valles, Soraya L.

    2016-01-01

    Ranolazine (Rn) is an antianginal agent used for the treatment of chronic angina pectoris when angina is not adequately controlled by other drugs. Rn also acts in the central nervous system and it has been proposed for the treatment of pain and epileptic disorders. Under the hypothesis that ranolazine could act as a neuroprotective drug, we studied its effects on astrocytes and neurons in primary culture. We incubated rat astrocytes and neurons in primary cultures for 24 hours with Rn (10−7, 10−6 and 10−5 M). Cell viability and proliferation were measured using trypan blue exclusion assay, MTT conversion assay and LDH release assay. Apoptosis was determined by Caspase 3 activity assay. The effects of Rn on pro-inflammatory mediators IL-β and TNF-α was determined by ELISA technique, and protein expression levels of Smac/Diablo, PPAR-γ, Mn-SOD and Cu/Zn-SOD by western blot technique. In cultured astrocytes, Rn significantly increased cell viability and proliferation at any concentration tested, and decreased LDH leakage, Smac/Diablo expression and Caspase 3 activity indicating less cell death. Rn also increased anti-inflammatory PPAR-γ protein expression and reduced pro-inflammatory proteins IL-1 β and TNFα levels. Furthermore, antioxidant proteins Cu/Zn-SOD and Mn-SOD significantly increased after Rn addition in cultured astrocytes. Conversely, Rn did not exert any effect on cultured neurons. In conclusion, Rn could act as a neuroprotective drug in the central nervous system by promoting astrocyte viability, preventing necrosis and apoptosis, inhibiting inflammatory phenomena and inducing anti-inflammatory and antioxidant agents. PMID:26950436

  8. Effects of Ranolazine on Astrocytes and Neurons in Primary Culture.

    PubMed

    Aldasoro, Martin; Guerra-Ojeda, Sol; Aguirre-Rueda, Diana; Mauricio, M Dolores; Vila, Jose M; Marchio, Patricia; Iradi, Antonio; Aldasoro, Constanza; Jorda, Adrian; Obrador, Elena; Valles, Soraya L

    2016-01-01

    Ranolazine (Rn) is an antianginal agent used for the treatment of chronic angina pectoris when angina is not adequately controlled by other drugs. Rn also acts in the central nervous system and it has been proposed for the treatment of pain and epileptic disorders. Under the hypothesis that ranolazine could act as a neuroprotective drug, we studied its effects on astrocytes and neurons in primary culture. We incubated rat astrocytes and neurons in primary cultures for 24 hours with Rn (10-7, 10-6 and 10-5 M). Cell viability and proliferation were measured using trypan blue exclusion assay, MTT conversion assay and LDH release assay. Apoptosis was determined by Caspase 3 activity assay. The effects of Rn on pro-inflammatory mediators IL-β and TNF-α was determined by ELISA technique, and protein expression levels of Smac/Diablo, PPAR-γ, Mn-SOD and Cu/Zn-SOD by western blot technique. In cultured astrocytes, Rn significantly increased cell viability and proliferation at any concentration tested, and decreased LDH leakage, Smac/Diablo expression and Caspase 3 activity indicating less cell death. Rn also increased anti-inflammatory PPAR-γ protein expression and reduced pro-inflammatory proteins IL-1 β and TNFα levels. Furthermore, antioxidant proteins Cu/Zn-SOD and Mn-SOD significantly increased after Rn addition in cultured astrocytes. Conversely, Rn did not exert any effect on cultured neurons. In conclusion, Rn could act as a neuroprotective drug in the central nervous system by promoting astrocyte viability, preventing necrosis and apoptosis, inhibiting inflammatory phenomena and inducing anti-inflammatory and antioxidant agents. PMID:26950436

  9. Astrocytes Enhance Streptococcus suis-Glial Cell Interaction in Primary Astrocyte-Microglial Cell Co-Cultures.

    PubMed

    Seele, Jana; Nau, Roland; Prajeeth, Chittappen K; Stangel, Martin; Valentin-Weigand, Peter; Seitz, Maren

    2016-06-13

    Streptococcus (S.) suis infections are the most common cause of meningitis in pigs. Moreover, S. suis is a zoonotic pathogen, which can lead to meningitis in humans, mainly in adults. We assume that glial cells may play a crucial role in host-pathogen interactions during S. suis infection of the central nervous system. Glial cells are considered to possess important functions during inflammation and injury of the brain in bacterial meningitis. In the present study, we established primary astrocyte-microglial cell co-cultures to investigate interactions of S. suis with glial cells. For this purpose, microglial cells and astrocytes were isolated from new-born mouse brains and characterized by flow cytometry, followed by the establishment of astrocyte and microglial cell mono-cultures as well as astrocyte-microglial cell co-cultures. In addition, we prepared microglial cell mono-cultures co-incubated with uninfected astrocyte mono-culture supernatants and astrocyte mono-cultures co-incubated with uninfected microglial cell mono-culture supernatants. After infection of the different cell cultures with S. suis, bacteria-cell association was mainly observed with microglial cells and most prominently with a non-encapsulated mutant of S. suis. A time-dependent induction of NO release was found only in the co-cultures and after co-incubation of microglial cells with uninfected supernatants of astrocyte mono-cultures mainly after infection with the capsular mutant. Only moderate cytotoxic effects were found in co-cultured glial cells after infection with S. suis. Taken together, astrocytes and astrocyte supernatants increased interaction of microglial cells with S. suis. Astrocyte-microglial cell co-cultures are suitable to study S. suis infections and bacteria-cell association as well as NO release by microglial cells was enhanced in the presence of astrocytes.

  10. Astrocytes Enhance Streptococcus suis-Glial Cell Interaction in Primary Astrocyte-Microglial Cell Co-Cultures

    PubMed Central

    Seele, Jana; Nau, Roland; Prajeeth, Chittappen K.; Stangel, Martin; Valentin-Weigand, Peter; Seitz, Maren

    2016-01-01

    Streptococcus (S.) suis infections are the most common cause of meningitis in pigs. Moreover, S. suis is a zoonotic pathogen, which can lead to meningitis in humans, mainly in adults. We assume that glial cells may play a crucial role in host-pathogen interactions during S. suis infection of the central nervous system. Glial cells are considered to possess important functions during inflammation and injury of the brain in bacterial meningitis. In the present study, we established primary astrocyte-microglial cell co-cultures to investigate interactions of S. suis with glial cells. For this purpose, microglial cells and astrocytes were isolated from new-born mouse brains and characterized by flow cytometry, followed by the establishment of astrocyte and microglial cell mono-cultures as well as astrocyte-microglial cell co-cultures. In addition, we prepared microglial cell mono-cultures co-incubated with uninfected astrocyte mono-culture supernatants and astrocyte mono-cultures co-incubated with uninfected microglial cell mono-culture supernatants. After infection of the different cell cultures with S. suis, bacteria-cell association was mainly observed with microglial cells and most prominently with a non-encapsulated mutant of S. suis. A time-dependent induction of NO release was found only in the co-cultures and after co-incubation of microglial cells with uninfected supernatants of astrocyte mono-cultures mainly after infection with the capsular mutant. Only moderate cytotoxic effects were found in co-cultured glial cells after infection with S. suis. Taken together, astrocytes and astrocyte supernatants increased interaction of microglial cells with S. suis. Astrocyte-microglial cell co-cultures are suitable to study S. suis infections and bacteria-cell association as well as NO release by microglial cells was enhanced in the presence of astrocytes. PMID:27304968

  11. Astrocytes.

    ERIC Educational Resources Information Center

    Kimelberg, Harold K.; Norenberg, Michael D.

    1989-01-01

    Describes the astrocytes' function as equal partners with neurons in both the normal and the abnormal brain. Discusses the developmental scaffolds, inert scar tissue, Huntington's disease, psychiatric disorders, and the identification of these brain cells. (RT)

  12. Energy-dependent volume regulation in primary cultured cerebral astrocytes.

    PubMed

    Olson, J E; Sankar, R; Holtzman, D; James, A; Fleischhacker, D

    1986-08-01

    Cell volume regulation and energy metabolism were studied in primary cultured cerebral astrocytes during exposure to media of altered osmolarity. Cells suspended in medium containing 1/2 the normal concentration of NaCl (hypoosmotic) swell immediately to a volume 40-50% larger than cells suspended in isoosmotic medium. The cell volume in hypoosmotic medium then decreases over 30 min to a volume approximately 25% larger than cells in isoosmotic medium. In hyperosmotic medium (containing twice the normal concentration of NaCl), astrocytes shrink by 29%. Little volume change occurs following this initial shrinkage. Cells resuspended in isoosmotic medium after a 30 min incubation in hypoosmotic medium shrink immediately to a volume 10% less than the volume of cells incubated continuously in isoosmotic medium. Thus, the regulatory volume decrease (RVD) in hypoosmotic medium involves a net reduction of intracellular osmoles. The RVD is partially blocked by inhibitors of mitochondrial electron transport but is unaffected by an inhibitor of glycolysis or by an uncoupler of oxidative phosphorylation. Inhibition of RVD by these metabolic agents is correlated with decreased cellular ATP levels. Ouabain, added immediately after hypoosmotic induced swelling, completely inhibits RVD, but does not alter cell volume if added after RVD has taken place. Ouabain also inhibits cell respiration 27% more in hypoosmotic medium than in isoosmotic medium indicating that the (Na,K)-ATPase-coupled ion pump is more active in the hypoosmotic medium. These data suggest that the cell volume response of astrocytes in hypoosmotic medium involves the net movement of osmoles by a mechanism dependent on cellular energy and tightly coupled to the (Na,K)-ATPase ion pump. This process may be important in the energy-dependent osmoregulation in the brain, a critical role attributed to the astrocyte in vivo. PMID:3015986

  13. Nanoparticle-mediated conversion of primary human astrocytes into neurons and oligodendrocytes.

    PubMed

    Li, Xiaowei; Kozielski, Kristen; Cheng, Yu-Hao; Liu, Huanhuan; Zamboni, Camila Gadens; Green, Jordan; Mao, Hai-Quan

    2016-06-21

    Central nervous system (CNS) diseases and injuries are accompanied by reactive gliosis and scarring involving the activation and proliferation of astrocytes to form hypertrophic and dense structures, which present a significant barrier to neural regeneration. Engineering astrocytes to functional neurons or oligodendrocytes may constitute a novel therapeutic strategy for CNS diseases and injuries. Such direct cellular programming has been successfully demonstrated using viral vectors via the transduction of transcriptional factors, such as Sox2, which could program resident astrocytes into neurons in the adult brain and spinal cord, albeit the efficiency was low. Here we report a non-viral nanoparticle-based transfection method to deliver Sox2 or Olig2 into primary human astrocytes and demonstrate the effective conversion of the astrocytes into neurons and oligodendrocyte progenitors following the transgene expression of Sox2 and Olig2, respectively. This approach is highly translatable for engineering astrocytes to repair injured CNS tissues. PMID:27328202

  14. Biological effects of inorganic arsenic on primary cultures of rat astrocytes.

    PubMed

    Catanzaro, Irene; Schiera, Gabriella; Sciandrello, Giulia; Barbata, Giusi; Caradonna, Fabio; Proia, Patrizia; Di Liegro, Italia

    2010-10-01

    It is well established that inorganic arsenic induces neurotoxic effects and neurological defects in humans and laboratory animals. The cellular and molecular mechanisms of its actions, however, remain elusive. Herein we report the effects of arsenite (NaAsO2) on primary cultures of rat astrocytes. Cells underwent induction of heat shock protein 70 only at the highest doses of inorganic arsenic (30 and 60 microM), suggesting a high threshold to respond to stress. We also investigated arsenic genotoxicity with the comet assay. Interestingly, although cells treated with 10 microM arsenite for 24 h maintained >70% viability, with respect to untreated cells, high DNA damage was already observed. Since arsenic is not known to be a direct-acting genotoxic agent, we investigated the possibility that its effects are due, in astrocytes as well, to ROS formation, as already described for other cell types. However, FACS analysis after CM-H2DCFDA staining did not evidence any significant increase of ROS production while, on the contrary, at the highest arsenite concentrations used, ROS production decreased. Concordantly, we found that, if most cells in the culture are still alive (i.e. up to 10 microM arsenite), they show a treatment-dependent increase in the concentration of SOD1. On the other hand, SOD2 concentration did not change. Finally, we found that astrocytes also synthesize PIPPin, an RNA-binding protein, the concentration of which was recently reported to change in response to stress induced by cadmium. Here we also report that, in cells exposed to high doses of arsenite, an anti-PIPPin antibody-positive faster migrating protein appears. PMID:20818482

  15. Diazinon and diazoxon impair the ability of astrocytes to foster neurite outgrowth in primary hippocampal neurons

    SciTech Connect

    Pizzurro, Daniella M.; Dao, Khoi; Costa, Lucio G.

    2014-02-01

    Evidence from in vivo and epidemiological studies suggests that organophosphorus insecticides (OPs) are developmental neurotoxicants, but possible underlying mechanisms are still unclear. Astrocytes are increasingly recognized for their active role in normal neuronal development. This study sought to investigate whether the widely-used OP diazinon (DZ), and its oxygen metabolite diazoxon (DZO), would affect glial–neuronal interactions as a potential mechanism of developmental neurotoxicity. Specifically, we investigated the effects of DZ and DZO on the ability of astrocytes to foster neurite outgrowth in primary hippocampal neurons. The results show that both DZ and DZO adversely affect astrocyte function, resulting in inhibited neurite outgrowth in hippocampal neurons. This effect appears to be mediated by oxidative stress, as indicated by OP-induced increased reactive oxygen species production in astrocytes and prevention of neurite outgrowth inhibition by antioxidants. The concentrations of OPs were devoid of cytotoxicity, and cause limited acetylcholinesterase inhibition in astrocytes (18 and 25% for DZ and DZO, respectively). Among astrocytic neuritogenic factors, the most important one is the extracellular matrix protein fibronectin. DZ and DZO decreased levels of fibronectin in astrocytes, and this effect was also attenuated by antioxidants. Underscoring the importance of fibronectin in this context, adding exogenous fibronectin to the co-culture system successfully prevented inhibition of neurite outgrowth caused by DZ and DZO. These results indicate that DZ and DZO increase oxidative stress in astrocytes, and this in turn modulates astrocytic fibronectin, leading to impaired neurite outgrowth in hippocampal neurons. - Highlights: • DZ and DZO inhibit astrocyte-mediated neurite outgrowth in rat hippocampal neurons. • Oxidative stress is involved in inhibition of neuritogenesis by DZ and DZO. • DZ and DZO decrease expression of the neuritogenic

  16. Astrocytes As the Main Players in Primary Degenerative Disorders of the Human Central Nervous System.

    PubMed

    Capani, Francisco; Quarracino, Cecilia; Caccuri, Roberto; Sica, Roberto E P

    2016-01-01

    Along the last years it has been demonstrated that non-neural cells play a major role in the pathogenesis of the primary degenerative disorders (PDDs) of the human central nervous system. Among them, astrocytes coordinate and participate in many different and complex metabolic processes, in close interaction with neurons. Moreover, increasing experimental evidence hints an early astrocytic dysfunction in these diseases. In this mini review we summarize the astrocytic behavior in PDDs, with special consideration to the experimental observations where astrocytic pathology precedes the development of neuronal dysfunction. We also suggest a different approach that could be consider in human investigations in Alzheimer's and Parkinson's disease. We believe that the study of PDDs with human brain samples may hold the key of a paradigmatic physiopathological process in which astrocytes might be the main players. PMID:26973519

  17. CPEB1 modulates lipopolysaccharide-mediated iNOS induction in rat primary astrocytes

    SciTech Connect

    Kim, Ki Chan; Hyun Joo, So; Shin, Chan Young

    2011-06-17

    Highlights: {yields} Expression and phosphorylation of CPEB1 is increased by LPS stimulation in rat primary astrocytes. {yields} JNK regulates expression and phosphorylation of CPEB1 in reactive astrocytes. {yields} Down-regulation of CPEB1 using siRNA inhibits oxidative stress and iNOS induction by LPS stimulation. {yields} CPEB1 may play an important role in regulating inflammatory responses in reactive astrocytes induced by LPS. -- Abstract: Upon CNS damage, astrocytes undergo a series of biological changes including increased proliferation, production of inflammatory mediators and morphological changes, in a response collectively called reactive gliosis. This process is an essential part of the brains response to injury, yet much is unknown about the molecular mechanism(s) that induce these changes. In this study, we investigated the role of cytoplasmic polyadenylation element binding protein 1 (CPEB1) in the regulation of inflammatory responses in a model of reactive gliosis, lipopolysaccharide-stimulated astrocytes. CPEB1 is an mRNA-binding protein recently shown to be expressed in astrocytes that may play a role in astrocytes migration. After LPS stimulation, the expression and phosphorylation of CPEB1 was increased in rat primary astrocytes in a JNK-dependent process. siRNA-induced knockdown of CPEB1 expression inhibited the LPS-induced up-regulation of iNOS as well as NO and ROS production, a hallmark of immunological activation of astrocytes. The results from the study suggest that CPEB1 is actively involved in the regulation of inflammatory responses in astrocytes, which might provide new insights into the regulatory mechanism after brain injury.

  18. Selective up-regulation of GLT-1 in cultured astrocytes exposed to soluble mediators released by activated microglia.

    PubMed

    Tilleux, Sébastien; Goursaud, Stéphanie; Hermans, Emmanuel

    2009-01-01

    Impaired glial glutamate uptake is commonly involved in neuronal damages observed in acute and chronic nervous disorders. As nervous insults are frequently associated with local inflammation involving microglia, this study aims at exploring the link between activated microglia and altered glutamate uptake in astrocytes. The regulation of the expression and activity of type 1 glutamate transporter (GLT-1) was examined after exposing cultures of rat astrocytes to conditioned medium from lipopolysaccharide-activated microglia cultures. Significant increases in GLT-1 mRNA expression and dihydrokainate sensitive uptake of aspartate were observed after 72h of treatment. These effects were reproduced by direct exposure of the astrocyte cultures to tumor necrosis factor alpha, a major cytokine released by activated microglia. The regulation of GLT-1 activity in response to inflammatory stimuli was also evidenced in cells exposed to dibutyryl cAMP, recognised as a model of reactive astrocytes in which the expression of this glutamate transporter is constitutively enhanced. Taken together, these results suggest that the GLT-1-dependent control of glutamate neurotransmission by either naive or chemically activated astrocytes is influenced by microglia-mediated inflammation. PMID:19428805

  19. Disparate effects of serum on basal and evoked NFAT activity in primary astrocyte cultures.

    PubMed

    Furman, Jennifer L; Artiushin, Irina A; Norris, Christopher M

    2010-01-29

    In astrocytes, the Ca(2+)-dependent protein phosphatase calcineurin (CN) strongly regulates neuro-immune/inflammatory cascades through activation of the transcription factor, nuclear factor of activated T cells (NFAT). While primary cell cultures provide a useful model system for investigating astrocytic CN/NFAT signaling, variable results may arise both within and across labs because of differences in culture conditions. Here, we determined the extent to which serum and cell confluency affect basal and evoked astrocytic NFAT activity in primary cortical astrocyte cultures. Cells were grown to either approximately 50% or >90% confluency, pre-loaded with an NFAT-luciferase reporter construct, and maintained for 16 h in medium with or without 10% fetal bovine serum (FBS). NFAT-dependent luciferase expression was then measured 5h after treatment with vehicle alone to assess basal NFAT activity, or with Ca(2+) mobilizers and IL-1 beta to assess evoked activity. The results revealed significantly higher levels of basal NFAT activity in FBS-containing medium, regardless of cell confluency. Conversely, evoked NFAT activation was significantly lower in serum-containing medium, with an even greater inhibition observed in confluent cultures. Application of 10% FBS to serum-free astrocyte cultures quickly evoked a roughly seven-fold increase in NFAT activity that was significantly reduced by co-delivery of neutralizing agents for IL-1 beta, TNFalpha, and/or IFN gamma, suggesting that serum occludes evoked NFAT activation through a cytokine-based mechanism. Together, the results demonstrate that the presence of serum and cell confluency have a major impact on CN/NFAT signaling in primary astrocyte cultures and therefore must be taken into consideration when using this model system.

  20. Role of porphyrin sequestration in the biogenesis of iron-laden astrocytic inclusions in primary culture.

    PubMed

    Schipper, H M; Small, L; Wang, X; Brawer, J R

    2002-01-01

    Astrocytes in subcortical regions of the mammalian brain progressively accumulate iron-rich, autofluorecent cytoplasmic inclusions as a function of aging. Cysteamine (CSH) accelerates the appearance of this senescent glial phenotype in situ and in primary rat astroglial cultures. Porphyrins have been implicated as the source of orange-red autofluorescence in these glial inclusions. Yet, CSH has been shown to suppress porphyrin-heme biosynthesis in cultured astroglia. To determine whether porphyrin biosynthesis or sequestration participates in the biogenesis of these glial inclusions, the porphyrin precursor, (3)H-delta-aminolevulinic acid ((3)H-ALA) was administered to CSH-exposed and control rat astroglial cultures followed by light and electron microscopic autoradiography. Control cultures exhibited faint orange-red autofluorescence, intense (3)H-ALA labeling, numerous normal mitochondria and few cytoplasmic inclusions. In these cells, (3)H-ALA labeling largely occurred over normal mitochondria. The CSH-treated astroglia exhibited diminished (3)H-ALA labeling and contained numerous orange-red autofluorescent inclusions. The latter manifested internal compartments delimited by double membranes characteristic of damaged mitochondria. The complement of normal mitochondria in the CSH-exposed cells was markedly reduced. In the CSH-treated cells, (3)H-ALA labeling predominated over the large multi-compartmental inclusions. CSH attenuates de novo porphyrin-heme biosynthesis in astroglia but may induce punctate orange-red autofluorescence in the cytoplasm of these cells by promoting large numbers of damaged, porphyrin-containing mitochondria to form tight aggregates within the nascent gliosomes.

  1. Astrocytes are the primary source of tissue factor in the murine central nervous system. A role for astrocytes in cerebral hemostasis.

    PubMed Central

    Eddleston, M; de la Torre, J C; Oldstone, M B; Loskutoff, D J; Edgington, T S; Mackman, N

    1993-01-01

    Hemostasis in the brain is of paramount importance because bleeding into the neural parenchyma can result in paralysis, coma, and death. Consistent with this sensitivity to hemorrhage, the brain contains large amounts of tissue factor (TF), the major cellular initiator of the coagulation protease cascades. However, to date, the cellular source for TF in the central nervous system has not been identified. In this study, analysis of murine brain sections by in situ hybridization demonstrated high levels of TF mRNA in cells that expressed glial fibrillary acidic protein, a specific marker for astrocytes. Furthermore, primary mouse astrocyte cultures and astrocyte cell lines from mouse, rat, and human constitutively expressed TF mRNA and functional protein. These data indicated that astrocytes are the primary source of TF in the central nervous system. We propose that astrocytes forming the glia limitans around the neural vasculature and deep to the meninges are intimately involved in controlling hemorrhage in the brain. Finally, we observed an increase in TF mRNA expression in the brains of scrapie-infected mice. This modulation of TF expression in the absence of hemorrhage suggested that TF may function in processes other than hemostasis by altering protease generation in normal and diseased brain. Images PMID:8326003

  2. Manganese accumulation in membrane fractions of primary astrocytes is associated with decreased γ-aminobutyric acid (GABA) uptake, and is exacerbated by oleic acid and palmitate.

    PubMed

    Fordahl, Steve C; Erikson, Keith M

    2014-05-01

    Manganese (Mn) exposure interferes with GABA uptake; however, the effects of Mn on GABA transport proteins (GATs) have not been identified. We sought to characterize how Mn impairs GAT function in primary rat astrocytes. Astrocytes exposed to Mn (500 μM) had significantly reduced (3)H-GABA uptake despite no change in membrane or cytosolic GAT3 protein levels. Co-treatment with 100 μM oleic or palmitic acids (both known to be elevated in Mn neurotoxicity), exacerbated the Mn-induced decline in (3)H-GABA uptake. Mn accumulation in the membrane fraction of astrocytes was enhanced with fatty acid administration, and was negatively correlated with (3)H-GABA uptake. Furthermore, control cells exposed to Mn only during the experimental uptake had significantly reduced (3)H-GABA uptake, and the addition of GABA (50 μM) blunted cytosolic Mn accumulation. These data indicate that reduced GAT function in astrocytes is influenced by Mn and fatty acids accumulating at or interacting with the plasma membrane.

  3. Ammonium increases Ca(2+) signalling and up-regulates expression of TRPC1 gene in astrocytes in primary cultures and in the in vivo brain.

    PubMed

    Liang, Chunguang; Du, Ting; Zhou, Jing; Verkhratsky, Alexei; Peng, Liang

    2014-11-01

    Rapid rise in ammonium concentration in the brain is the major pathogenic factor in hepatic encephalopathy that is manifested by state of confusion, forgetfulness and irritability, psychotic symptoms, delusions, lethargy, somnolence and, in the terminal stages, coma. Primary cultures of mouse astrocytes were used to investigate effects of chronic treatment (3 days) with ammonium chloride (ammonium) at 3 mM, this being a relevant concentration for hepatic encephalopathy condition, on metabotropic receptor agonist-induced increases in free cytosolic Ca(2+) concentration [(Ca(2+))i], measured with fura-2 based microfluorimetry and on store-operated Ca(2+) entry (SOCE) activated following treatment with the SERCA inhibitor thapsigargin. The agonists used were the β-adrenergic agonist isoproterenol, the α2-adrenergic agonist dexmedetomidine, the InsP3 receptor (InsP3R) agonist adenophostin A and ryanodine receptor agonist 4-Chloro-m-cresol (4-CMC). Agonist-induced [Ca(2+)]i responses were significantly increased in astrocytes chronically exposed to ammonium. Similarly, the SOCE, meditated by the transient receptor potential channel 1 (TRPC1), was significantly augmented. The ammonium-induced increase in SOCE was a result of an up-regulation of mRNA and protein expression of TRPC1 in astrocytes. Increase in TRPC1 expression and in SOCE were both prevented by ouabain antagonist canrenone. Similar up-regulation of TRPC1 gene expression was found in the brain of adult mice subjected to intraperitoneal injection of urease for 3 days. In transgenic mice tagged with an astrocyte-specific or a neurone-specific markers and treated with intraperitoneal injections of urease for 3 days, the fluorescence-activated cell sorting of neurones and astrocytes demonstrated that TRPC1 mRNA expression was up-regulated in astrocytes, but not in neurones. PMID:25113123

  4. Rapid Reprogramming of Primary Human Astrocytes into Potent Tumor-Initiating Cells with Defined Genetic Factors.

    PubMed

    Li, Fang; Liu, Xinjian; Sampson, John H; Bigner, Darell D; Li, Chuan-Yuan

    2016-09-01

    Cancer stem-like cells (CSC) are thought to drive brain cancer, but their cellular and molecular origins remain uncertain. Here, we report the successful generation of induced CSC (iCSC) from primary human astrocytes through the expression of defined genetic factors. Combined transduction of four factors, Myc, Oct-4, p53DD, and Ras, induced efficient transformation of primary human astrocytes into malignant cells with powerful tumor-initiating capabilities. Notably, transplantation of 100 transduced cells into nude mice was sufficient for tumor formation. The cells showed unlimited self-renewal ability with robust telomerase activities. In addition, they expressed typical glioma stem-like cell markers, such as CD133, CD15, and CD90. Moreover, these cells could form spheres in culture and differentiate into neuron-like, astrocyte-like, and oligodendrocyte-like cells. Finally, they also displayed resistance to the widely used brain cancer drug temozolomide. These iCSCs could provide important tools for studies of glioma biology and therapeutics development. Cancer Res; 76(17); 5143-50. ©2016 AACR. PMID:27364552

  5. Primary neuronal-astrocytic co-culture platform for neurotoxicity assessment of di-(2-ethylhexyl) phthalate.

    PubMed

    Wu, Yang; Li, Ke; Zuo, Haoxiao; Yuan, Ye; Sun, Yi; Yang, Xu

    2014-05-01

    Plastics such as polyvinyl chlorides (PVC) are widely used in many indoor constructed environments; however, their unbound chemicals, such as di-(2-ethylhexyl) phthalates (DEHP), can leach into the surrounding environment. This study focused on DEHP's effect on the central nervous system by determining the precise DEHP content in mice brain tissue after exposure to the chemical, to evaluate the specific exposure range. Primary neuronal-astrocyte co-culture systems were used as in vitro models for chemical hazard identification of DEHP. Oxidative stress was hypothesized as a probable mechanism involved, and therefore the total reactive oxygen species (ROS) concentration was determined as a biomarker of oxidative stress. In addition, NeuriteTracer, a neurite tracing plugin with ImageJ, was used to develop an assay for neurotoxicity to provide quantitative measurements of neurological parameters, such as neuronal number, neuron count and neurite length, all of which could indicate neurotoxic effects. The results showed that with 1 nmol/L DEHP exposure, there was a significant increase in ROS concentrations, indicating that the neuronal-astrocyte cultures were injured due to exposure to DEHP. In response, astrocyte proliferation (gliosis) was initiated, serving as a mechanism to maintain a homeostatic environment for neurons and protect neurons from toxic chemicals. There is a need to assess the cumulative effects of DEHP in animals to evaluate the possible uptake and effects on the human neuronal system from exposure to DEHP in the indoor environment.

  6. Primary adult human astrocytes as an ex vivo vehicle for beta-glucuronidase delivery in the brain.

    PubMed

    Serguera, C; Sarkis, C; Ridet, J L; Colin, P; Moullier, P; Mallet, J

    2001-06-01

    Astrocytes are a good candidate cell type for brain transplantation: They are endogenous to the CNS, they have efficient secretory machinery, and they play a major role in neuronal support. We assessed the potential of genetically modified primary adult human astrocytes as vehicles for the delivery of secreted molecules in the mammalian CNS. We report that such cells can be efficiently transduced by a recombinant adenoviral vector carrying the human beta-glucuronidase cDNA (Ad/CMV*beta-glu) and that the transduced astrocytes produce large amounts of the enzyme. Released beta-glucuronidase could be captured, in vitro, by primary neurons and astrocytes and by a neuroblastoma cell line and beta-glucuronidase-deficient fibroblasts. Following grafting into the mouse striatum, adult human astrocytes survived and expressed the transgene for at least 8 weeks. Moreover, the dosage of beta-glucuronidase activity within the grafted brains revealed high enzymatic levels at a long distance from the graft. These experiments document the grafting of engineered primary adult human astrocytes, allowing the release of a secreted therapeutic factor throughout the brain.

  7. Neurotoxic potential and cellular uptake of T-2 toxin in human astrocytes in primary culture.

    PubMed

    Weidner, Maria; Lenczyk, Marlies; Schwerdt, Gerald; Gekle, Michael; Humpf, Hans-Ulrich

    2013-03-18

    The trichothecene mycotoxin T-2 toxin, which is produced by fungi of the Fusarium species, is a worldwide occurring contaminant of cereal based food and feed. The cytotoxic properties of T-2 toxin are already well described with apoptosis being a major mechanism of action in various cell lines as well as in primary cells of different origin. However, only few data on neurotoxic properties of T-2 toxin are reported so far, but in vivo studies showed different effects of T-2 toxin on behavior as well as on levels of brain amines in animals. To further investigate the cytotoxic properties of T-2 toxin on cells derived from brain tissue, normal human astrocytes in primary culture (NHA) were used in this study. Besides studies of cytotoxicity, apoptosis (caspase-3-activation, Annexin V) and necrosis (LDH-release), the cellular uptake and metabolism of T-2 toxin in NHA was analyzed and compared to the uptake in an established human cell line (HT-29). The results show that human astrocytes were highly sensitive to the cytotoxic properties of T-2 toxin, and apoptosis, induced at low concentrations, was identified for the first time as the mechanism of toxic action in NHA. Furthermore, a strong accumulation of T-2 toxin in NHA and HT-29 cells was detected, and T-2 toxin was subjected to metabolism leading to HT-2 toxin, a commonly found metabolite after T-2 toxin incubation in both cell types. This formation seems to occur within the cells since incubations of T-2 toxin with cell depleted culture medium did not lead to any degradation of the parent toxin. The results of this study emphasize the neurotoxic potential of T-2 toxin in human astrocytes at low concentrations after short incubation times. PMID:23363530

  8. Arctigenin Increases Hemeoxygenase-1 Gene Expression by Modulating PI3K/AKT Signaling Pathway in Rat Primary Astrocytes.

    PubMed

    Jeong, Yeon-Hui; Park, Jin-Sun; Kim, Dong-Hyun; Kim, Hee-Sun

    2014-11-01

    In the present study, we found that the natural compound arctigenin inhibited hydrogen peroxide-induced reactive oxygen species (ROS) production in rat primary astrocytes. Since hemeoxygenase-1 (HO-1) plays a critical role as an antioxidant defense factor in the brain, we examined the effect of arctigenin on HO-1 expression in rat primary astrocytes. We found that arctigenin increased HO-1 mRNA and protein levels. Arctigenin also increases the nuclear translocation and DNA binding of Nrf2/c-Jun to the antioxidant response element (ARE) on HO-1 promoter. In addition, arctigenin increased ARE-mediated transcriptional activities in rat primary astrocytes. Further mechanistic studies revealed that arctigenin increased the phosphorylation of AKT, a downstream substrate of phosphatidylinositol 3-kinase (PI3K). Treatment of cells with a PI3K-specific inhibitor, LY294002, suppressed the HO-1 expression, Nrf2 DNA binding and ARE-mediated transcriptional activities in arctigenin-treated astrocyte cells. The results collectively suggest that PI3K/AKT signaling pathway is at least partly involved in HO-1 expression by arctigenin via modulation of Nrf2/ARE axis in rat primary astrocytes.

  9. Arctigenin Increases Hemeoxygenase-1 Gene Expression by Modulating PI3K/AKT Signaling Pathway in Rat Primary Astrocytes.

    PubMed

    Jeong, Yeon-Hui; Park, Jin-Sun; Kim, Dong-Hyun; Kim, Hee-Sun

    2014-11-01

    In the present study, we found that the natural compound arctigenin inhibited hydrogen peroxide-induced reactive oxygen species (ROS) production in rat primary astrocytes. Since hemeoxygenase-1 (HO-1) plays a critical role as an antioxidant defense factor in the brain, we examined the effect of arctigenin on HO-1 expression in rat primary astrocytes. We found that arctigenin increased HO-1 mRNA and protein levels. Arctigenin also increases the nuclear translocation and DNA binding of Nrf2/c-Jun to the antioxidant response element (ARE) on HO-1 promoter. In addition, arctigenin increased ARE-mediated transcriptional activities in rat primary astrocytes. Further mechanistic studies revealed that arctigenin increased the phosphorylation of AKT, a downstream substrate of phosphatidylinositol 3-kinase (PI3K). Treatment of cells with a PI3K-specific inhibitor, LY294002, suppressed the HO-1 expression, Nrf2 DNA binding and ARE-mediated transcriptional activities in arctigenin-treated astrocyte cells. The results collectively suggest that PI3K/AKT signaling pathway is at least partly involved in HO-1 expression by arctigenin via modulation of Nrf2/ARE axis in rat primary astrocytes. PMID:25489416

  10. Arctigenin Increases Hemeoxygenase-1 Gene Expression by Modulating PI3K/AKT Signaling Pathway in Rat Primary Astrocytes

    PubMed Central

    Jeong, Yeon-Hui; Park, Jin-Sun; Kim, Dong-Hyun; Kim, Hee-Sun

    2014-01-01

    In the present study, we found that the natural compound arctigenin inhibited hydrogen peroxide-induced reactive oxygen species (ROS) production in rat primary astrocytes. Since hemeoxygenase-1 (HO-1) plays a critical role as an antioxidant defense factor in the brain, we examined the effect of arctigenin on HO-1 expression in rat primary astrocytes. We found that arctigenin increased HO-1 mRNA and protein levels. Arctigenin also increases the nuclear translocation and DNA binding of Nrf2/c-Jun to the antioxidant response element (ARE) on HO-1 promoter. In addition, arctigenin increased ARE-mediated transcriptional activities in rat primary astrocytes. Further mechanistic studies revealed that arctigenin increased the phosphorylation of AKT, a downstream substrate of phosphatidylinositol 3-kinase (PI3K). Treatment of cells with a PI3K-specific inhibitor, LY294002, suppressed the HO-1 expression, Nrf2 DNA binding and ARE-mediated transcriptional activities in arctigenin-treated astrocyte cells. The results collectively suggest that PI3K/AKT signaling pathway is at least partly involved in HO-1 expression by arctigenin via modulation of Nrf2/ARE axis in rat primary astrocytes. PMID:25489416

  11. Propofol treatment modulates neurite extension regulated by immunologically challenged rat primary astrocytes: a possible role of PAI-1.

    PubMed

    Ko, Hyun Myung; Joo, So Hyun; Lee, Sung Hoon; Kim, Hee Jin; Lee, Seung-Hyun; Cheong, Jae Hoon; Ryu, Jong Hoon; Kim, Jeong Min; Koo, Bon-Nyeo; Shin, Chan Young

    2015-04-01

    Propofol, a widely used anesthetic, regulates neurological processes including neurotoxicity, neuroprotection, glial activation, synaptic plasticity and neuronal maturation. Tissue plasminogen activator/tissue plasminogen activator inhibitor-1 (tPA/PAI-1) in CNS acts as a neuromodulator regulating synaptic plasticity, neurite outgrowth, seizure spreading and cell survival. Here, we investigated the effects of propofol on tPA/PAI-1 system using cultured neurons and astrocytes and their role in the regulation of neurite extension. Cultured rat primary astrocytes were treated with propofol (1-10 µM) and LPS (10 ng/ml). The expression of functional tPA/PAI-1 was examined by casein zymography, Western blot and RT-PCR. Alternatively, culture supernatants were added to cultured rat primary neuron to investigate the effects on neurite extension. Propofol alone did not affect tPA activity in rat primary cortical neuron. Similarly, propofol alone changed neither tPA nor PAI-1 activity in rat primary astrocytes. In immunologically challenged situation using LPS, propofol synergistically increased expression of PAI-1 in rat primary astrocytes without affecting tPA expression in a manner dependent on MAPKs activation. Increased expression of PAI-1 reduced tPA activity in LPS plus propofol-treated rat primary astrocytes. Consistent with the critical role of tPA activity in the regulation of neurite extension (Cho et al. 2013), the diminished tPA activity in astrocyte culture supernatants resulted in decreased neurite extension when administered to cultured rat primary cortical neuron. The results from the present study suggest that propofol, especially in immunologically-challenged situation, dysregulates tPA/PAI-1 system in brain. Whether the dysregulated tPA/PAI-1 activity adversely affects neural differentiation as well as regeneration of neuron in vivo should be empirically determined in the future.

  12. GABA uptake in astrocytes in primary cultures: coupling with two sodium ions.

    PubMed

    Larsson, O M; Hertz, L; Schousboe, A

    1980-01-01

    The influence of sodium ions on GABA uptake into astrocytes in primary cultures has been investigated performing kinetic analysis of GABA uptake at different sodium concentrations in the range 16 to 151 mM. These investigations reveal that sodium affects both the Km and the Vmax of the saturable component of the astroglial GABA uptake. Uptake rates as a function of the sodium concentration at high GABA concentrations (greater than or equal to 50 microM) were clearly sigmoid whereas at lower GABA concentrations this sigmoid shape was not obvious. Accordingly, Hill plots of the sodium dependency at high GABA concentrations exhibited straight lines with slopes of 2.0 to 2.5, suggesting that the coupling ratio between sodium and GABA is at least two. Corresponding Hill plots at lower GABA concentrations exhibited slopes of 1.6 to 1.8. Moreover, plots of 1/v versus 1/Na2 gave better fits to straight lines than plots of 1/v versus 1/Na which were curvilinear upward. Again, this curvilinearity was more pronounced at high GABA concentrations that at low GABA concentrations. From these results it is concluded that GABA uptake into astrocytes in primary cultures requires the binding of at least two sodium ions per GABA molecule transported.

  13. Trafficking of astrocytic vesicles in hippocampal slices

    SciTech Connect

    Potokar, Maja; Kreft, Marko; Lee, So-Young; Takano, Hajime; Haydon, Philip G.; Zorec, Robert

    2009-12-25

    The increasingly appreciated role of astrocytes in neurophysiology dictates a thorough understanding of the mechanisms underlying the communication between astrocytes and neurons. In particular, the uptake and release of signaling substances into/from astrocytes is considered as crucial. The release of different gliotransmitters involves regulated exocytosis, consisting of the fusion between the vesicle and the plasma membranes. After fusion with the plasma membrane vesicles may be retrieved into the cytoplasm and may continue to recycle. To study the mobility implicated in the retrieval of secretory vesicles, these structures have been previously efficiently and specifically labeled in cultured astrocytes, by exposing live cells to primary and secondary antibodies. Since the vesicle labeling and the vesicle mobility properties may be an artifact of cell culture conditions, we here asked whether the retrieving exocytotic vesicles can be labeled in brain tissue slices and whether their mobility differs to that observed in cell cultures. We labeled astrocytic vesicles and recorded their mobility with two-photon microscopy in hippocampal slices from transgenic mice with fluorescently tagged astrocytes (GFP mice) and in wild-type mice with astrocytes labeled by Fluo4 fluorescence indicator. Glutamatergic vesicles and peptidergic granules were labeled by the anti-vesicular glutamate transporter 1 (vGlut1) and anti-atrial natriuretic peptide (ANP) antibodies, respectively. We report that the vesicle mobility parameters (velocity, maximal displacement and track length) recorded in astrocytes from tissue slices are similar to those reported previously in cultured astrocytes.

  14. Protective effects of parecoxib on rat primary astrocytes from oxidative stress induced by hydrogen peroxide* #

    PubMed Central

    Ling, Yun-zhi; Li, Xiao-hong; Yu, Li; Zhang, Ye; Liang, Qi-sheng; Yang, Xiao-di; Wang, Hong-tao

    2016-01-01

    Objective: To investigate the protective effects of parecoxib from oxidative stress induced by hydrogen peroxide (H2O2) in rat astrocytes in vitro. Methods: All experiments included 4 groups: (1) negative control (NC) group, without any treatment; (2) H2O2 treatment group, 100 μmol/L H2O2 treatment for 24 h; (3) and (4) parecoxib pretreatment groups, 80 and 160 μmol/L parecoxib treatment for 24 h, respectively, and then treated with 100 μmol/L H2O2. Several indices were investigated, and the expressions of Bax, Bcl-2, and brain-derived neurotrophic factor (BDNF) were quantified. Results: Compared to the NC group, exposure to H2O2 resulted in significant morphological changes, which could be reversed by pretreatment of parecoxib. In addition, H2O2 treatment led to loss of viability (P=0.026) and increased intracellular reactive oxygen species (ROS) levels (P<0.001), and induced apoptosis (P<0.01) in the primary astrocytes relative to the NC group. However, in the parecoxib pretreatment groups, all the above changes reversed significantly (P<0.05) as compared to the H2O2 treatment group, and were nearly unchanged when compared to the NC group. Mechanical investigation showed that dysregulated Bax, Bcl-2, and BDNF could be implicated in these changes. Conclusions: Our results indicated that parecoxib provided a protective effect from oxidative stress induced by exposure to H2O2. PMID:27604861

  15. PPAR-γ Impairment Alters Peroxisome Functionality in Primary Astrocyte Cell Cultures

    PubMed Central

    Di Cesare Mannelli, Lorenzo; Zanardelli, Matteo; Micheli, Laura; Ghelardini, Carla

    2014-01-01

    Peroxisomes provide glial cells with protective functions against the harmful effects of H2O2 on neurons and peroxisome impairment results in nervous lesions. Agonists of the γ-subtype of the Peroxisome-Proliferator-Activated-Receptors (PPAR) have been proposed as neuroprotective agents in neurodegenerative disorders. Nevertheless, the role of PPAR-γ alterations in pathophysiological mechanisms and the relevance of peroxisome functions in the PPAR-γ effects are not yet clear. In a primary cell culture of rat astrocytes, the irreversible PPAR-γ antagonist GW9662 concentration-dependently decreased the activity of catalase, the most important antioxidant defense enzyme in peroxisomes. Catalase functionality recovered in a few days and the PPAR-γ agonist rosiglitazone promoted reversal of enzymatic damage. The reversible antagonist G3335 reduced both the activity and expression of catalase in a rosiglitazone-prevented manner. G3335 reduced also the glutathione reductase expression, indicating that enzyme involved in glutathione regeneration was compromised. Neither the PPAR-α target gene Acyl-Coenzyme-A-oxidase-1 nor the mitochondrial detoxifying enzyme NADH:ubiquinone-oxidoreductase (NDFUS3) was altered by PPAR-γ inhibition. In conclusion, PPAR-γ inhibition induced impairment of catalase in astrocytes. A general decrease of the antioxidant defenses of the cell suggests that a PPAR-γ hypofunction could participate in neurodegenerative mechanisms through peroxisomal damage. This series of experiments could be a useful model for studying compounds able to restore peroxisome functionality. PMID:24729976

  16. Substance P receptors in primary cultures of cortical astrocytes from the mouse.

    PubMed Central

    Torrens, Y; Beaujouan, J C; Saffroy, M; Daguet de Montety, M C; Bergström, L; Glowinski, J

    1986-01-01

    Binding sites for substance P were labeled on intact cortical glial cells from newborn mice in primary culture using 125I-labeled Bolton-Hunter-labeled substance P. Maximal specific binding (95% of total binding) was reached after 2-3 weeks in culture. The binding was saturable, reversible, and temperature dependent. Scatchard and Hill analysis revealed a single population of noninteracting high-affinity binding sites (Kd, 0.33 nM; Bmax, 14.4 fmol per dish). Competition studies made with tachykinins and substance P analogues indicated that the characteristics of the 125I-labeled Bolton-Hunter labeled substance P binding sites on glial cells were identical to those on rat brain synaptosomes. 125I-labeled Bolton-Hunter labeled substance P binding sites were visualized by autoradiography, and differences in the intensity of labeling were seen among astrocytes. Substance P was found to stimulate phosphatidylinositol turnover; the EC50 value (0.36 nM) was identical to the IC50 value (0.38 nM) determined in binding studies. 125I-labeled Bolton-Hunter labeled substance P binding sites were also found on astrocytes derived from other brain structures and from the spinal cord of mice. Images PMID:2431412

  17. Cytotoxicity Study on Luminescent Nanocrystals Containing Phospholipid Micelles in Primary Cultures of Rat Astrocytes.

    PubMed

    Latronico, Tiziana; Depalo, Nicoletta; Valente, Gianpiero; Fanizza, Elisabetta; Laquintana, Valentino; Denora, Nunzio; Fasano, Anna; Striccoli, Marinella; Colella, Matilde; Agostiano, Angela; Curri, M Lucia; Liuzzi, Grazia Maria

    2016-01-01

    Luminescent colloidal nanocrystals (NCs) are emerging as a new tool in neuroscience field, representing superior optical probes for cellular imaging and medical diagnosis of neurological disorders with respect to organic fluorophores. However, only a limited number of studies have, so far, explored NC applications in primary neurons, glia and related cells. Indeed astrocytes, as resident cells in the central nervous system (CNS), play an important pathogenic role in several neurodegenerative and neuroinflammatory diseases, therefore enhanced imaging tools for their thorough investigation are strongly amenable. Here, a comprehensive and systematic study on the in vitro toxicological effect of core-shell type luminescent CdSe@ZnS NCs incorporated in polyethylene glycol (PEG) terminated phospholipid micelles on primary cultures of rat astrocytes was carried out. Cytotoxicity response of empty micelles based on PEG modified phospholipids was compared to that of their NC containing counterpart, in order to investigate the effect on cell viability of both inorganic NCs and micelles protecting NC surface. Furthermore, since the surface charge and chemistry influence cell interaction and toxicity, effect of two different functional groups terminating PEG-modified phospholipid micelles, namely amine and carboxyl group, respectively, was evaluated against bare micelles, showing that carboxyl group was less toxic. The ability of PEG-lipid micelles to be internalized into the cells was qualitatively and quantitatively assessed by fluorescence microscopy and photoluminescence (PL) assay. The results of the experiments clearly demonstrate that, once incorporated into the micelles, a low, not toxic, concentration of NCs is sufficient to be distinctly detected within cells. The overall study provides essential indications to define the optimal experimental conditions to effectively and profitably use the proposed luminescent colloidal NCs as optical probe for future in vivo

  18. Cytotoxicity Study on Luminescent Nanocrystals Containing Phospholipid Micelles in Primary Cultures of Rat Astrocytes

    PubMed Central

    Valente, Gianpiero; Fanizza, Elisabetta; Laquintana, Valentino; Denora, Nunzio; Fasano, Anna; Striccoli, Marinella; Colella, Matilde; Agostiano, Angela; Curri, M. Lucia; Liuzzi, Grazia Maria

    2016-01-01

    Luminescent colloidal nanocrystals (NCs) are emerging as a new tool in neuroscience field, representing superior optical probes for cellular imaging and medical diagnosis of neurological disorders with respect to organic fluorophores. However, only a limited number of studies have, so far, explored NC applications in primary neurons, glia and related cells. Indeed astrocytes, as resident cells in the central nervous system (CNS), play an important pathogenic role in several neurodegenerative and neuroinflammatory diseases, therefore enhanced imaging tools for their thorough investigation are strongly amenable. Here, a comprehensive and systematic study on the in vitro toxicological effect of core-shell type luminescent CdSe@ZnS NCs incorporated in polyethylene glycol (PEG) terminated phospholipid micelles on primary cultures of rat astrocytes was carried out. Cytotoxicity response of empty micelles based on PEG modified phospholipids was compared to that of their NC containing counterpart, in order to investigate the effect on cell viability of both inorganic NCs and micelles protecting NC surface. Furthermore, since the surface charge and chemistry influence cell interaction and toxicity, effect of two different functional groups terminating PEG-modified phospholipid micelles, namely amine and carboxyl group, respectively, was evaluated against bare micelles, showing that carboxyl group was less toxic. The ability of PEG-lipid micelles to be internalized into the cells was qualitatively and quantitatively assessed by fluorescence microscopy and photoluminescence (PL) assay. The results of the experiments clearly demonstrate that, once incorporated into the micelles, a low, not toxic, concentration of NCs is sufficient to be distinctly detected within cells. The overall study provides essential indications to define the optimal experimental conditions to effectively and profitably use the proposed luminescent colloidal NCs as optical probe for future in vivo

  19. Cytotoxicity Study on Luminescent Nanocrystals Containing Phospholipid Micelles in Primary Cultures of Rat Astrocytes.

    PubMed

    Latronico, Tiziana; Depalo, Nicoletta; Valente, Gianpiero; Fanizza, Elisabetta; Laquintana, Valentino; Denora, Nunzio; Fasano, Anna; Striccoli, Marinella; Colella, Matilde; Agostiano, Angela; Curri, M Lucia; Liuzzi, Grazia Maria

    2016-01-01

    Luminescent colloidal nanocrystals (NCs) are emerging as a new tool in neuroscience field, representing superior optical probes for cellular imaging and medical diagnosis of neurological disorders with respect to organic fluorophores. However, only a limited number of studies have, so far, explored NC applications in primary neurons, glia and related cells. Indeed astrocytes, as resident cells in the central nervous system (CNS), play an important pathogenic role in several neurodegenerative and neuroinflammatory diseases, therefore enhanced imaging tools for their thorough investigation are strongly amenable. Here, a comprehensive and systematic study on the in vitro toxicological effect of core-shell type luminescent CdSe@ZnS NCs incorporated in polyethylene glycol (PEG) terminated phospholipid micelles on primary cultures of rat astrocytes was carried out. Cytotoxicity response of empty micelles based on PEG modified phospholipids was compared to that of their NC containing counterpart, in order to investigate the effect on cell viability of both inorganic NCs and micelles protecting NC surface. Furthermore, since the surface charge and chemistry influence cell interaction and toxicity, effect of two different functional groups terminating PEG-modified phospholipid micelles, namely amine and carboxyl group, respectively, was evaluated against bare micelles, showing that carboxyl group was less toxic. The ability of PEG-lipid micelles to be internalized into the cells was qualitatively and quantitatively assessed by fluorescence microscopy and photoluminescence (PL) assay. The results of the experiments clearly demonstrate that, once incorporated into the micelles, a low, not toxic, concentration of NCs is sufficient to be distinctly detected within cells. The overall study provides essential indications to define the optimal experimental conditions to effectively and profitably use the proposed luminescent colloidal NCs as optical probe for future in vivo

  20. Modulation of copper accumulation and copper-induced toxicity by antioxidants and copper chelators in cultured primary brain astrocytes.

    PubMed

    Bulcke, Felix; Santofimia-Castaño, Patricia; Gonzalez-Mateos, Antonio; Dringen, Ralf

    2015-10-01

    Copper is essential for several important cellular processes, but an excess of copper can also lead to oxidative damage. In brain, astrocytes are considered to play a pivotal role in the copper homeostasis and antioxidative defence. To investigate whether antioxidants and copper chelators can modulate the uptake and the toxicity of copper ions in brain astrocytes, we used primary astrocytes as cell culture model. These cells accumulated substantial amounts of copper during exposure to copper chloride. Copper accumulation was accompanied by a time- and concentration-dependent loss in cell viability, as demonstrated by a lowering in cellular MTT reduction capacity and by an increase in membrane permeability for propidium iodide. During incubations in the presence of the antioxidants ascorbate, trolox or ebselen, the specific cellular copper content and the toxicity in copper chloride-treated astrocyte cultures were strongly increased. In contrast, the presence of the copper chelators bathocuproine disulfonate or tetrathiomolybdate lowered the cellular copper accumulation and the copper-induced as well as the ascorbate-accelerated copper toxicity was fully prevented. These data suggest that predominantly the cellular content of copper determines copper-induced toxicity in brain astrocytes. PMID:26302925

  1. Modulation of copper accumulation and copper-induced toxicity by antioxidants and copper chelators in cultured primary brain astrocytes.

    PubMed

    Bulcke, Felix; Santofimia-Castaño, Patricia; Gonzalez-Mateos, Antonio; Dringen, Ralf

    2015-10-01

    Copper is essential for several important cellular processes, but an excess of copper can also lead to oxidative damage. In brain, astrocytes are considered to play a pivotal role in the copper homeostasis and antioxidative defence. To investigate whether antioxidants and copper chelators can modulate the uptake and the toxicity of copper ions in brain astrocytes, we used primary astrocytes as cell culture model. These cells accumulated substantial amounts of copper during exposure to copper chloride. Copper accumulation was accompanied by a time- and concentration-dependent loss in cell viability, as demonstrated by a lowering in cellular MTT reduction capacity and by an increase in membrane permeability for propidium iodide. During incubations in the presence of the antioxidants ascorbate, trolox or ebselen, the specific cellular copper content and the toxicity in copper chloride-treated astrocyte cultures were strongly increased. In contrast, the presence of the copper chelators bathocuproine disulfonate or tetrathiomolybdate lowered the cellular copper accumulation and the copper-induced as well as the ascorbate-accelerated copper toxicity was fully prevented. These data suggest that predominantly the cellular content of copper determines copper-induced toxicity in brain astrocytes.

  2. Testosterone Protects Mitochondrial Function and Regulates Neuroglobin Expression in Astrocytic Cells Exposed to Glucose Deprivation.

    PubMed

    Toro-Urrego, Nicolas; Garcia-Segura, Luis M; Echeverria, Valentina; Barreto, George E

    2016-01-01

    Testosterone is a hormone that has been shown to confer neuroprotection from different insults affecting the central nervous system (CNS). Testosterone induces this protection by different mechanisms that include the activation of anti-apoptotic pathways that are directly implicated in neuronal survival. However, little attention has been devoted to its actions on glial cells. In the present study, we have assessed whether testosterone exerts protection in a human astrocyte cell model, the T98G cells. Our results indicate that testosterone improves cell survival and mitochondrial membrane potential and reduces nuclear fragmentation and reactive oxygen species (ROS) generation. These effects were accompanied by a positive regulation of neuroglobin, an oxygen-binding and sensor protein, which may serve as a regulator of ROS and nitrogen reactive species (NOS), and these protective effects of testosterone may be at least in part mediated by estradiol and DHT. In conclusion, these findings suggest that astroglia may mediate some of the protective actions of testosterone in the brain upon pathological conditions. PMID:27445795

  3. Testosterone Protects Mitochondrial Function and Regulates Neuroglobin Expression in Astrocytic Cells Exposed to Glucose Deprivation.

    PubMed

    Toro-Urrego, Nicolas; Garcia-Segura, Luis M; Echeverria, Valentina; Barreto, George E

    2016-01-01

    Testosterone is a hormone that has been shown to confer neuroprotection from different insults affecting the central nervous system (CNS). Testosterone induces this protection by different mechanisms that include the activation of anti-apoptotic pathways that are directly implicated in neuronal survival. However, little attention has been devoted to its actions on glial cells. In the present study, we have assessed whether testosterone exerts protection in a human astrocyte cell model, the T98G cells. Our results indicate that testosterone improves cell survival and mitochondrial membrane potential and reduces nuclear fragmentation and reactive oxygen species (ROS) generation. These effects were accompanied by a positive regulation of neuroglobin, an oxygen-binding and sensor protein, which may serve as a regulator of ROS and nitrogen reactive species (NOS), and these protective effects of testosterone may be at least in part mediated by estradiol and DHT. In conclusion, these findings suggest that astroglia may mediate some of the protective actions of testosterone in the brain upon pathological conditions.

  4. Testosterone Protects Mitochondrial Function and Regulates Neuroglobin Expression in Astrocytic Cells Exposed to Glucose Deprivation

    PubMed Central

    Toro-Urrego, Nicolas; Garcia-Segura, Luis M.; Echeverria, Valentina; Barreto, George E.

    2016-01-01

    Testosterone is a hormone that has been shown to confer neuroprotection from different insults affecting the central nervous system (CNS). Testosterone induces this protection by different mechanisms that include the activation of anti-apoptotic pathways that are directly implicated in neuronal survival. However, little attention has been devoted to its actions on glial cells. In the present study, we have assessed whether testosterone exerts protection in a human astrocyte cell model, the T98G cells. Our results indicate that testosterone improves cell survival and mitochondrial membrane potential and reduces nuclear fragmentation and reactive oxygen species (ROS) generation. These effects were accompanied by a positive regulation of neuroglobin, an oxygen-binding and sensor protein, which may serve as a regulator of ROS and nitrogen reactive species (NOS), and these protective effects of testosterone may be at least in part mediated by estradiol and DHT. In conclusion, these findings suggest that astroglia may mediate some of the protective actions of testosterone in the brain upon pathological conditions. PMID:27445795

  5. Silver nanoparticles induce tight junction disruption and astrocyte neurotoxicity in a rat blood–brain barrier primary triple coculture model

    PubMed Central

    Xu, Liming; Dan, Mo; Shao, Anliang; Cheng, Xiang; Zhang, Cuiping; Yokel, Robert A; Takemura, Taro; Hanagata, Nobutaka; Niwa, Masami; Watanabe, Daisuke

    2015-01-01

    Background Silver nanoparticles (Ag-NPs) can enter the brain and induce neurotoxicity. However, the toxicity of Ag-NPs on the blood–brain barrier (BBB) and the underlying mechanism(s) of action on the BBB and the brain are not well understood. Method To investigate Ag-NP suspension (Ag-NPS)-induced toxicity, a triple coculture BBB model of rat brain microvascular endothelial cells, pericytes, and astrocytes was established. The BBB permeability and tight junction protein expression in response to Ag-NPS, NP-released Ag ions, and polystyrene-NP exposure were investigated. Ultrastructural changes of the microvascular endothelial cells, pericytes, and astrocytes were observed using transmission electron microscopy (TEM). Global gene expression of astrocytes was measured using a DNA microarray. Results A triple coculture BBB model of primary rat brain microvascular endothelial cells, pericytes, and astrocytes was established, with the transendothelial electrical resistance values >200 Ω·cm2. After Ag-NPS exposure for 24 hours, the BBB permeability was significantly increased and expression of the tight junction (TJ) protein ZO-1 was decreased. Discontinuous TJs were also observed between microvascular endothelial cells. After Ag-NPS exposure, severe mitochondrial shrinkage, vacuolations, endoplasmic reticulum expansion, and Ag-NPs were observed in astrocytes by TEM. Global gene expression analysis showed that three genes were upregulated and 20 genes were downregulated in astrocytes treated with Ag-NPS. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the 23 genes were associated with metabolic processes, biosynthetic processes, response to stimuli, cell death, the MAPK pathway, and so on. No GO term and KEGG pathways were changed in the released-ion or polystyrene-NP groups. Ag-NPS inhibited the antioxidant defense of the astrocytes by increasing thioredoxin interacting protein, which inhibits the Trx system, and

  6. Glucose deprivation increases hydrogen peroxide level in immunostimulated rat primary astrocytes.

    PubMed

    Choi, Ji Woong; Shin, Chan Young; Yoo, Byoung Kwon; Choi, Min Sik; Lee, Woo Jong; Han, Byoung Hee; Kim, Won-Ki; Kim, Hyoung-Chun; Ko, Kwang Ho

    2004-03-01

    Activated astrocytes produce a large amount of bioactive molecules, including reactive oxygen and nitrogen species. Astrocytes are in general resistant to those reactive species. However, we previously reported that immunostimulated astrocytes became highly vulnerable to metabolic insults, such as glucose deprivation. In this study, we investigated whether H(2)O(2) production was associated with the increased vulnerability. Glucose deprivation for up to 8 hr did not change the intracellular level of H(2)O(2) in astrocytes. Treatment with lipopolysaccharide plus interferon-gamma for 48 hr evoked astroglial H(2)O(2) production; however, no apparent death or injury was observed in immunostimulated astrocytes. Glucose deprivation after 48 hr of immunostimulation markedly increased H(2)O(2) level, depleted adenosine triphosphate (ATP), and enhanced lactate dehydrogenase (LDH) release. The ATP depletion and LDH release were in part prevented by catalase, mannitol, and N-acetyl-L-cysteine. The enhanced level of H(2)O(2) in glucose-deprived immunostimulated astrocytes appeared to be secondary to the depletion of reduced glutathione. 4-(2-Aminoethyl)bebzenesulfonyl fluoride (AEBSF), an inhibitor of NADPH oxidase, reduced H(2)O(2) level and LDH release in glucose-deprived immunostimulated astrocytes. H(2)O(2), either endogenously produced or exogenously added, depolarized mitochondrial transmembrane potential in glucose-deprived astrocytes, leading to their ATP depletion and death. The present results strongly indicate that glucose deprivation causes deterioration of immunostimulated astrocytes by increasing the intracellular concentration of H(2)O(2).

  7. Phosphorylation in the C-terminal domain of Aquaporin-4 is required for Golgi transition in primary cultured astrocytes

    SciTech Connect

    Kadohira, Ikuko; Abe, Yoichiro Nuriya, Mutsuo; Sano, Kazumi; Tsuji, Shoji; Arimitsu, Takeshi; Yoshimura, Yasunori; Yasui, Masato

    2008-12-12

    Aquaporin-4 (AQP4) is expressed in the perivascular and subpial astrocytes end-feet in mammalian brain, and plays a critical component of an integrated water and potassium homeostasis. Here we examine whether AQP4 is phosphorylated in primary cultured mouse astrocytes. Astrocytes were metabolically labeled with [{sup 32}P]phosphoric acid, then AQP4 was immunoprecipitated with anti-AQP4 antibody. We observed that AQP4 was constitutively phosphorylated, which is reduced by treatment with protein kinase CK2 inhibitors. To elucidate the phosphorylation of AQP4 by CK2, myc-tagged wild-type or mutant AQP4 was transiently transfected in primary cultured astrocytes. Substitution of Ala residues for four putative CK2 phosphorylation sites in the C terminus abolished the phosphorylation of AQP4. Immunofluorescent microscopy revealed that the quadruple mutant was localized in the Golgi apparatus. These observations indicate that the C-terminal domain of AQP4 is constitutively phosphorylated at least in part by protein kinase CK2 and it is required for Golgi transition.

  8. LCAT synthesized by primary astrocytes esterifies cholesterol on glia-derived lipoproteins

    PubMed Central

    Hirsch-Reinshagen, Veronica; Donkin, James; Stukas, Sophie; Chan, Jennifer; Wilkinson, Anna; Fan, Jianjia; Parks, John S.; Kuivenhoven, Jan Albert; Lütjohann, Dieter; Pritchard, Haydn; Wellington, Cheryl L.

    2009-01-01

    Lipid trafficking in the brain is essential for the maintenance and repair of neuronal membranes, especially after neurotoxic insults. However, brain lipid metabolism is not completely understood. In plasma, LCAT catalyses the esterification of free cholesterol on circulating lipoproteins, a key step in the maturation of HDL. Brain lipoproteins are apolipoprotein E (apoE)-containing, HDL-like particles secreted initially as lipid-poor discs by glial cells. LCAT is synthesized within the brain, suggesting that it may play a key role in the maturation of these lipoproteins. Here we demonstrate that astrocytes are the primary producers of brain LCAT. This LCAT esterifies free cholesterol on nascent apoE-containing lipopoproteins secreted from glia. ApoE is the major LCAT activator in glia-conditioned media (GCM), and both the cholesterol transporter ABCA1 and apoE are required to generate glial LCAT substrate particles. LCAT deficiency leads to the appearance of abnormal ∼8 nm particles in GCM, and exogenous LCAT restores the lipoprotein particle distribution to the wild-type (WT) pattern. In vivo, complete LCAT deficiency results in a dramatic increase in apoE-HDL and reduced apolipoprotein A-I (apoA-I)-HDL in murine cerebrospinal fluid (CSF). These data show that brain LCAT esterifies cholesterol on glial-derived apoE-lipoproteins, and influences CSF apoE and apoA-I levels. PMID:19065001

  9. Actin is not required for nanotubular protrusions of primary astrocytes grown on metal nano-lawn.

    PubMed

    Gimsa, Ulrike; Iglic, Ales; Fiedler, Stefan; Zwanzig, Michael; Kralj-Iglic, Veronika; Jonas, Ludwig; Gimsa, Jan

    2007-01-01

    We used sub-micron metal rod decorated surfaces, 'nano-lawn' structures, as a substrate to study cell-to-cell and cell-to-surface interactions of primary murine astrocytes. These cells form thin membranous tubes with diameters of less than 100 nm and a length of several microns, which make contact to neighboring cells and the substrate during differentiation. While membrane protrusions grow on top of the nano-lawn pillars, nuclei sink to the bottom of the substrate. We observed gondola-like structures along those tubes, suggestive of their function as transport vehicles. Elements of the cytoskeleton such as actin fibers are commonly believed to be essential for triggering the onset and growth of tubular membrane protrusions. A rope-pulling mechanism along actin fibers has recently been proposed to account for the transport or exchange of cellular material between cells. We present evidence for a complementary mechanism that promotes growth and stabilization of the observed tubular protrusions of cell membranes. This mechanism does not require active involvement of actin fibers as the formation of membrane protrusions could not be prevented by suppressing polymerization of actin by latrunculin B. Also theoretically, actin fibers are not essential for the growing and stability of nanotubes since curvature-driven self-assembly of interacting anisotropic raft elements is sufficient for the spontaneous formation of thin nano-tubular membrane protrusions. PMID:17520481

  10. Aquaporin 4-dependent expression of glial fibrillary acidic protein and tenascin-C in activated astrocytes in stab wound mouse brain and in primary culture.

    PubMed

    Ikeshima-Kataoka, Hiroko; Abe, Yoichiro; Yasui, Masato

    2015-01-01

    We previously reported that aquaporin 4 (AQP4) has a neuroimmunological function via astrocytes and microglial cells involving osteopontin. AQP4 is a water channel localized in the endofoot of astrocytes in the brain, and its expression is upregulated after a stab wound to the mouse brain or the injection of methylmercury in common marmosets. In this study, the correlation between the expression of AQP4 and the expression of glial fibrillary acidic protein (GFAP) or tenascin-C (TN-C) in reactive astrocytes was examined in primary cultures and brain tissues of AQP4-deficient mice (AQP4/KO). In the absence of a stab wound to the brain or of any stimulation of the cells, the expressions of both GFAP and TN-C were lower in astrocytes from AQP4/KO mice than in those from wild-type (WT) mice. High levels of GFAP and TN-C expression were observed in activated astrocytes after a stab wound to the brain in WT mice; however, the expressions of GFAP and TN-C were insignificant in AQP4/KO mice. Furthermore, lipopolysaccharide (LPS) stimulation activated primary culture of astrocytes and upregulated GFAP and TN-C expression in cells from WT mice, whereas the expressions of GFAP and TN-C were slightly upregulated in cells from AQP4/KO mice. Moreover, the stimulation of primary culture of astrocytes with LPS also upregulated inflammatory cytokines in cells from WT mice, whereas modest increases were observed in cells from AQP4/KO mice. These results suggest that AQP4 expression accelerates GFAP and TN-C expression in activated astrocytes induced by a stab wound in the mouse brain and LPS-stimulated primary culture of astrocytes.

  11. Inhibition of astrocyte metabolism is not the primary mechanism for anaesthetic hypnosis.

    PubMed

    Voss, Logan J; Harvey, Martyn G; Sleigh, James W

    2016-01-01

    Astrocytes have been promoted as a possible mechanistic target for anaesthetic hypnosis. The aim of this study was to explore this using the neocortical brain slice preparation. The methods were in two parts. Firstly, multiple general anaesthetic compounds demonstrating varying in vivo hypnotic potency were analysed for their effect on "zero-magnesium" seizure-like event (SLE) activity in mouse neocortical slices. Subsequently, the effect of astrocyte metabolic inhibition was investigated in neocortical slices, and compared with that of the anaesthetic drugs. The rationale was that, if suppression of astrocytes was both necessary and sufficient to cause hypnosis in vivo, then inhibition of astrocytic metabolism in slices should mimic the anaesthetic effect. In vivo anaesthetic potency correlated strongly with the magnitude of reduction in SLE frequency in neocortical slices (R(2) 37.7 %, p = 0.002). Conversely, SLE frequency and length were significantly enhanced during exposure to both fluoroacetate (23 and 20 % increase, respectively, p < 0.01) and aminoadipate (12 and 38 % increase, respectively, p < 0.01 and p < 0.05). The capacity of an anaesthetic agent to reduce SLE frequency in the neocortical slice is a good indicator of its in vivo hypnotic potency. The results do not support the hypothesis that astrocytic metabolic inhibition is a mechanism of anaesthetic hypnosis. PMID:27462489

  12. Role of autophagy in methylmercury-induced neurotoxicity in rat primary astrocytes

    PubMed Central

    Yuntao, Fang; Chenjia, Guo; Panpan, Zhang; Wenjun, Zhao; Suhua, Wang; Guangwei, Xing; Haifeng, Shi; Wanxin, Peng; Aschner, Michael; Rongzhu, Lu

    2016-01-01

    Autophagy is an evolutionarily conserved process in which cytoplasmic proteins and organelles are degraded and recycled for reuse. There are numerous reports on the role of autophagy in cell growth and death; however, the role of autophagy in methylmercury (MeHg)-induced neurotoxicity has yet to be identified. We studied the role of autophagy in MeHg-induced neurotoxicity in astrocytes. MeHg reduced astrocytic viability in a concentration- and time-dependent manner, and induced apoptosis. Pharmacological inhibition of autophagy with 3-methyladenine (3-MA) or chloroquine (CQ), as well as the silencing of the autophagy-related protein 5 (Atg5), increased MeHg-induced cytotoxicity and the ratio of apoptotic astrocytes. Conversely, Rapamycin, an autophagy inducer, along with as N-acetyl-L-cysteine (NAC), a precursor of reduced glutathione (GSH), decreased MeHg-induced toxicity and the ratio of apoptotic astrocytes. These results indicated that MeHg-induced neurotoxicity was reduced, at least in part, through the activation of autophagy. Accordingly, modulation of autophagy may offer a new avenue for attenuating MeHg-induced neurotoxicity. PMID:25488884

  13. IL-17 induces MIP-1α expression in primary mouse astrocytes via TRPC channel.

    PubMed

    Zhang, Yuan; Huang, Rongrong; Zhang, Yanhong; Yi, Hongwei; Bai, Ying; Chao, Jie; Yao, Honghong

    2016-02-01

    Our previous study demonstrated IL-17-mediated induction of MIP-1α through its binding to the cognate IL-17RA and MIP-1α was involved in astrocyte activation. Transient receptor potential canonical (TRPC) channel was involved in astrocyte activation, however, whether TRPC channel regulates MIP-1α expression in the context of multiple sclerosis (MS) remains largely unknown. In this study we identify the essential role of TRPC channel in IL-17-mediated MIP-1α expression and astrocyte activation. Moreover, treatment of astrocytes with IL-17 activated MAPKs and PI3K/Akt signaling pathways with downstream NF-κB pathways. Interestingly, the TRPC blocker-SKF96365 (10 μM) and Norgestimate (10 μM) significantly inhibited the increased expression of MIP-1α via suppression of IL-17-mediated ERK, p38 and JNK MAPKs and PI3K/Akt pathway activation, thereby underscoring the role of TRPC channel in this process. Together these data underpin the role of TRPC channel as a novel target that regulates MIP-1α expression and cell activation-mediated by IL-17 with implications for therapeutic intervention for reversal of neuroinflammation inflicted by IL-17. Understanding the regulation of MIP-1α expression may provide insights into the development of potential therapeutic targets for neuroinflammation associated with MS.

  14. Induction of inducible nitric oxide synthase expression in ammonia-exposed cultured astrocytes is coupled to increased arginine transport by upregulated y(+)LAT2 transporter.

    PubMed

    Zielińska, Magdalena; Milewski, Krzysztof; Skowrońska, Marta; Gajos, Anna; Ziemińska, Elżbieta; Beręsewicz, Andrzej; Albrecht, Jan

    2015-12-01

    One of the aspects of ammonia toxicity to brain cells is increased production of nitric oxide (NO) by NO synthases (NOSs). Previously we showed that ammonia increases arginine (Arg) uptake in cultured rat cortical astrocytes specifically via y(+)L amino acid transport system, by activation of its member, a heteromeric y(+)LAT2 transporter. Here, we tested the hypothesis that up-regulation of y(+)LAT2 underlies ammonia-dependent increase of NO production via inducible NOS (iNOS) induction, and protein nitration. Treatment of rat cortical astrocytes for 48 with 5 mM ammonium chloride ('ammonia') (i) increased the y(+)L-mediated Arg uptake, (ii) raised the expression of iNOS and endothelial NOS (eNOS), (iii) stimulated NO production, as manifested by increased nitrite+nitrate (Griess) and/or nitrite alone (chemiluminescence), and consequently, (iv) evoked nitration of tyrosine residues of proteins in astrocytes. Except for the increase of eNOS, all the above described effects of ammonia were abrogated by pre-treatment of astrocytes with either siRNA silencing of the Slc7a6 gene coding for y(+)LAT2 protein, or antibody to y(+)LAT2, indicating their strict coupling to y(+)LAT2 activity. Moreover, induction of y(+)LAT2 expression by ammonia was sensitive to Nf-κB inhibitor, BAY 11-7085, linking y(+)LAT2 upregulation to the Nf-κB activation in this experimental setting as reported earlier and here confirmed. Importantly, ammonia did not affect y(+)LAT2 expression nor y(+)L-mediated Arg uptake activity in the cultured cerebellar neurons, suggesting astroglia-specificity of the above described mechanism. The described coupling of up-regulation of y(+)LAT2 transporter with iNOS in ammonia-exposed astrocytes may be considered as a mechanism to ensure NO supply for protein nitration. Ammonia (NH4(+)) increases the expression and activity of the L-arginine (Arg) transporter (Arg/neutral amino acids [NAA] exchanger) y(+)LAT2 in cultured rat cortical astrocytes by a mechanism

  15. Induction of inducible nitric oxide synthase expression in ammonia-exposed cultured astrocytes is coupled to increased arginine transport by upregulated y(+)LAT2 transporter.

    PubMed

    Zielińska, Magdalena; Milewski, Krzysztof; Skowrońska, Marta; Gajos, Anna; Ziemińska, Elżbieta; Beręsewicz, Andrzej; Albrecht, Jan

    2015-12-01

    One of the aspects of ammonia toxicity to brain cells is increased production of nitric oxide (NO) by NO synthases (NOSs). Previously we showed that ammonia increases arginine (Arg) uptake in cultured rat cortical astrocytes specifically via y(+)L amino acid transport system, by activation of its member, a heteromeric y(+)LAT2 transporter. Here, we tested the hypothesis that up-regulation of y(+)LAT2 underlies ammonia-dependent increase of NO production via inducible NOS (iNOS) induction, and protein nitration. Treatment of rat cortical astrocytes for 48 with 5 mM ammonium chloride ('ammonia') (i) increased the y(+)L-mediated Arg uptake, (ii) raised the expression of iNOS and endothelial NOS (eNOS), (iii) stimulated NO production, as manifested by increased nitrite+nitrate (Griess) and/or nitrite alone (chemiluminescence), and consequently, (iv) evoked nitration of tyrosine residues of proteins in astrocytes. Except for the increase of eNOS, all the above described effects of ammonia were abrogated by pre-treatment of astrocytes with either siRNA silencing of the Slc7a6 gene coding for y(+)LAT2 protein, or antibody to y(+)LAT2, indicating their strict coupling to y(+)LAT2 activity. Moreover, induction of y(+)LAT2 expression by ammonia was sensitive to Nf-κB inhibitor, BAY 11-7085, linking y(+)LAT2 upregulation to the Nf-κB activation in this experimental setting as reported earlier and here confirmed. Importantly, ammonia did not affect y(+)LAT2 expression nor y(+)L-mediated Arg uptake activity in the cultured cerebellar neurons, suggesting astroglia-specificity of the above described mechanism. The described coupling of up-regulation of y(+)LAT2 transporter with iNOS in ammonia-exposed astrocytes may be considered as a mechanism to ensure NO supply for protein nitration. Ammonia (NH4(+)) increases the expression and activity of the L-arginine (Arg) transporter (Arg/neutral amino acids [NAA] exchanger) y(+)LAT2 in cultured rat cortical astrocytes by a mechanism

  16. Estrogen protects against dopamine neuron toxicity in primary mesencephalic cultures through an indirect P13K/Akt mediated astrocyte pathway.

    PubMed

    Bains, Mona; Roberts, James L

    2016-01-01

    Astrocytes regulate neuronal homeostasis and have been implicated in affecting the viability and functioning of surrounding neurons under stressed and injured conditions. Previous data from our lab suggests indirect actions of estrogen through ERα in neighboring astroglia to protect dopamine neurons against 1-methyl-4-phenylpyridinium (MPP(+)) toxicity in mouse mesencephalic cultures. We further evaluate estrogen signaling in astrocytes and the mechanism of estrogen's indirect neuroprotective effects on dopamine neurons. Primary mesencephalic cultures pre-treated with 17β-estradiol and the membrane impermeable estrogen, E2-BSA, were both neuroprotective against MPP(+) -induced dopamine neuron toxicity, suggesting membrane-initiated neuroprotection. ERα was found in the plasma membrane of astrocyte cultures and colocalized with the lipid raft marker, flotillin-1. A 17β-estradiol time course revealed a significant increase in Akt, which was inhibited by the PI3 kinase inhibitor, LY294004. Estrogen conditioned media collected from pure astrocyte cultures rescued glial deficient mesencephalic cultures from MPP(+). This indirect estrogen-mediated neuroprotective effect in mesencephalic cultures was significantly reduced when PI3 kinase signaling in astrocytes was blocked prior to collecting estrogen-conditioned media using the irreversible PI3 kinase inhibitor, Wortmannin. Estrogen signaling via astrocytes is rapidly initiated at the membrane level and requires PI3 kinase signaling in order to protect primary mesencephalic dopamine neurons from MPP(+) neurotoxicity. PMID:26520464

  17. Inhibition of glycogen synthase kinase-3β by lithium chloride suppresses 6-hydroxydopamine-induced inflammatory response in primary cultured astrocytes.

    PubMed

    Wang, Hong-Mei; Zhang, Ting; Li, Qiang; Huang, Jian-Kang; Chen, Rong-Fu; Sun, Xiao-Jiang

    2013-11-01

    An increasing amount of evidence has emerged to suggest that neuroinflammatory process is involved in the pathogenesis of Parkinson's disease (PD). Activated microglia and astrocytes are found in the substantia nigra (SN) of Parkinson's disease brains as well as in animal models of Parkinson's disease. Although reactive astrocytes are involved in the progression of PD, the role of reactive astrocytes in neuroinflammation of PD has received limited attention to date. Recently, Glycogen synthase kinase-3β (GSK-3β) was identified as a crucial regulator of the inflammatory response. The purpose of this study was to explore the mechanism by which 6-hydroxydopamine (6-OHDA) induces inflammatory response in astrocytes and observe the anti-inflammatory effect of lithium chloride (LiCl) on 6-OHDA-treated astrocytes. In the present study, we found that glial fibrillary acidic protein (GFAP) was markedly upregulated in the presence of 6-OHDA. Moreover, our results revealed that proinflammatory molecules including inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase-2(COX-2), prostaglandins E2 (PGE2), and tumor necrosis factor-α (TNF-α) were obviously increased in astrocytes exposed to 6-OHDA. Western blot analysis revealed that 6-OHDA significantly increased dephosphorylation/activation of GSK-3β as well as the nuclear translocation of nuclear factor-κB (NF-κB) p65. Besides, GSK-3β inhibitor LiCl and SB415286 inhibited the GSK-3β/NF-κB signaling pathway, leading to the reduction of proinflammatory molecules in 6-OHDA-activated astrocytes. These results confirmed that GSK-3β inhibitor LiCl and SB415286 provide protection against neuroinflammation in 6-OHDA-treated astrocytes. Therefore, GSK-3β may be a potential therapeutic target for the treatment of PD.

  18. Effect of topography of an electrospun nanofiber on modulation of activity of primary rat astrocytes.

    PubMed

    Min, Seul Ki; Kim, Sung Hoon; Kim, Cho Rong; Paik, Sang-Min; Jung, Sang-Myung; Shin, Hwa Sung

    2013-02-01

    Several biomaterials for neural tissue engineering have recently been proposed for regeneration of damaged tissue and promotion of axonal guidance following CNS injury. When implanted into damaged nerve tissue, biomaterials should favorably induce cell infiltration and axonal guiding while suppressing inflammation. Nanofiber scaffolds are regarded as adequate materials to meet the above requirements; however, most studies of these materials conducted to date have targeted neuronal cells, not glial cells, despite their important function in the injured CNS. In this study, an electrospun nanofibrous scaffold of polycaprolactone (PCL) was investigated with respect to its topographic effects on astrocyte behavior and expression of GFAP. The results revealed that the PCL nanofiber topograghy promoted adhesion, but GFAP expression was down-regulated, leading to reduced astrocytes activity. Taken together, these results indicate that the topographic structure of electrospun nanofibers provides a scaffold that is favorable to neural regeneration via alleviation of astrogliosis. PMID:23178191

  19. Impact of Heat Shock Protein A 12B Overexpression on Spinal Astrocyte Survival Against Oxygen-Glucose-Serum Deprivation/Restoration in Primary Cultured Astrocytes.

    PubMed

    Xia, Xun; Ma, Yuan; Yang, Li-Bin; Cheng, Jing-Ming; Yang, Tao; Fan, Ke-Xia; Li, Yun-Ming; Liu, En-Yu; Cheng, Lin; Huang, Hai-Dong; Gu, Jian-Wen; Kuang, Yong-Qin

    2016-08-01

    Heat shock protein A 12B (HSPA12B) is a newly discovered member of the heat shock protein 70 family. Preclinical evidence indicates that HSPA12B helps protect the brain from ischemic injury, although its specific function remains unclear. The aim of this study is to investigate whether HSPA12B overexpression can protect astrocytes from oxygen-glucose-serum deprivation/restoration (OGD/R) injury. We analyzed the effects of HSPA12B overexpression on spinal cord ischemia-reperfusion injury and spinal astrocyte survival. After ischemia-reperfusion injury, we found that HSPA12B overexpression decreased spinal cord water content and infarct volume. MTT assay showed that HSPA12B overexpression increased astrocyte survival after OGD/R treatment. Flow cytometry results showed a marked inhibition of OGD/R-induced astrocyte apoptosis. Western blot assay showed that HSPA12B overexpression significantly increased regulatory protein B-cell lymphocyte 2 (Bcl-2) levels, whereas it decreased expression of the Bax protein, which forms a heterodimer with Bcl-2. Measurements of the level of activation of caspase-3 by Caspase-Glo®3/7 Assay kit showed that HSPA12B overexpression markedly inhibited caspase-3 activation. Notably, we demonstrated that the effects of HSPA12B on spinal astrocyte survival depended on activation of the PI3K/Akt signal pathway. These findings indicate that HSPA12B protects against spinal cord ischemia-reperfusion injury and may represent a potential treatment target. PMID:27179807

  20. Sex differences and effects of estrogenic compounds on the expression of inflammatory molecules by astrocytes exposed to the insecticide dimethoate.

    PubMed

    Astiz, Mariana; Acaz-Fonseca, Estefania; Garcia-Segura, Luis M

    2014-04-01

    A low dose of the organophosphorus insecticide dimethoate (DMT) produces oxidation of lipids and proteins and impairs mitochondrial function in the brain of male rats, together with a reduction of gonadal hormones in plasma. Here, we have assessed whether DMT affected the expression of inflammatory molecules, the production of reactive oxygen species (ROS), and the expression of steroidogenic proteins and estrogen receptors in cortical astrocyte-enriched cultures obtained separately from male and female CD1 mice pups. Furthermore, we have analyzed whether estradiol may counteract the effects of DMT. A dose of DMT (2 μg/mL) did not affect cell viability, increased interleukin (IL) 6, IL1β, tumor necrosis factor (TNF)α, interferon-γ-inducible protein 10 (IP10), ERβ, steroidogenic acute regulatory protein, and aromatase mRNA levels and ERα protein levels in male but not in female cultures. Estradiol decreased the mRNA levels of IL6, IP10, TNFα, and IL1β in male but not in female cultures treated with DMT. The effect of estradiol was prevented by the ER antagonist ICI 182,780, fully imitated by an ERβ agonist and partially imitated by an ERα agonist. Furthermore, DMT increased the production of ROS in male astrocytes while estradiol reduced ROS production to control levels. These findings indicate that a sublethal dose of DMT alters astrocyte function. The selective action of estradiol on male astrocytes and the sexually dimorphic action of DMT suggest that the pesticide may have different neurological outcomes in males and females.

  1. Regulation of the system x(C)- cystine/glutamate exchanger by intracellular glutathione levels in rat astrocyte primary cultures.

    PubMed

    Seib, Todd Michael; Patel, Sarjubhai Amratbhai; Bridges, Richard James

    2011-10-01

    The system x(C)- (Sx(C)-) transporter functions to mediate the exchange of extracellular cystine (L-Cys(2)) and intracellular glutamate (L-Glu). Internalized L-Cys(2) serves as a rate-limiting precursor for the biosynthesis of glutathione (GSH), while the externalized L-Glu can contribute to either excitatory signaling or excitotoxicity. In the present study the influence of culture conditions (with and without dibutyryl-cAMP) and GSH levels on the expression of Sx(C)- were investigated in primary rat astrocyte cultures. Sx(C)- activity in dbcAMP-treated cells was nearly sevenfold greater than in untreated astrocytes and increased further (∼threefold) following the depletion of intracellular GSH with buthionine sulfoximine. This increase in Sx(C)- triggered by GSH depletion was only observed in the dbcAMP-treated phenotype and was distinct from the Nrf2-mediated response initiated by exposure to electrophiles. Changes in Sx(C)- activity correlated with increases in both protein and mRNA levels of the xCT subunit of the Sx(C)- heterodimer, an increase in the V(max) for L-Glu uptake and was linked temporally to GSH levels. This induction of Sx(C)- was not mimicked by hydrogen peroxide nor attenuated by nonspecific antioxidants but was partially prevented by the co-administration of the cell-permeant thiols GSH-ethyl ester and N-acetylcysteine. These findings demonstrate that the expression of Sx(C)- on astrocytes is dynamically regulated by intracellular GSH levels in a cell- and phenotype-dependent manner. The presence of this pathway likely reflects the inherent vulnerability of the CNS to oxidative damage and raises interesting questions as to the functional consequences of changes in Sx(C)- activity in CNS injury and disease.

  2. Enhancing NAD+ Salvage Pathway Reverts the Toxicity of Primary Astrocytes Expressing Amyotrophic Lateral Sclerosis-linked Mutant Superoxide Dismutase 1 (SOD1).

    PubMed

    Harlan, Benjamin A; Pehar, Mariana; Sharma, Deep R; Beeson, Gyda; Beeson, Craig C; Vargas, Marcelo R

    2016-05-13

    Nicotinamide adenine dinucleotide (NAD(+)) participates in redox reactions and NAD(+)-dependent signaling pathways. Although the redox reactions are critical for efficient mitochondrial metabolism, they are not accompanied by any net consumption of the nucleotide. On the contrary, NAD(+)-dependent signaling processes lead to its degradation. Three distinct families of enzymes consume NAD(+) as substrate: poly(ADP-ribose) polymerases, ADP-ribosyl cyclases (CD38 and CD157), and sirtuins (SIRT1-7). Because all of the above enzymes generate nicotinamide as a byproduct, mammalian cells have evolved an NAD(+) salvage pathway capable of resynthesizing NAD(+) from nicotinamide. Overexpression of the rate-limiting enzyme in this pathway, nicotinamide phosphoribosyltransferase, increases total and mitochondrial NAD(+) levels in astrocytes. Moreover, targeting nicotinamide phosphoribosyltransferase to the mitochondria also enhances NAD(+) salvage pathway in astrocytes. Supplementation with the NAD(+) precursors nicotinamide mononucleotide and nicotinamide riboside also increases NAD(+) levels in astrocytes. Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Superoxide dismutase 1 (SOD1) mutations account for up to 20% of familial ALS and 1-2% of apparently sporadic ALS cases. Primary astrocytes isolated from mutant human superoxide dismutase 1-overexpressing mice as well as human post-mortem ALS spinal cord-derived astrocytes induce motor neuron death in co-culture. Increasing total and mitochondrial NAD(+) content in ALS astrocytes increases oxidative stress resistance and reverts their toxicity toward co-cultured motor neurons. Taken together, our results suggest that enhancing the NAD(+) salvage pathway in astrocytes could be a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS. PMID:27002158

  3. GM-CSF reduces expression of chondroitin sulfate proteoglycan (CSPG) core proteins in TGF-β-treated primary astrocytes.

    PubMed

    Choi, Jung-Kyoung; Park, Sang-Yoon; Kim, Kil Hwan; Park, So Ra; Lee, Seok-Geun; Choi, Byung Hyune

    2014-12-01

    GM-CSF plays a role in the nervous system, particularly in cases of injury. A therapeutic effect of GM-CSF has been reported in rat models of various central nervous system injuries. We previously showed that GM-CSF could enhance long-term recovery in a rat spinal cord injury model, inhibiting glial scar formation and increasing the integrity of axonal structure. Here, we investigated molecular the mechanism(s) by which GM-CSF suppressed glial scar formation in an in vitro system using primary astrocytes treated with TGF-β. GM-CSF repressed the expression of chondroitin sulfate proteoglycan (CSPG) core proteins in astrocytes treated with TGF-β. GM-CSF also inhibited the TGF-β-induced Rho-ROCK pathway, which is important in CSPG expression. Finally, the inhibitory effect of GM-CSF was blocked by a JAK inhibitor. These results may provide the basis for GM-CSF's effects in glial scar inhibition and ultimately for its therapeutic effect on neural cell injuries.

  4. ZnO nanoparticle-induced oxidative stress triggers apoptosis by activating JNK signaling pathway in cultured primary astrocytes

    NASA Astrophysics Data System (ADS)

    Wang, Jieting; Deng, Xiaobei; Zhang, Fang; Chen, Deliang; Ding, Wenjun

    2014-03-01

    It has been documented in in vitro studies that zinc oxide nanoparticles (ZnO NPs) are capable of inducing oxidative stress, which plays a crucial role in ZnO NP-mediated apoptosis. However, the underlying molecular mechanism of apoptosis in neurocytes induced by ZnO NP exposure was not fully elucidated. In this study, we investigated the potential mechanisms of apoptosis provoked by ZnO NPs in cultured primary astrocytes by exploring the molecular signaling pathways triggered after ZnO NP exposure. ZnO NP exposure was found to reduce cell viability in MTT assays, increase lactate dehydrogenase (LDH) release, stimulate intracellular reactive oxygen species (ROS) generation, and elicit caspase-3 activation in a dose- and time-dependent manner. Apoptosis occurred after ZnO NP exposure as evidenced by nuclear condensation and poly(ADP-ribose) polymerase-1 (PARP) cleavage. A decrease in mitochondrial membrane potential (MMP) with a concomitant increase in the expression of Bax/Bcl-2 ratio suggested that the mitochondria also mediated the pathway involved in ZnO NP-induced apoptosis. In addition, exposure of the cultured cells to ZnO NPs led to phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK), and p38 mitogen-activated protein kinase (p38 MAPK). Moreover, JNK inhibitor (SP600125) significantly reduced ZnO NP-induced cleaved PARP and cleaved caspase-3 expression, but not ERK inhibitor (U0126) or p38 MAPK inhibitor (SB203580), indicating that JNK signaling pathway is involved in ZnO NP-induced apoptosis in primary astrocytes.

  5. CDK5 knockdown in astrocytes provide neuroprotection as a trophic source via Rac1.

    PubMed

    Posada-Duque, Rafael Andrés; Palacio-Castañeda, Valentina; Cardona-Gómez, Gloria Patricia

    2015-09-01

    Astrocytes perform metabolic and structural support functions in the brain and contribute to the integrity of the blood-brain barrier. Astrocytes influence neuronal survival and prevent gliotoxicity by capturing glutamate (Glu), reactive oxygen species, and nutrients. During these processes, astrocytic morphological changes are supported by actin cytoskeleton remodeling and require the involvement of Rho GTPases, such as Rac1. The protein cyclin-dependent kinase 5 (CDK5) may have a dual effect on astrocytes because it has been shown to be involved in migration, senescence, and the dysfunction of glutamate recapture; however, its role in astrocytes remains unclear. Treating a possible deregulation of CDK5 with RNAi is a strategy that has been proposed as a therapy for neurodegenerative diseases. Models of glutamate gliotoxicity in the C6 astroglioma cell line, primary cultures of astrocytes, and co-cultures with neurons were used to analyze the effects of CDK5 RNAi in astrocytes and the role of Rac1 in neuronal viability. In C6 cells and primary astrocytes, CDK5 RNAi prevented the cell death generated by glutamate-induced gliotoxicity, and this finding was corroborated by pharmacological inhibition with roscovitine. This effect was associated with the appearance of lamellipodia, protrusions, increased cell area, stellation, Rac1 activation, BDNF release, and astrocytic protection in neurons that were exposed to glutamate excitotoxicity. Interestingly, Rac1 inhibition in astrocytes blocked BDNF upregulation and the astrocyte-mediated neuroprotection. Actin cytoskeleton remodeling and stellation may be a functional phenotype for BDNF release that promotes neuroprotection. In summary, our findings suggest that CDK5- knockdown in astrocytes acts as a trophic source for neuronal protection in a Rac1-dependent manner. PMID:26160434

  6. Effects of arsenite in astrocytes on neuronal signaling transduction.

    PubMed

    Wang, Yan; Zhao, Fenghong; Liao, Yingjun; Jin, Yaping; Sun, Guifan

    2013-01-01

    The main purpose of this study was to test the hypothesis that arsenite induces neurotoxicity via effects on astrocytes. Astrocytes were exposed to 0, 5 or 10 μM arsenite in medium for 24 h, and then astrocyte-conditioned medium (ACM) was collected after incubation with fresh medium for 6 h. Primary neuron cultures were divided into four groups due to ACM, which were neurons without ACM exposure (group I) and neurons exposed to ACM from 0, 5 or 10 μM arsenite treated astrocytes (group II-IV). Protein expression of N-methyl-d-aspartate receptors (NR1, NR2A, NR2B), calmodulin-dependent protein kinase II (CaMKII) and adenylate cyclase (AC) in neurons were measured after incubation with ACM for 4, 8 or 12 h. Morphological changes and synaptic formation were observed after a 72 h-incubation with ACM. Compared to group II, synaptic formation and protein expression of NR2A, NR2B, CaMKII and AC in group III and IV were significantly suppressed. Moreover, synaptic formation and protein expression of CaMKII and AC in group II were significantly enhanced when compared with group I. Taken together, findings from this study suggested that arsenic in astrocytes might impair synaptic formation through disturbing astrocytic effects on neuronal signal transduction.

  7. IL-17A induces MIP-1α expression in primary astrocytes via Src/MAPK/PI3K/NF-kB pathways: implications for multiple sclerosis.

    PubMed

    Yi, Hongwei; Bai, Ying; Zhu, Xinjian; Lin, Lin; Zhao, Lei; Wu, Xiaodong; Buch, Shilpa; Wang, Longxin; Chao, Jie; Yao, Honghong

    2014-12-01

    Neuroinflammation plays critical roles in multiple sclerosis (MS). In addition to the part played by the lymphocytes, the underlying mechanisms could, in part, be also attributed to activation mediated by astrocytes. Macrophage inflammatory protein-1α (MIP-1α) has been implicated in a number of pathological conditions, specifically attributable to its potent chemottractant effects. Its modulation by IL-17, however, has received very little attention. In the present study, we demonstrated IL-17-mediated induction of MIP-1α in rat primary astroctyes through its binding to the cognate IL-17RA. Furthermore, this effect was mediated via the activation of Src, mitogen-activated protein kinases (MAPKs), PI3K/Akt and NF-kB pathways, culminating ultimately into increased expression of MIP-1α. Exposure of primary mouse astrocytes to IL-17 resulted in increased expression of glial fibrillary acidic protein and, this effect was abrogated in cells cultured in presence of the MIP-1α neutralizing antibody, thus underscoring its role in the activation of astrocytes. In vivo relevance of these findings was further corroborated in experimental autoimmune encephalomyelitis mice that demonstrated significantly increased activation of astrocytes with concomitant increased expression of MIP-1α in the corpus callosum compared with control group. Understanding the regulation of MIP-1α expression may provide insights into the development of potential therapeutic targets for neuroinflammation associated with multiple sclerosis.

  8. WIN 55,212-2, Agonist of Cannabinoid Receptors, Prevents Amyloid β1-42 Effects on Astrocytes in Primary Culture

    PubMed Central

    Aguirre-Rueda, Diana; Guerra-Ojeda, Sol; Aldasoro, Martin; Iradi, Antonio; Obrador, Elena; Mauricio, Maria D.; Vila, Jose Mª; Marchio, Patricia; Valles, Soraya L.

    2015-01-01

    Alzheimer´s disease (AD), a neurodegenerative illness involving synaptic dysfunction with extracellular accumulation of Aβ1-42 toxic peptide, glial activation, inflammatory response and oxidative stress, can lead to neuronal death. Endogenous cannabinoid system is implicated in physiological and physiopathological events in central nervous system (CNS), and changes in this system are related to many human diseases, including AD. However, studies on the effects of cannabinoids on astrocytes functions are scarce. In primary cultured astrocytes we studied cellular viability using MTT assay. Inflammatory and oxidative stress mediators were determined by ELISA and Western-blot techniques both in the presence and absence of Aβ1-42 peptide. Effects of WIN 55,212-2 (a synthetic cannabinoid) on cell viability, inflammatory mediators and oxidative stress were also determined. Aβ1-42 diminished astrocytes viability, increased TNF-α and IL-1β levels and p-65, COX-2 and iNOS protein expression while decreased PPAR-γ and antioxidant enzyme Cu/Zn SOD. WIN 55,212-2 pretreatment prevents all effects elicited by Aβ1-42. Furthermore, cannabinoid WIN 55,212-2 also increased cell viability and PPAR-γ expression in control astrocytes. In conclusion cannabinoid WIN 55,212-2 increases cell viability and anti-inflammatory response in cultured astrocytes. Moreover, WIN 55,212-2 increases expression of anti-oxidant Cu/Zn SOD and is able to prevent inflammation induced by Aβ1-42 in cultured astrocytes. Further studies would be needed to assess the possible beneficial effects of cannabinoids in Alzheimer's disease patients. PMID:25874692

  9. Astrocytes produce interferon that enhances the expression of H-2 antigens on a subpopulation of brain cells

    PubMed Central

    1986-01-01

    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. PMID:2423537

  10. Enhanced GLT-1 mediated glutamate uptake and migration of primary astrocytes directed by fibronectin-coated electrospun poly-L-lactic acid fibers

    PubMed Central

    Zuidema, Jonathan M.; Hyzinski-García, María C.; Vlasselaer, Kristien Van; Zaccor, Nicholas; Plopper, George E.; Mongin, Alexander A.; Gilbert, Ryan J.

    2014-01-01

    Bioengineered fiber substrates are increasingly studied as a means to promote regeneration and remodeling in the injured central nervous system (CNS). Previous reports largely focused on the ability of oriented scaffolds to bridge injured regions and direct outgrowth of axonal projections. In the present work, we explored the effects of electrospun microfibers on the migration and physiological properties of brain astroglial cells. Primary rat astrocytes were cultured on either fibronectin-coated poly-l-lactic acid (PLLA) films, fibronectin-coated randomly oriented PLLA electrospun fibers, or fibronectin-coated aligned PLLA electrospun fibers. Aligned PLLA fibers strongly altered astrocytic morphology, orienting cell processes, actin microfilaments, and microtubules along the length of the fibers. On aligned fibers, astrocytes also significantly increased their migration rates in the direction of fiber orientation. We further investigated if fiber topography modifies astrocytic neuroprotective properties, namely glutamate and glutamine transport and metabolism. This was done by quantifying changes in mRNA expression (qRT-PCR) and protein levels (Western blotting) for a battery of relevant biomolecules. Interestingly, we found that cells grown on random and/or aligned fibers increased the expression levels of two glutamate transporters, GLAST and GLT-1, and an important metabolic enzyme, glutamine synthetase, as compared to the fibronectin-coated films. Functional assays revealed increases in glutamate transport rates due to GLT-1 mediated uptake, which was largely determined by the dihydrokainate-sensitive GLT-1. Overall, this study suggests that aligned PLLA fibers can promote directed astrocytic migration, and, of most importance, our in vitro results indicate for the first time that electrospun PLLA fibers can positively modify neuroprotective properties of glial cells by increasing rates of glutamate uptake. PMID:24246642

  11. Interleukin 1β Regulation of the System xc- Substrate-specific Subunit, xCT, in Primary Mouse Astrocytes Involves the RNA-binding Protein HuR.

    PubMed

    Shi, Jingxue; He, Yan; Hewett, Sandra J; Hewett, James A

    2016-01-22

    System xc(-) is a heteromeric amino acid cystine/glutamate antiporter that is constitutively expressed by cells of the CNS, where it functions in the maintenance of intracellular glutathione and extracellular glutamate levels. We recently determined that the cytokine, IL-1β, increases the activity of system xc(-) in CNS astrocytes secondary to an up-regulation of its substrate-specific light chain, xCT, and that this occurs, in part, at the level of transcription. However, an in silico analysis of the murine xCT 3'-UTR identified numerous copies of adenine- and uridine-rich elements, raising the possibility that undefined trans-acting factors governing mRNA stability and translation may also contribute to xCT expression. Here we show that IL-1β increases the level of mRNA encoding xCT in primary cultures of astrocytes isolated from mouse cortex in association with an increase in xCT mRNA half-life. Additionally, IL-1β induces HuR translocation from the nucleus to the cytoplasm. RNA immunoprecipitation analysis reveals that HuR binds directly to the 3'-UTR of xCT in an IL-1β-dependent manner. Knockdown of endogenous HuR protein abrogates the IL-1β-mediated increase in xCT mRNA half-life, whereas overexpression of HuR in unstimulated primary mouse astrocytes doubles the half-life of constitutive xCT mRNA. This latter effect is accompanied by an increase in xCT protein levels, as well as a functional increase in system xc(-) activity. Altogether, these data support a critical role for HuR in mediating the IL-1β-induced stabilization of astrocyte xCT mRNA.

  12. Adrenergic activation attenuates astrocyte swelling induced by hypotonicity and neurotrauma.

    PubMed

    Vardjan, Nina; Horvat, Anemari; Anderson, Jamie E; Yu, Dou; Croom, Deborah; Zeng, Xiang; Lužnik, Zala; Kreft, Marko; Teng, Yang D; Kirov, Sergei A; Zorec, Robert

    2016-06-01

    Edema in the central nervous system can rapidly result in life-threatening complications. Vasogenic edema is clinically manageable, but there is no established medical treatment for cytotoxic edema, which affects astrocytes and is a primary trigger of acute post-traumatic neuronal death. To test the hypothesis that adrenergic receptor agonists, including the stress stimulus epinephrine protects neural parenchyma from damage, we characterized its effects on hypotonicity-induced cellular edema in cortical astrocytes by in vivo and in vitro imaging. After epinephrine administration, hypotonicity-induced swelling of astrocytes was markedly reduced and cytosolic 3'-5'-cyclic adenosine monophosphate (cAMP) was increased, as shown by a fluorescence resonance energy transfer nanosensor. Although, the kinetics of epinephrine-induced cAMP signaling was slowed in primary cortical astrocytes exposed to hypotonicity, the swelling reduction by epinephrine was associated with an attenuated hypotonicity-induced cytosolic Ca(2+) excitability, which may be the key to prevent astrocyte swelling. Furthermore, in a rat model of spinal cord injury, epinephrine applied locally markedly reduced neural edema around the contusion epicenter. These findings reveal new targets for the treatment of cellular edema in the central nervous system. PMID:27018061

  13. A High Throughput Screen Identifies Chemical Modulators of the Laminin-Induced Clustering of Dystroglycan and Aquaporin-4 in Primary Astrocytes

    PubMed Central

    Noël, Geoffroy; Stevenson, Sarah; Moukhles, Hakima

    2011-01-01

    Background Aquaporin-4 (AQP4) constitutes the principal water channel in the brain and is clusteredat the perivascular astrocyte endfeet. This specific distribution of AQP4 plays a major role in maintaining water homeostasis in the brain. A growing body of evidence points to a role ofthe dystroglycan complex and its interaction with perivascular laminin in the clusteringof AQP4 atperivascular astrocyte endfeet. Indeed, mice lacking components of this complex or in which laminin-dystroglycan interaction is disrupted show a delayed onset of brain edema due to a redistribution of AQP4 away from astrocyte endfeet. It is therefore important to identify inhibitory drugs of laminin-dependent AQP4 clustering which may prevent or reduce brain edema. Methodolgy/Principal Findings In the present study we used primary rat astrocyte cultures toscreen a library of >3,500 chemicals and identified 6 drugs that inhibit the laminin-induced clustering of dystroglycan and AQP4. Detailed analysis of the inhibitory drug, chloranil, revealed that its inhibition of the clustering is due to the metalloproteinase-2-mediated ß-dystroglycan shedding and subsequent loss of laminin interaction with dystroglycan. Furthermore, chemical variants of chloranil induced a similar effect on ß-dystroglycan and this was prevented by the antioxidant N-acetylcysteine. Conclusion/Significance These findings reveal the mechanism of action of chloranil in preventing the laminin-induced clustering of dystroglycan and AQP4 and validate the use of high-throughput screening as a tool to identify drugs that modulate AQP4 clustering and that could be tested in models of brain edema. PMID:21408176

  14. Primary stress responses in Arabidopsis thaliana exposed to gamma radiation.

    PubMed

    Vanhoudt, Nathalie; Horemans, Nele; Wannijn, Jean; Nauts, Robin; Van Hees, May; Vandenhove, Hildegarde

    2014-03-01

    As the environment is inevitably exposed to ionizing radiation from natural and anthropogenic sources, it is important to evaluate gamma radiation induced stress responses in plants. The objective of this research is therefore to investigate radiation effects in Arabidopsis thaliana on individual and subcellular level by exposing 2-weeks-old seedlings for 7 days to total doses of 3.9 Gy, 6.7 Gy, 14.8 Gy and 58.8 Gy and evaluating growth, photosynthesis, chlorophyll a, chlorophyll b and carotenoid concentrations and antioxidative enzyme capacities. While the capacity of photosystem II (PSII measured as Fv/Fm) remained intact, plants started optimizing their photosynthetic process at the lower radiation doses by increasing the PSII efficiency (φPSII) and the maximal electron transport rate (ETRmax) and by decreasing the non-photochemical quenching (NPQ). At the highest radiation dose, photosynthetic parameters resembled those of control conditions. On subcellular level, roots showed increased superoxide dismutase (SOD) and ascorbate peroxidase (APX) capacities under gamma irradiation but catalase (CAT), syringaldazine peroxidase (SPX) and guaiacol peroxidase (GPX) activities, on the other hand, decreased. In the leaves no alterations were observed in SOD, CAT and SPX capacities, but GPX was highly affected. Based on these results it seems that roots are more sensitive for oxidative stress under gamma radiation exposure than leaves.

  15. 1,3-Dinitrobenzene neurotoxicity - Passage effect in immortalized astrocytes.

    PubMed

    Maurer, Laura L; Latham, Jackelyn D; Landis, Rory W; Song, Dong Hoon; Epstein, Tamir; Philbert, Martin A

    2016-03-01

    Age-related disturbances in astrocytic mitochondrial function are linked to loss of neuroprotection and decrements in neurological function. The immortalized rat neocortical astrocyte-derived cell line, DI-TNC1, provides a convenient model for the examination of cellular aging processes that are difficult to study in primary cell isolates from aged brain. Successive passages in culture may serve as a surrogate of aging in which time-dependent adaptation to culture conditions may result in altered responses to xenobiotic challenge. To investigate the hypothesis that astrocytic mitochondrial homeostatic function is decreased with time in culture, low passage DI-TNC1 astrocytes (LP; #2-8) and high passage DI-TNC1 astrocytes (HP; #17-28) were exposed to the mitochondrial neurotoxicant 1,3-dinitrobenzene (DNB). Cells were exposed in either monoculture or in co-culture with primary cortical neurons. Astrocyte mitochondrial membrane potential, morphology, ATP production and proliferation were monitored in monoculture, and the ability of DI-TNC1 cells to buffer K(+)-induced neuronal depolarization was examined in co-cultures. In HP DI-TNC1 cells, DNB exposure decreased proliferation, reduced mitochondrial membrane potential and significantly decreased mitochondrial form factor. Low passage DI-TNC1 cells effectively attenuated K(+)-induced neuronal depolarization in the presence of DNB whereas HP counterparts were unable to buffer K(+) in DNB challenge. Following DNB challenge, LP DI-TNC1 cells exhibited greater viability in co-culture than HP. The data provide compelling evidence that there is an abrupt phenotypic change in DI-TNC1 cells between passage #9-16 that significantly diminishes the ability of DI-TNC1 cells to compensate for neurotoxic challenge and provide neuroprotective spatial buffering. Whether or not these functional changes have an in vivo analog in aging brain remains to be determined. PMID:26769196

  16. Regulation of hemeoxygenase-1 gene expression by Nrf2 and c-Jun in tertiary butylhydroquinone-stimulated rat primary astrocytes

    SciTech Connect

    Park, Jin-Sun; Kim, Hee-Sun

    2014-05-16

    Highlights: • tBHQ increased HO-1 mRNA and protein levels in rat primary astrocytes. • tBHQ enhanced HO-1 gene transcription in an ARE-dependent manner. • tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. • Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. • Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction. - Abstract: Hemeoxygenase-1 (HO-1) is a phase II antioxidant enzyme that is primarily involved in detoxification and cytoprotection in a variety of tissues. However, the mechanism underlying HO-1 gene expression remains unclear. In the present study, we investigated the regulation of HO-1 expression in primary cultured astrocytes by using the natural antioxidant compound tertiary butylhydroquinone (tBHQ). We found that tBHQ increased HO-1 mRNA and protein levels. Promoter analysis revealed that tBHQ enhanced HO-1 gene transcription in an antioxidant response element (ARE)-dependent manner. In addition, tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. Small interfering RNA (siRNA) experiments demonstrated that Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. Thus, Nrf2 knockdown reduced the basal level of HO-1 expression but did not affect the fold induction by tBHQ. On the other hand, knockdown of c-Jun diminished tBHQ-mediated induction of HO-1 without affecting basal expression. The data suggest that Nrf2 generally modulates the basal expression of HO-1, while c-Jun mediates HO-1 induction in response to tBHQ. The results of co-immunoprecipitation assays demonstrated a physical interaction between Nrf2 and c-Jun in tBHQ-treated astrocytes. The results suggest that Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction in tBHQ-treated rat primary astrocytes.

  17. Astrocytes Prevent Ethanol Induced Apoptosis of Nrf2 Depleted Neurons by Maintaining GSH Homeostasis.

    PubMed

    Narasimhan, Madhusudhanan; Rathinam, Marylatha; Patel, Dhyanesh; Henderson, George; Mahimainathan, Lenin

    2012-07-01

    Glutathione (GSH), a major cellular antioxidant protects cells against oxidative stress injury. Nuclear factor erythroid 2-related factor 2 (NFE2L2/Nrf2) is a redox sensitive master regulator of battery of antioxidant enzymes including those involved in GSH antioxidant machinery. Earlier we reported that ethanol (ETOH) elicits apoptotic death of primary cortical neurons (PCNs) which in partly due to depletion of intracellular GSH levels. Further a recent report from our laboratory illustrated that ETOH exacerbated the dysregulation of GSH and caspase mediated cell death of cortical neurons that are compromised in Nrf2 machinery (Narasimhan et al., 2011). In various experimental models of neurodegeneration, neuronal antioxidant defenses mainly GSH has been shown to be supported by astrocytes. We therefore sought to determine whether astrocytes can render protection to neurons against ETOH toxicity, particularly when the function of Nrf2 is compromised in neurons. The experimental model consisted of co-culturing primary cortical astrocytes (PCA) with Nrf2 downregulated PCNs that were exposed with 4 mg/mL ETOH for 24 h. Monochlorobimane (MCB) staining followed by FACS analysis showed that astrocytes blocked ETOH induced GSH decrement in Nrf2-silenced neurons as opposed to exaggerated GSH depletion in Nrf2 downregulated PCNs alone. Similarly, the heightened activation of caspase 3/7 observed in Nrf2-compromised neurons was attenuated when co-cultured with astrocytes as measured by luminescence based caspase Glo assay. Furthermore, annexin-V-FITC staining followed by FACS analysis revealed that Nrf2 depleted neurons showed resistance to ETOH induced neuronal apoptosis when co-cultured with astrocytes. Thus, the current study identifies ETOH induced dysregulation of GSH and associated apoptotic events observed in Nrf2-depleted neurons can be blocked by astrocytes. Further our results suggest that this neuroprotective effect of astrocyte despite dysfunctional Nrf2 system

  18. Effects of potassium on the anion and cation contents of primary cultures of mouse astrocytes and neurons.

    PubMed

    Chow, S Y; Yen-Chow, Y C; White, H S; Hertz, L; Woodbury, D M

    1991-12-01

    In astrocytes, as [K+]o was increased from 1.2 to 10 mM, [K+]i and [Cl-]i were increased, whereas [Na+]i was decreased. As [K+]o was increased from 10 to 60 mM, intracellular concentration of these three ions showed no significant change. When [K+]o was increased from 60 to 122 mM, an increase in [K+]i and [Cl-]i and a decrease in [Na+]i were observed. In neurons, as [K+]o was increased from 1.2 to 2.8 mM, [Na+]i and [Cl-]i were decreased, whereas [K+]i was increased. As [K+]o was increased from 2.8 to 30 mM, [K+]i, [Na+]i and [Cl-]i showed no significant change. When [K+]o was increased from 30 to 122 mM, [K+]i and [Cl-]i were increased, whereas [Na+]i was decreased. In astrocytes, pHi increased when [K+]o was increased. In neurons, there was a biphasic change in pHi. In lower [K+]o (1.2-2.8 mM) pHi decreased as [K+]o increased, whereas in higher [K+]o (2.8-122 mM) pHi was directly related to [K+]o. In both astrocytes and neurons, changes in [K+]o did not affect the extracellular water content, whereas the intracellular water content increased as the [K+]o increased. Transmembrane potential (Em) as measured with Tl-204 was inversely related to [K+]o between 1.2 and 90 mM, a ten-fold increase in [K+]o depolarized the astrocytes by about 56 mV and the neurons about 52 mV. The Em values measured with Tl-204 were close to the potassium equilibrium potential (Ek) except those in neurons at lower [K+]o. However, they were not equal to the chloride equilibrium potential (ECl) at [K+]o lower than 30 mM in both astrocytes and neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Levetiracetam inhibits oligomeric Aβ-induced glutamate release from human astrocytes.

    PubMed

    Sanz-Blasco, Sara; Piña-Crespo, Juan C; Zhang, Xiaofei; McKercher, Scott R; Lipton, Stuart A

    2016-06-15

    A recently identified mechanism for oligomeric Aβ-induced glutamate release from astrocytes involves intracellular Ca elevation, potentially by Ca-dependent vesicular release. Evidence suggests that levetiracetam (LEV; Keppra), an antiepileptic drug, can improve cognitive performance in both humans with mild cognitive impairment and animal models of Alzheimer disease. Because LEV acts by modulating neurotransmitter release from neurons by interaction with synaptic vesicles, we tested the effect of LEV on Aβ-induced astrocytic release of glutamate. We used a fluorescence resonance energy transfer-based glutamate sensor (termed SuperGluSnFR), whose structure is based on the ligand-binding site of glutamate receptors, to monitor glutamate release from primary cultures of human astrocytes exposed to oligomeric amyloid-β peptide 1-42 (Aβ42). We found that LEV (10 µM) inhibited oligomeric Aβ-induced astrocytic glutamate release. In addition, we show that this Aβ-induced glutamate release from astrocytes is sensitive to tetanus neurotoxin, an inhibitor of the vesicle release machinery. Taken together, our evidence suggests that LEV inhibits Aβ-induced vesicular glutamate release from astrocytes and thus may underlie, at least in part, the ability of LEV to reduce hyperexcitability in Alzheimer disease. PMID:27183239

  20. Valproate and Amitriptyline Exert Common and Divergent Influences on Global and Gene Promoter-Specific Chromatin Modifications in Rat Primary Astrocytes

    PubMed Central

    Perisic, Tatjana; Zimmermann, Nicole; Kirmeier, Thomas; Asmus, Maria; Tuorto, Francesca; Uhr, Manfred; Holsboer, Florian; Rein, Theo; Zschocke, Jürgen

    2010-01-01

    Aberrant biochemical processes in the brain frequently go along with subtle shifts of the cellular epigenetic profile that might support the pathogenic progression of psychiatric disorders. Although recent reports have implied the ability of certain antidepressants and mood stabilizers to modulate epigenetic parameters, studies comparing the actions of these compounds under the same conditions are lacking. In this study, we screened amitriptyline (AMI), venlafaxine, citalopram, as well as valproic acid (VPA), carbamazepine, and lamotrigine for their potential actions on global and local epigenetic modifications in rat primary astrocytes. Among all drugs, VPA exposure evoked the strongest global chromatin modifications, including histone H3/H4 hyperacetylation, 2MeH3K9 hypomethylation, and DNA demethylation, as determined by western blot and luminometric methylation analysis, respectively. CpG demethylation occurred independently of DNA methyltransferase (DNMT) suppression. Strikingly, AMI also induced slight cytosine demethylation, paralleled by the reduction in DNMT enzymatic activity, without affecting the global histone acetylation status. Locally, VPA-induced chromatin modifications were reflected at the glutamate transporter (GLT-1) promoter as shown by bisulfite sequencing and acetylated histone H4 chromatin immunoprecipitation analysis. Distinct CpG sites in the distal part of the GLT-1 promoter were demethylated and enriched in acetylated histone H4 in response to VPA. For the first time, we could show that these changes were associated with an enhanced transcription of this astrocyte-specific gene. In contrast, AMI failed to stimulate GLT-1 transcription and to alter promoter methylation levels. In conclusion, VPA and AMI globally exerted chromatin-modulating activities using different mechanisms that divergently precipitated at an astroglial gene locus. PMID:19924110

  1. Comparing the effects of two neurotoxins in cortical astrocytes obtained from rats of different ages: involvement of oxidative damage.

    PubMed

    Alarcón-Aguilar, Adriana; González-Puertos, Viridiana Yazmín; Luna-López, Armando; López-Macay, Ambar; Morán, Julio; Santamaría, Abel; Königsberg, Mina

    2014-02-01

    Oxidative stress has been recognized as a potential mediator of cell death. Astrocytes play an active role in brain physiology responding to harmful stimuli by activating astrogliosis, which in turn has been associated either with survival or degenerative events. The characterization of the mechanistic actions exerted by different toxins in astrocytes is essential to understand the brain function and pathology. As age plays a critical role in degenerative processes, the aim of this study was to determine whether the administration of equimolar concentrations of two neurotoxins evoking different toxic patterns can induce differential effects on primary astrocytes obtained either from newborn or adult rats, with particular emphasis on those events linked to oxidative stress as a potential source of damage. Primary cortical astrocyte cultures derived from rat brains were exposed to 1-methyl-4-phenylpyridinium (MPP+) or beta-amyloid peptide (β-amyloid). Mitochondrial functionality and cell viability were determined as physiological parameters, whereas lipid and protein oxidation were used as markers of oxidative damage. The results of these experiments pointed towards a higher vulnerability to MPP + over β-amyloid, on most of the tested markers. Hence, in order to allow a comprehensive evaluation of astrocytic responses against MPP + intoxication, a third astrocyte group was included for dose-response experiments: astrocytes derived from aged rats. The present data indicate that the differences associated with age were mainly found in astrocytes exposed to MPP + (25 and 50 μM) at 1-h treatment. Results are discussed in terms of the differential mechanisms involved in each model.

  2. Interleukin-1-mediated febrile responses in mice and interleukin-1 beta activation of NFkappaB in mouse primary astrocytes, involves the interleukin-1 receptor accessory protein.

    PubMed

    Zetterström, M; Lundkvist, J; Malinowsky, D; Eriksson, G; Bartfai, T

    1998-06-01

    The endogenous pyrogen interleukin-1 (IL-1) is considered as one of the key molecules in orchestrating the host response of injury and inflammation. IL-1 exerts its effects upon binding to the type I IL-1 receptor (IL-1RI). The IL-1-IL-1RI complex is further thought to associate with the IL-1 receptor accessory protein (IL-1RAcP), which is suggested to be important for most IL-1 signal transduction pathways. With the aim of investigating the importance of the IL-1RAcP in IL-1 signalling, IL-1alpha and IL-1beta induced febrile responses and IL-1beta-mediated activation of NFkappaB in primary astrocyte cultures were examined using IL-1RAcP-deficient (IL-1RAcP KO) and wild type mice, respectively. It was shown that neither recombinant rat IL-1alpha (rrIL-1alpha, 25 microg/kg), recombinant rat IL-1beta (rrIL-1beta, 40 microg/kg) nor recombinant human IL-1beta (rhIL-1beta, 50 microg/kg) injected i.p. could elicit febrile responses in the IL-1RAcP-deficient mice, while the same doses of rrIL-1alpha/beta or rhIL-1beta injected into wild type mice caused normal fever responses. A febrile response could be induced in the IL-1RAcP-deficient mice by i.p. administration of E. coli lipopolysaccharide (LPS, 50 microg/kg) and this response was similar to that obtained in wild type mice. Furthermore, it was shown that rhIL-1beta activated, in a concentration-dependent manner, nuclear translocation of the transcriptional nuclear factor kappa B (NFkappaB) in primary astrocyte cultures prepared from wild type mice, whereas no IL-1beta-induced translocation of NFkappaB could be detected in cultures prepared from IL-1RAcP-deficient mice, as revealed by electrophoretic mobility shift assay (EMSA). The rhIL-1beta-induced NFkappaB complexes were shown to contain p50 but no, or very little, p65 and cRel immunoreactive proteins.

  3. Mechanisms of CDDO-imidazolide-mediated cytoprotection against acrolein-induced neurocytotoxicity in SH-SY5Y cells and primary human astrocytes.

    PubMed

    Speen, Adam; Jones, Colton; Patel, Ruby; Shah, Halley; Nallasamy, Palanisamy; Brooke, Elizabeth A S; Zhu, Hong; Li, Y Robert; Jia, Zhenquan

    2015-10-01

    Acrolein is a ubiquitous unsaturated aldehyde has been implicated in the pathogenesis of various neurological disorders. However, limited study has been conducted into potential therapeutic protection and underlying mechanism against acrolein-induced cytotoxicity via upregulation of cellular aldehyde-detoxification defenses. In this study we have utilized RA-differentiated human SH-SY5Y cells and primary human astrocytes to investigate the induction of glutathione (GSH) by the synthetic triterpenoid 2-cyano-3,12-dixooleana-1,9-dien-28-imidazolide (CDDO-Im) and the protective effects CDDO-Im-mediated antioxidant defenses on acrolein toxicity. Acrolein exposure to RA-differentiated SH-SY5Y cells resulted in a significant time dependent depletion of cellular GSH preceding a reduction in cell viability and LDH release. Further, we demonstrated the predominance of cellular GSH in protection against acrolein-induced cytotoxicity. Buthionine sulfoximine (BSO) at 25μM dramatically depleted GSH and significantly potentiated acrolein-induced cytotoxicity. Pretreatment of the cells with 100nM CDDO-Im afforded a dramatic protection against acrolein-induced cytotoxicity. Pretreatment of BSO and CDDO was found to prevent the CDDO-Im-mediated GSH induction and partially reversed the cytoprotective effects of CDDO-Im against acrolein cytotoxicity. Overall, this study represents for the first time the CDDO-Im mediated upregulation of GSH is a predominant mechanism against acrolein-induced neurotoxicity.

  4. Primary liver tumors in beagle dogs exposed by inhalation to aerosols of plutonium-238 dioxide

    SciTech Connect

    Gillett, N.A.; Muggenburg, B.A.; Mewhinney, J.A.; Hahn, F.F.; Seiler, F.A.; Boecker, B.B.; McClellan, R.O.

    1988-11-01

    Primary liver tumors developed in Beagle dogs exposed by inhalation to aerosols of /sup 238/PuO/sub 2/. Initial deposition of /sup 238/PuO/sub 2/ in the respiratory tract was followed by translocation of a portion of the /sup 238/Pu to the liver and skeleton, which resulted in a large dose commitment and tumor risk to all three tissues. In a population of 144 dogs exposed to /sup 238/PuO/sub 2/, 112 dogs died or were killed 4000 days after /sup 238/Pu exposure, 100 dogs had osteosarcoma, and 28 dogs had lung cancers. At increasing times after exposure, however, liver lesions have become more pronounced. Ten primary liver tumors in nine animals were diagnosed in the dogs dying before 4000 days after exposure. An additional five primary liver tumors in three dogs occurred in 9 animals killed after 4000 days after exposure. The majority of these tumors have been fibrosarcomas. The liver tumors were usually not the cause of death, and rarely metastasized. The occurrence of liver tumors in this study indicates that /sup 238/Pu is an effective hepatic carcinogen. Liver carcinogenesis is assuming an increasing importance in this study at late times after inhalation exposure. These results suggest that the liver may be an important organ at risk for the development of neoplasia in humans at time periods long after inhalation of /sup 238/Pu.

  5. Effects of homocysteine on metabolic pathways in cultured astrocytes.

    PubMed

    Jin, Ying; Brennan, Lorraine

    2008-06-01

    Homocysteine is an amino acid that is an important risk factor for several neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Increased homocysteine levels induce neuronal cell death in a variety of neuronal types. However, very few studies have probed the effects of homocysteine in astrocytes. The present study investigated the effects of homocysteine on primary cultures of astrocytes by exposing astrocytes to 400 microM homocysteine for 20 h. Metabolic extracts of cells were prepared following a 4-h incubation in minimum medium with 5.5 mM [U-(13)C]glucose in the presence or absence of homocysteine and analysed using (13)C NMR. The expression level of pyruvate dehydrogenase kinase isoform 2 (PDK-2), NAD(P)H levels and mitochondrial membrane potential responses were investigated following culture with homocysteine. Metabolomic analysis was performed using (1)H NMR spectroscopy and pattern recognition analysis. Following incubation with homocysteine there was a significant decrease (48%) in the ratio of flux through pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH) which was due to an increased flux through PDH. In addition, homocysteine culture resulted in a significant reduction in PDK-2 protein expression. Following stimulation with glucose there was a significant increase in NAD(P)H levels and an impaired hyperpolarisation of the mitochondrial membrane in homocysteine-treated cells. Metabolomic analysis showed that the most discriminating metabolites following homocysteine treatment were choline and hypotaurine. In summary, the results demonstrated that sub-lethal concentrations of homocysteine caused significant metabolic changes and altered mitochondrial function in primary cultures of astrocytes. PMID:18417255

  6. A novel co-culture model of the blood-retinal barrier based on primary retinal endothelial cells, pericytes and astrocytes.

    PubMed

    Wisniewska-Kruk, Joanna; Hoeben, Kees A; Vogels, Ilse M C; Gaillard, Pieter J; Van Noorden, Cornelis J F; Schlingemann, Reinier O; Klaassen, Ingeborg

    2012-03-01

    Loss of blood-retinal barrier (BRB) properties is an important feature in the pathology of diabetic macular edema (DME), but cellular mechanisms underlying BRB dysfunction are poorly understood. Therefore, we developed and characterized a novel in vitro BRB model, based on primary bovine retinal endothelial cells (BRECs). These cells were shown to maintain specific in vivo BRB properties by expressing high levels of the endothelial junction proteins occludin, claudin-5, VE-cadherin and ZO-1 at cell borders, and the specific pumps glucose transporter-1 (GLUT1) and efflux transporter P-glycoprotein (MDR1). To investigate the influence of pericytes and astrocytes on BRB maintenance in vitro, we compared five different co-culture BRB models, based on BRECs, bovine retinal pericytes (BRPCs) and rat glial cells. Co-cultures of BRECs with BRPCs and glial cells showed the highest trans-endothelial resistance (TEER) as well as decreased permeability of tracers after vascular endothelial growth factor (VEGF) stimulation, suggesting a major role for these cell types in maintaining barrier properties. To mimic the in vivo situation of DME, we stimulated BRECs with VEGF, which downregulated MDR1 and GLUT1 mRNA levels, transiently reduced expression levels of endothelial junctional proteins and altered their organization, increased the number of intercellular gaps in BRECs monolayers and influence the permeability of the model to differently-sized molecular tracers. Moreover, as has been shown in vivo, expression of plasmalemma vesicle-associated protein (PLVAP) was increased in endothelial cells in the presence of VEGF. This in vitro model is the first co-culture model of the BRB that mimicks in vivo VEGF-dependent changes occurring in DME. PMID:22200486

  7. Isolation and Characterization of Ischemia-Derived Astrocytes (IDAs) with Ability to Transactivate Quiescent Astrocytes

    PubMed Central

    Villarreal, Alejandro; Rosciszewski, Gerardo; Murta, Veronica; Cadena, Vanesa; Usach, Vanina; Dodes-Traian, Martin M.; Setton-Avruj, Patricia; Barbeito, Luis H.; Ramos, Alberto J.

    2016-01-01

    Reactive gliosis involving activation and proliferation of astrocytes and microglia, is a widespread but largely complex and graded glial response to brain injury. Astroglial population has a previously underestimated high heterogeneity with cells differing in their morphology, gene expression profile, and response to injury. Here, we identified a subset of reactive astrocytes isolated from brain focal ischemic lesions that show several atypical characteristics. Ischemia-derived astrocytes (IDAs) were isolated from early ischemic penumbra and core. IDA did not originate from myeloid precursors, but rather from pre-existing local progenitors. Isolated IDA markedly differ from primary astrocytes, as they proliferate in vitro with high cell division rate, show increased migratory ability, have reduced replicative senescence and grow in the presence of macrophages within the limits imposed by the glial scar. Remarkably, IDA produce a conditioned medium that strongly induced activation on quiescent primary astrocytes and potentiated the neuronal death triggered by oxygen-glucose deprivation. When re-implanted into normal rat brains, eGFP-IDA migrated around the injection site and induced focal reactive gliosis. Inhibition of gamma secretases or culture on quiescent primary astrocytes monolayers facilitated IDA differentiation to astrocytes. We propose that IDA represent an undifferentiated, pro-inflammatory, highly replicative and migratory astroglial subtype emerging from the ischemic microenvironment that may contribute to the expansion of reactive gliosis. Main Points: Ischemia-derived astrocytes (IDA) were isolated from brain ischemic tissue IDA show reduced replicative senescence, increased cell division and spontaneous migration IDA potentiate death of oxygen-glucose deprived cortical neurons IDA propagate reactive gliosis on quiescent astrocytes in vitro and in vivo Inhibition of gamma secretases facilitates IDA differentiation to astrocytes PMID:27313509

  8. α1-Antitrypsin reduces rhinovirus infection in primary human airway epithelial cells exposed to cigarette smoke

    PubMed Central

    Berman, Reena; Jiang, Di; Wu, Qun; Chu, Hong Wei

    2016-01-01

    Human rhinovirus (HRV) infections target airway epithelium and are the leading cause of acute exacerbations of COPD. Cigarette smoke (CS) increases the severity of viral infections, but there is no effective therapy for HRV infection. We determined whether α1-antitrypsin (A1AT) reduces HRV-16 infection in CS-exposed primary human airway epithelial cells. Brushed bronchial epithelial cells from normal subjects and patients diagnosed with COPD were cultured at air–liquid interface to induce mucociliary differentiation. These cells were treated with A1AT or bovine serum albumin for 2 hours and then exposed to air or whole cigarette smoke (WCS) with or without HRV-16 (5×104 50% Tissue Culture Infective Dose [TCID50]/transwell) infection for 24 hours. WCS exposure significantly increased viral load by an average of fivefold and decreased the expression of antiviral genes interferon-λ1, OAS1, and MX1. When A1AT was added to WCS-exposed cells, viral load significantly decreased by an average of 29-fold. HRV-16 infection significantly increased HRV-16 receptor intercellular adhesion molecule-1 messenger RNA expression in air-exposed cells, which was decreased by A1AT. A1AT-mediated reduction of viral load was not accompanied by increased epithelial antiviral gene expression or by inhibiting the activity of 3C protease involved in viral replication or maturation. Our findings demonstrate that A1AT treatment prevents a WCS-induced increase in viral load and for the first time suggest a therapeutic effect of A1AT on HRV infection. PMID:27354786

  9. Redox-sensitive protein phosphatase activity regulates the phosphorylation state of p38 protein kinase in primary astrocyte culture.

    PubMed

    Robinson, K A; Stewart, C A; Pye, Q N; Nguyen, X; Kenney, L; Salzman, S; Floyd, R A; Hensley, K

    1999-03-15

    Reactive oxygen species (ROS) have been implicated as second messengers that activate protein kinase cascades, although the means by which ROS regulate signal transduction remains unclear. In the present study, we show that interleukin 1beta (IL1beta), H2O2, and sorbitol-induced hyperosmolarity mediate a 5- to 10-fold increase in phosphorylation (activation) of the p38 protein kinase in rat primary glial cells as measured by analyses of Western blots using an antibody directed against the dually phosphorylated (active) p38. Additionally, IL1beta was found to elicit H2O2 synthesis in these cells. Concurrent with p38 phosphorylation, all three stimulation paradigms caused an inhibition of protein phosphatase activity. Phenyl-tert-butyl nitrone (PBN), a nitrone-based free radical trap and N-acetyl-cysteine (NAC), a thiol reducing agent, were examined for their effects on the phosphorylation of p38 as well as phosphatase activity. Pretreatment of cells with either PBN or NAC at 1.0 mM suppressed IL1beta H2O2, and sorbitol-mediated activation of p38 and significantly increased phosphatase activity. These data suggest that ROS, particularly H2O2, are used as second messenger substances that activate p38 in part via the transient inactivation of regulatory protein phosphatases.

  10. Spatial and temporal single-cell volume estimation by a fluorescence imaging technique with application to astrocytes in primary culture

    NASA Astrophysics Data System (ADS)

    Khatibi, Siamak; Allansson, Louise; Gustavsson, Tomas; Blomstrand, Fredrik; Hansson, Elisabeth; Olsson, Torsten

    1999-05-01

    Cell volume changes are often associated with important physiological and pathological processes in the cell. These changes may be the means by which the cell interacts with its surrounding. Astroglial cells change their volume and shape under several circumstances that affect the central nervous system. Following an incidence of brain damage, such as a stroke or a traumatic brain injury, one of the first events seen is swelling of the astroglial cells. In order to study this and other similar phenomena, it is desirable to develop technical instrumentation and analysis methods capable of detecting and characterizing dynamic cell shape changes in a quantitative and robust way. We have developed a technique to monitor and to quantify the spatial and temporal volume changes in a single cell in primary culture. The technique is based on two- and three-dimensional fluorescence imaging. The temporal information is obtained from a sequence of microscope images, which are analyzed in real time. The spatial data is collected in a sequence of images from the microscope, which is automatically focused up and down through the specimen. The analysis of spatial data is performed off-line and consists of photobleaching compensation, focus restoration, filtering, segmentation and spatial volume estimation.

  11. ASTROCYTES: EMERGING STARS IN LEUKODYSTROPHY PATHOGENESIS

    PubMed Central

    Lanciotti, Angela; Brignone, Maria Stefania; Bertini, Enrico; Petrucci, Tamara C.; Aloisi, Francesca; Ambrosini, Elena

    2013-01-01

    Astrocytes are the predominant glial cell population in the central nervous system (CNS). Once considered only passive scaffolding elements, astrocytes are now recognised as cells playing essential roles in CNS development and function. They control extracellular water and ion homeostasis, provide substrates for energy metabolism, and regulate neurogenesis, myelination and synaptic transmission. Due to these multiple activities astrocytes have been implicated in almost all brain pathologies, contributing to various aspects of disease initiation, progression and resolution. Evidence is emerging that astrocyte dysfunction can be the direct cause of neurodegeneration, as shown in Alexander’s disease where myelin degeneration is caused by mutations in the gene encoding the astrocyte-specific cytoskeleton protein glial fibrillary acidic protein. Recent studies point to a primary role for astrocytes in the pathogenesis of other genetic leukodystrophies such as megalencephalic leukoencephalopathy with subcortical cysts and vanishing white matter disease. The aim of this review is to summarize current knowledge of the pathophysiological role of astrocytes focusing on their contribution to the development of the above mentioned leukodystrophies and on new perspectives for the treatment of neurological disorders. PMID:24340223

  12. 4-Hydroxyhexenal (HHE) impairs glutamate transport in astrocyte cultures.

    PubMed

    Lovell, Mark A; Bradley, Melissa A; Fister, Shuling X

    2012-01-01

    Multiple studies show elevations of α,β-unsaturated aldehydic by-products of lipid peroxidation including 4-hydroxynonenal and acrolein in vulnerable brain regions of subjects throughout the progression of Alzheimer's disease (AD). More recently 4-hydroxyhexenal (HHE), a diffusible α,β-unsaturated aldehyde resulting from peroxidation of ω-3 polyunsaturated fatty acids, was shown to be elevated in the hippocampus/parahippocampal gyrus (HPG) of subjects with preclinical AD (PCAD) and in late stage AD (LAD). HHE treatment of primary rat cortical neuron cultures led to a time- and concentration-dependent decrease in survival and glucose uptake. To determine if HHE also impairs glutamate uptake, primary rat astrocyte cultures were exposed to HHE for 4 hours and glutamate transport measured. Results show subtoxic (2.5 μM) HHE concentrations significantly (p < 0.05) impair glutamate uptake in primary astrocytes. Immunoprecipitation of excitatory amino acid transporter-2 (EAAT-2), the primary glutamate transporter in brain, from normal control, mild cognitive impairment (MCI), PCAD, and LAD HPG followed by quantification of HHE immunolabeling showed a significant increase in HHE positive EAAT-2 in MCI and LAD HPG. Together these data suggest HHE can significantly impair glutamate uptake and may play a role in the pathogenesis of AD. PMID:22766736

  13. Primary cultured astrocytes from old rats are capable to activate the Nrf2 response against MPP+ toxicity after tBHQ pretreatment.

    PubMed

    Alarcón-Aguilar, Adriana; Luna-López, Armando; Ventura-Gallegos, José L; Lazzarini, Roberto; Galván-Arzate, Sonia; González-Puertos, Viridiana Y; Morán, Julio; Santamaría, Abel; Königsberg, Mina

    2014-08-01

    Astrocytes are key players for brain physiology, protecting neurons by releasing antioxidant enzymes; however, they are also susceptible to damage by neurotoxins. Nuclear factor erythroid-derived 2-like 2 (Nrf2) is a central regulator of the antioxidant response, and therefore, pharmacologic inducers are often used to activate this transcription factor to induce cellular protection. To date, it still remains unknown if cells from aged animals are capable of developing this response. Therefore, the purpose of this work was to determine if cortical astrocytes derived from old rats are able to respond to tertbuthyl-hydroquinene (tBHQ) pretreatment and stimulate the Nrf2-antioxidant response pathway to induce an antioxidant strategy against MPP+ toxicity, one of the most used molecules to model Parkinson's disease. Our results show that, although astrocytes from adult and old rats were more susceptible to MPP+ toxicity than astrocytes from newborn rats, when pretreated with tertbuthyl-hydroquinene, they were able to transactivate Nrf2, increasing antioxidant enzymes and developing cellular protection. These results are discussed in terms of the doses used to create protective responses.

  14. Methylene blue protects astrocytes against glucose oxygen deprivation by improving cellular respiration.

    PubMed

    Roy Choudhury, Gourav; Winters, Ali; Rich, Ryan M; Ryou, Myoung-Gwi; Gryczynski, Zygmunt; Yuan, Fang; Yang, Shao-Hua; Liu, Ran

    2015-01-01

    Astrocytes outnumber neurons and serve many metabolic and trophic functions in the mammalian brain. Preserving astrocytes is critical for normal brain function as well as for protecting the brain against various insults. Our previous studies have indicated that methylene blue (MB) functions as an alternative electron carrier and enhances brain metabolism. In addition, MB has been shown to be protective against neurodegeneration and brain injury. In the current study, we investigated the protective role of MB in astrocytes. Cell viability assays showed that MB treatment significantly protected primary astrocytes from oxygen-glucose deprivation (OGD) & reoxygenation induced cell death. We also studied the effect of MB on cellular oxygen and glucose metabolism in primary astrocytes following OGD-reoxygenation injury. MB treatment significantly increased cellular oxygen consumption, glucose uptake and ATP production in primary astrocytes. In conclusion our study demonstrated that MB protects astrocytes against OGD-reoxygenation injury by improving astrocyte cellular respiration.

  15. Modulation of neuritogenesis by astrocyte muscarinic receptors.

    PubMed

    Guizzetti, Marina; Moore, Nadia H; Giordano, Gennaro; Costa, Lucio G

    2008-11-14

    Astrocytes have been shown to release factors that have promoting or inhibiting effects on neuronal development. However, mechanisms controlling the release of such factors from astrocytes are not well established. Astrocytes express muscarinic receptors whose activation stimulates a robust intracellular signaling, although the role of these receptors in glial cells is not well understood. Acetylcholine and acetylcholine receptors are present in the brain before synaptogenesis occurs and are believed to be involved in neuronal maturation. The present study was undertaken to investigate whether stimulation of muscarinic receptors in astrocytes would modulate neurite outgrowth in hippocampal neurons. Rat hippocampal neurons, co-cultured with rat cortical astrocytes previously exposed to the cholinergic agonist carbachol, displayed longer neurites. The effect of carbachol in astrocytes was due to the activation of M3 muscarinic receptors. Exposure of astrocytes to carbachol increased the expression of the extracellular matrix proteins fibronectin and laminin-1 in these cells. This effect was mediated in part by an increase in laminin-1 and fibronectin mRNA levels and in part by the up-regulation of the production and release of plasminogen activator inhibitor-1, an inhibitor of the proteolytic degradation of the extracellular matrix. The inhibition of fibronectin activity strongly reduced the effect of carbachol on the elongation of all the neurites, whereas inhibition of laminin-1 activity reduced the elongation of minor neurites only. Plasminogen activator inhibitor-1 also induced neurite elongation through a direct effect on neurons. Taken together, these results demonstrate that cholinergic muscarinic stimulation of astrocytes induces the release of permissive factors that accelerate neuronal development.

  16. Growth of purified astrocytes in a chemically defined medium

    SciTech Connect

    Morrison, R.S.; De Vellis, J.

    1981-11-01

    Astrocytes purified from primary cultures of neonatal rat cerebrum can not be grown in a synthetic medium supplemented with putrescine, prostaglandin F/sub 2//sub ..cap alpha../, insulin, fibroblast growth factor, and hydrocortisone. These five supplements have a marked synergistic effect on growth when used in combination but have little effect when used individually. Astrocytes grown in the defined medium exhibit dramatic changes in morphological characteristics in comparison to cells grown in serum-free or serum-supplemented medium. In addition, these cells express the astrocyte-specific marker glial fibrillary acidic protein and are estimated by several criteria to be greater than 95% astrocytes.

  17. Astrocyte glutamine synthetase: pivotal in health and disease.

    PubMed

    Rose, Christopher F; Verkhratsky, Alexei; Parpura, Vladimir

    2013-12-01

    The multifunctional properties of astrocytes signify their importance in brain physiology and neurological function. In addition to defining the brain architecture, astrocytes are primary elements of brain ion, pH and neurotransmitter homoeostasis. GS (glutamine synthetase), which catalyses the ATP-dependent condensation of ammonia and glutamate to form glutamine, is an enzyme particularly found in astrocytes. GS plays a pivotal role in glutamate and glutamine homoeostasis, orchestrating astrocyte glutamate uptake/release and the glutamate-glutamine cycle. Furthermore, astrocytes bear the brunt of clearing ammonia in the brain, preventing neurotoxicity. The present review depicts the central function of astrocytes, concentrating on the importance of GS in glutamate/glutamine metabolism and ammonia detoxification in health and disease.

  18. Caffeic acid phenethyl ester induces adrenoleukodystrophy (Abcd2) gene in human X-ALD fibroblasts and inhibits the proinflammatory response in Abcd1/2 silenced mouse primary astrocytes.

    PubMed

    Singh, Jaspreet; Khan, Mushfiquddin; Singh, Inderjit

    2013-04-01

    X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder caused by mutations in the ABCD1 gene. Accumulation of very long chain fatty acids (VLCFA) that have been attributed to reduced peroxisomal VLCFA β-oxidation activity are the hallmark of the disease. Overexpression of ABCD2 gene, the closest homolog of ABCD1, has been shown to compensate for ABCD1, thus correcting the VLCFA derangement. The accumulation of VLCFA leads to a neuroinflammatory disease process associated with demyelination of the cerebral white matter. The present study underlines the importance of caffeic acid phenethyl ester (CAPE) in inducing the expression of ABCD2 (ALDRP), and normalizing the peroxisomal β-oxidation as well as the levels of saturated and monounsaturated VLCFAs in cultured human skin fibroblasts of X-ALD patients. The expression of ELOVL1, the single elongase catalyzing the synthesis of both saturated VLCFA (C26:0) and mono-unsaturated VLCFA (C26:1), was also reduced by CAPE treatment. Importantly, CAPE upregulated Abcd2 expression and peroxisomal β-oxidation and lowered the VLCFA levels in Abcd1-deficient U87 astrocytes and B12 oligodendrocytes. In addition, using Abcd1/Abcd2-silenced mouse primary astrocytes we examined the effects of CAPE in VLCFA-induced inflammatory response. CAPE treatment decreased the inflammatory response as the expression of inducible nitric oxide synthase, inflammatory cytokine, and activation of NF-κB in Abcd1/Abcd2-silenced mouse primary astrocytes was reduced. The observations indicate that CAPE corrects both the metabolic disease of VLCFA as well as secondary inflammatory disease; therefore, it may be a potential drug candidate to be tested for X-ALD therapy in humans.

  19. Membrane damage and repair in primary monocytes exposed to human β-defensin-3

    PubMed Central

    Lioi, Anthony B.; Reyes Rodriguez, Angel L.; Funderburg, Nicholas T.; Feng, Zhimin; Weinberg, Aaron; Sieg, Scott F.

    2012-01-01

    Interactions of AMPs with plasma membranes of primary human immune cells are poorly characterized. Analysis of PI exclusion as a measure of membrane integrity indicated that hBD-3 caused membrane perturbations in monocytes but not T or B cells at concentrations typically used to kill bacteria or to induce activation of APCs. Bleb-like structures were observed in monocytes exposed to hBD-3. These cells also increased surface expression of LAMP1, a membrane repair marker after exposure to hBD-3. Furthermore, cell death was enhanced by adding an inhibitor of membrane repair. Removal of cholesterol from membranes resulted in greater susceptibility of cells to hBD-3, but cholesterol content was not different between the cell types, as assessed by filipin staining. Freshly isolated monocytes expressed higher levels of the negatively charged phospholipid, PS, on their outer leaflet compared with B or T cells. Preincubation of monocytes with molecules that bind PS protected these cells from hBD-3-induced membrane damage, suggesting that outer-membrane PS expression can at least partially explain monocyte susceptibility to hBD-3. The potential for membrane disruption caused by AMPs should be evaluated in various cell types when considering these molecules for therapeutic applications in humans. PMID:22837529

  20. Endothelin, Astrocytes and Glaucoma

    PubMed Central

    Prasanna, Ganesh; Krishnamoorthy, Raghu; Yorio, Thomas

    2010-01-01

    It has become increasingly clear that astrocytes may play an important role in the genesis of glaucoma. Astrogliosis occurs in response to ocular stress or the presence of noxious stimuli. Agents that appear to stimulate reactive gliosis are becoming increasingly clear. One class of agents that is emerging is the endothelins (ETs; specifically, ET-1). In this review we examine the interactions of ET-1 with astrocytes and provide examples where ET-1 appears to contribute to activation of astrocytes and play a role in the neurodegenerative effects that accompany such reactivation resulting in astrogliosis. These actions are presented in the context of glaucoma although information is also presented with respect to ET-1's role in the central nervous system and brain. While much has been learned with respect to ET-1/astrocyte interactions, there are still a number of questions concerning the potential therapeutic implications of these findings. Hopefully this review will stimulate others to examine this potential. PMID:20849847

  1. Inhibition of Astrocytic Glutamine Synthetase by Lead is Associated with a Slowed Clearance of Hydrogen Peroxide by the Glutathione System

    PubMed Central

    Robinson, Stephen R.; Lee, Alan; Bishop, Glenda M.; Czerwinska, Hania; Dringen, Ralf

    2015-01-01

    Lead intoxication in humans is characterized by cognitive impairments, particularly in the domain of memory, where evidence indicates that glutamatergic neurotransmission may be impacted. Animal and cell culture studies have shown that lead decreases the expression and activity of glutamine synthetase (GS) in astrocytes, yet the basis of this effect is uncertain. To investigate the mechanism responsible, the present study exposed primary astrocyte cultures to a range of concentrations of lead acetate (0–330 μM) for up to 24 h. GS activity was significantly reduced in cells following 24 h incubation with 100 or 330 μM lead acetate. However, no reduction in GS activity was detected when astrocytic lysates were co-incubated with lead acetate, suggesting that the mechanism is not due to a direct interaction and involves intact cells. Since GS is highly sensitive to oxidative stress, the capacity of lead to inhibit the clearance of hydrogen peroxide (H2O2) was investigated. It was found that exposure to lead significantly diminished the capacity of astrocytes to degrade H2O2, and that this was due to a reduction in the effectiveness of the glutathione system, rather than to catalase. These results suggest that the inhibition of GS activity in lead poisoning is a consequence of slowed H2O2 clearance, and supports the glutathione pathway as a primary therapeutic target. PMID:26696846

  2. Spinal astrocytes produce and secrete dynorphin neuropeptides.

    PubMed

    Wahlert, Andrew; Funkelstein, Lydiane; Fitzsimmons, Bethany; Yaksh, Tony; Hook, Vivian

    2013-04-01

    Dynorphin peptide neurotransmitters (neuropeptides) have been implicated in spinal pain processing based on the observations that intrathecal delivery of dynorphin results in proalgesic effects and disruption of extracellular dynorphin activity (by antisera) prevents injury evoked hyperalgesia. However, the cellular source of secreted spinal dynorphin has been unknown. For this reason, this study investigated the expression and secretion of dynorphin-related neuropeptides from spinal astrocytes (rat) in primary culture. Dynorphin A (1-17), dynorphin B, and α-neoendorphin were found to be present in the astrocytes, illustrated by immunofluorescence confocal microscopy, in a discrete punctate pattern of cellular localization. Measurement of astrocyte cellular levels of these dynorphins by radioimmunoassays confirmed the expression of these three dynorphin-related neuropeptides. Notably, BzATP (3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate) and KLA (di[3-deoxy-D-manno-octulosonyl]-lipid A) activation of purinergic and toll-like receptors, respectively, resulted in stimulated secretion of dynorphins A and B. However, α-neoendorphin secretion was not affected by BzATP or KLA. These findings suggest that dynorphins A and B undergo regulated secretion from spinal astrocytes. These findings also suggest that spinal astrocytes may provide secreted dynorphins that participate in spinal pain processing.

  3. Decreased face primary motor cortex (face-M1) excitability induced by noxious stimulation of the rat molar tooth pulp is dependent on the functional integrity of face-M1 astrocytes.

    PubMed

    Awamleh, L; Pun, H; Lee, J-C; Avivi-Arber, L

    2015-04-01

    Acute inflammatory dental pain is a prevalent condition often associated with limited jaw movements. Mustard oil (MO, a small-fiber excitant/inflammatory irritant) application to the rat molar tooth pulp induces increased excitability (i.e., central sensitization) of trigeminal medullary dorsal horn (MDH) nociceptive neurons that can be modulated by MDH application of the astrocytic inhibitor methionine sulfoximine (MSO). The objectives of the study were to determine whether MO application to the rat right maxillary first molar tooth pulp affects left face-M1 excitability manifested as altered intracortical microstimulation thresholds for evoking electromyographic activity in the right anterior digastric (RAD, jaw-opening muscle), and whether MSO application to face-M1 can modulate this MO effect. Under Ketamine general anesthesia, Sprague-Dawley male rats had a microelectrode positioned at a low-threshold (≤30 μA) face-M1 site. Then MO (n = 16) or control solution (n = 16) was applied to the previously exposed tooth pulp, and RAD threshold was monitored for 15 min. MSO (0.1 mM, n = 8) or saline (n = 8) was then applied to the face-M1, and RAD thresholds were monitored every 15 min for 120 min. ANOVA followed by post hoc Bonferroni was used to analyze data (p < 0.05). Within 15 min of MO (but not control) pulp application, RAD thresholds increased significantly (p < 0.001) as compared to baseline. One hour following MSO (but not saline) application to the face-M1, RAD thresholds decreased significantly (p = 0.005) toward baseline. These novel findings suggest that acute inflammatory dental pain is associated with decreased face-M1 excitability that may be dependent on the functional integrity of face-M1 astrocytes and related to mechanisms underlying limited jaw movements in acute orofacial pain conditions. PMID:25618005

  4. Ghrelin Regulates Glucose and Glutamate Transporters in Hypothalamic Astrocytes

    PubMed Central

    Fuente-Martín, Esther; García-Cáceres, Cristina; Argente-Arizón, Pilar; Díaz, Francisca; Granado, Miriam; Freire-Regatillo, Alejandra; Castro-González, David; Ceballos, María L.; Frago, Laura M.; Dickson, Suzanne L.; Argente, Jesús; Chowen, Julie A.

    2016-01-01

    Hypothalamic astrocytes can respond to metabolic signals, such as leptin and insulin, to modulate adjacent neuronal circuits and systemic metabolism. Ghrelin regulates appetite, adiposity and glucose metabolism, but little is known regarding the response of astrocytes to this orexigenic hormone. We have used both in vivo and in vitro approaches to demonstrate that acylated ghrelin (acyl-ghrelin) rapidly stimulates glutamate transporter expression and glutamate uptake by astrocytes. Moreover, acyl-ghrelin rapidly reduces glucose transporter (GLUT) 2 levels and glucose uptake by these glial cells. Glutamine synthetase and lactate dehydrogenase decrease, while glycogen phosphorylase and lactate transporters increase in response to acyl-ghrelin, suggesting a change in glutamate and glucose metabolism, as well as glycogen storage by astrocytes. These effects are partially mediated through ghrelin receptor 1A (GHSR-1A) as astrocytes do not respond equally to desacyl-ghrelin, an isoform that does not activate GHSR-1A. Moreover, primary astrocyte cultures from GHSR-1A knock-out mice do not change glutamate transporter or GLUT2 levels in response to acyl-ghrelin. Our results indicate that acyl-ghrelin may mediate part of its metabolic actions through modulation of hypothalamic astrocytes and that this effect could involve astrocyte mediated changes in local glucose and glutamate metabolism that alter the signals/nutrients reaching neighboring neurons. PMID:27026049

  5. Ghrelin Regulates Glucose and Glutamate Transporters in Hypothalamic Astrocytes.

    PubMed

    Fuente-Martín, Esther; García-Cáceres, Cristina; Argente-Arizón, Pilar; Díaz, Francisca; Granado, Miriam; Freire-Regatillo, Alejandra; Castro-González, David; Ceballos, María L; Frago, Laura M; Dickson, Suzanne L; Argente, Jesús; Chowen, Julie A

    2016-03-30

    Hypothalamic astrocytes can respond to metabolic signals, such as leptin and insulin, to modulate adjacent neuronal circuits and systemic metabolism. Ghrelin regulates appetite, adiposity and glucose metabolism, but little is known regarding the response of astrocytes to this orexigenic hormone. We have used both in vivo and in vitro approaches to demonstrate that acylated ghrelin (acyl-ghrelin) rapidly stimulates glutamate transporter expression and glutamate uptake by astrocytes. Moreover, acyl-ghrelin rapidly reduces glucose transporter (GLUT) 2 levels and glucose uptake by these glial cells. Glutamine synthetase and lactate dehydrogenase decrease, while glycogen phosphorylase and lactate transporters increase in response to acyl-ghrelin, suggesting a change in glutamate and glucose metabolism, as well as glycogen storage by astrocytes. These effects are partially mediated through ghrelin receptor 1A (GHSR-1A) as astrocytes do not respond equally to desacyl-ghrelin, an isoform that does not activate GHSR-1A. Moreover, primary astrocyte cultures from GHSR-1A knock-out mice do not change glutamate transporter or GLUT2 levels in response to acyl-ghrelin. Our results indicate that acyl-ghrelin may mediate part of its metabolic actions through modulation of hypothalamic astrocytes and that this effect could involve astrocyte mediated changes in local glucose and glutamate metabolism that alter the signals/nutrients reaching neighboring neurons.

  6. Astrocytes are central in the pathomechanisms of vanishing white matter

    PubMed Central

    Dooves, Stephanie; Bugiani, Marianna; Postma, Nienke L.; Polder, Emiel; Land, Niels; Horan, Stephen T.; van Deijk, Anne-Lieke F.; van de Kreeke, Aleid; Jacobs, Gerbren; Vuong, Caroline; Klooster, Jan; Kamermans, Maarten; Wortel, Joke; Wisse, Lisanne E.; Scheper, Gert C.; Abbink, Truus E.M.; Heine, Vivi M.; van der Knaap, Marjo S.

    2016-01-01

    Vanishing white matter (VWM) is a fatal leukodystrophy that is caused by mutations in genes encoding subunits of eukaryotic translation initiation factor 2B (eIF2B). Disease onset and severity are codetermined by genotype. White matter astrocytes and oligodendrocytes are almost exclusively affected; however, the mechanisms of VWM development remain unclear. Here, we used VWM mouse models, patients’ tissue, and cell cultures to investigate whether astrocytes or oligodendrocytes are the primary affected cell type. We generated 2 mouse models with mutations (Eif2b5Arg191His/Arg191His and Eif2b4Arg484Trp/Arg484Trp) that cause severe VWM in humans and then crossed these strains to develop mice with various mutation combinations. Phenotypic severity was highly variable and dependent on genotype, reproducing the clinical spectrum of human VWM. In all mutant strains, impaired maturation of white matter astrocytes preceded onset and paralleled disease severity and progression. Bergmann glia and retinal Müller cells, nonforebrain astrocytes that have not been associated with VWM, were also affected, and involvement of these cells was confirmed in VWM patients. In coculture, VWM astrocytes secreted factors that inhibited oligodendrocyte maturation, whereas WT astrocytes allowed normal maturation of VWM oligodendrocytes. These studies demonstrate that astrocytes are central in VWM pathomechanisms and constitute potential therapeutic targets. Importantly, astrocytes should also be considered in the pathophysiology of other white matter disorders. PMID:26974157

  7. A mental health intervention strategy for low-income, trauma-exposed Latina immigrants in primary care

    PubMed Central

    Kaltman, Stacey; de Mendoza, Alejandra Hurtado; Serrano, Adriana; Gonzales, Felisa A.

    2016-01-01

    Latinos in the United States face significant mental health disparities related to access to care, quality of care, and outcomes. Prior research suggests that Latinos prefer to receive care for common mental health problems (e.g., depression and anxiety disorders) in primary care settings, suggesting a need for evidence-based mental health services designed for delivery in these settings. This study sought to develop and preliminarily evaluate a mental health intervention for trauma-exposed Latina immigrants with depression and/or PTSD for primary care clinics that serve the uninsured. The intervention was designed to be simultaneously responsive to patients’ preferences for individual psychotherapy, to the needs of safety-net primary care clinics for efficient services, and to address the social isolation that is common to the Latina immigrant experience. Developed based on findings from the research team’s formative research, the resulting intervention incorporated individual and group sessions and combined evidence-based interventions to reduce depression and PTSD symptoms, increase group readiness, and improve perceived social support. Twenty-eight trauma-exposed low-income Latina immigrant women who screened positive for depression and/or PTSD participated in an open pilot trial of the intervention at a community primary care clinic. Results indicated that the intervention was feasible, acceptable, and safe. A randomized controlled trial of the intervention is warranted. PMID:26913774

  8. Cardiac arrhythmia is the primary response of embryonic Pacific herring (Clupea pallasi) exposed to crude oil during weathering.

    PubMed

    Incardona, John P; Carls, Mark G; Day, Heather L; Sloan, Catherine A; Bolton, Jennie L; Collier, Tracy K; Scholz, Nathaniel L

    2009-01-01

    Teleost embryos develop a syndrome characterized by edema when exposed to water that weathers substrates contaminated with crude oil. Previous studies using zebrafish demonstrated that crude oil exposure causes cardiogenic edema, and that the most abundant polycyclic aromatic hydrocarbons (PAHs) in weathered crude oils (tricyclic fluorenes, dibenzothiophenes, and phenanthrenes) are cardiotoxic, causing arrhythmia through a pathway that does not require activation of the aryl hydrocarbon receptor (AHR). We demonstrate here for Pacific herring, a species impacted by the Exxon Valdez oil spill, that the developing heart is the primary target of crude oil exposure. Herring embryos exposed to the effluent of oiled gravel columns developed dose-dependent edema and irregular cardiac arrhythmia soon afterthe heartbeat was established. At a dose that produced cardiac dysfunction in 100% of exposed embryos, tissue levels of tricyclic PAHs were below 1 micromol/kg, suggesting a specific, high affinity target in the heart. These findings have implications for understanding the mechanism of tricyclic PAH cardiotoxicity, the development of biomarkers for the effects of PAH exposure in fish, and understanding the long-term impacts of oil spills and other sources of PAH pollution in aquatic environments. PMID:19209607

  9. Cardiac arrhythmia is the primary response of embryonic Pacific herring (Clupea pallasi) exposed to crude oil during weathering.

    PubMed

    Incardona, John P; Carls, Mark G; Day, Heather L; Sloan, Catherine A; Bolton, Jennie L; Collier, Tracy K; Scholz, Nathaniel L

    2009-01-01

    Teleost embryos develop a syndrome characterized by edema when exposed to water that weathers substrates contaminated with crude oil. Previous studies using zebrafish demonstrated that crude oil exposure causes cardiogenic edema, and that the most abundant polycyclic aromatic hydrocarbons (PAHs) in weathered crude oils (tricyclic fluorenes, dibenzothiophenes, and phenanthrenes) are cardiotoxic, causing arrhythmia through a pathway that does not require activation of the aryl hydrocarbon receptor (AHR). We demonstrate here for Pacific herring, a species impacted by the Exxon Valdez oil spill, that the developing heart is the primary target of crude oil exposure. Herring embryos exposed to the effluent of oiled gravel columns developed dose-dependent edema and irregular cardiac arrhythmia soon afterthe heartbeat was established. At a dose that produced cardiac dysfunction in 100% of exposed embryos, tissue levels of tricyclic PAHs were below 1 micromol/kg, suggesting a specific, high affinity target in the heart. These findings have implications for understanding the mechanism of tricyclic PAH cardiotoxicity, the development of biomarkers for the effects of PAH exposure in fish, and understanding the long-term impacts of oil spills and other sources of PAH pollution in aquatic environments.

  10. Exposed human pulp caused by trauma and/or caries in primary dentition: a histological evaluation.

    PubMed

    Raslan, Nabih; Wetzel, Willi-Eckhard

    2006-06-01

    The aim of this study was to compare the pulpal reactions after exposure due to trauma and/or caries and to suggest the appropriate treatment options for the management of pulp, in the light of the histological findings in the primary teeth. Fifteen primary maxillary incisors with pulpal exposure were examined with light microscope in a blind study. After the histological evaluation, teeth were divided into three groups for the etiological factors due to the exposure according to the patient's files: trauma group, caries group and caries and trauma group. The inflammatory infiltration was diminished from the pulp chamber towards the apical third of the root canal in all of the examined teeth. Teeth in the trauma group presented fewer inflammatory cells in the root canal in comparison with other groups. It was concluded that the teeth with traumatic pulp exposure were considered as more likely to respond positively to pulpotomy technique. Pulpectomy or extraction seemed to be indicated for the decayed or decayed and traumatically injured teeth.

  11. Lead induces increased water permeability in astrocytes expressing aquaporin 4.

    PubMed

    Gunnarson, E; Axehult, G; Baturina, G; Zelenin, S; Zelenina, M; Aperia, A

    2005-01-01

    The water channel aquaporin 4 (AQP4) is abundantly expressed in astrocytes. There is now compelling evidence that AQP4 may contribute to an unfavorable course in brain edema. Acute lead intoxication is a condition that causes brain damage preceded by brain edema. Here we report that lead increases AQP4 water permeability (P(f)) in astrocytes. A rat astrocyte cell line that does not express aquaporin 4 was transiently transfected with aquaporin 4 tagged with green fluorescent protein (GFP). Using confocal laser scanning microscopy we measured water permeability in these cells and in AQP4-negative cells located on the same plate. AQP4-expressing astrocytes had a three-fold higher water permeability than astrocytes not expressing AQP4. Lead exposure induced a significant, 40%, increase in water permeability in astrocytes expressing AQP4, but had no effect on P(f) in astrocytes not expressing AQP4. The increase in water permeability persisted after lead washout, while treatment with a lead chelator, meso-2,3-dimercaptosuccinic acid, abolished the lead-induced increase in P(f). The effect of lead was attenuated in the presence of a calcium (Ca(2+))/calmodulin-dependent protein kinase II (CaMKII) inhibitor, but not in the presence of a protein kinase C inhibitor. In cells expressing AQP4 where the consensus site for CaMKII phosphorylation was mutated, lead failed to increase water permeability. Lead exposure also increased P(f) in rat astroglial cells in primary culture, which express endogenous AQP4. Lead had no effect on P(f) in astrocytes transfected with aquaporin 3. In situ hybridization studies on rat brain after oral lead intake for three days showed no change in distribution of AQP4 mRNA. It is suggested that lead-triggered stimulation of water transport in AQP4-expressing astrocytes may contribute to the pathology of acute lead intoxication.

  12. p53 isoforms regulate astrocyte-mediated neuroprotection and neurodegeneration.

    PubMed

    Turnquist, C; Horikawa, I; Foran, E; Major, E O; Vojtesek, B; Lane, D P; Lu, X; Harris, B T; Harris, C C

    2016-09-01

    Bidirectional interactions between astrocytes and neurons have physiological roles in the central nervous system and an altered state or dysfunction of such interactions may be associated with neurodegenerative diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Astrocytes exert structural, metabolic and functional effects on neurons, which can be either neurotoxic or neuroprotective. Their neurotoxic effect is mediated via the senescence-associated secretory phenotype (SASP) involving pro-inflammatory cytokines (e.g., IL-6), while their neuroprotective effect is attributed to neurotrophic growth factors (e.g., NGF). We here demonstrate that the p53 isoforms Δ133p53 and p53β are expressed in astrocytes and regulate their toxic and protective effects on neurons. Primary human astrocytes undergoing cellular senescence upon serial passaging in vitro showed diminished expression of Δ133p53 and increased p53β, which were attributed to the autophagic degradation and the SRSF3-mediated alternative RNA splicing, respectively. Early-passage astrocytes with Δ133p53 knockdown or p53β overexpression were induced to show SASP and to exert neurotoxicity in co-culture with neurons. Restored expression of Δ133p53 in near-senescent, otherwise neurotoxic astrocytes conferred them with neuroprotective activity through repression of SASP and induction of neurotrophic growth factors. Brain tissues from AD and ALS patients possessed increased numbers of senescent astrocytes and, like senescent astrocytes in vitro, showed decreased Δ133p53 and increased p53β expression, supporting that our in vitro findings recapitulate in vivo pathology of these neurodegenerative diseases. Our finding that Δ133p53 enhances the neuroprotective function of aged and senescent astrocytes suggests that the p53 isoforms and their regulatory mechanisms are potential targets for therapeutic intervention in neurodegenerative diseases. PMID:27104929

  13. DISC1, astrocytes and neuronal maturation: a possible mechanistic link with implications for mental disorders.

    PubMed

    Xia, Meng; Zhu, Shanshan; Shevelkin, Alexey; Ross, Christopher A; Pletnikov, Mikhail

    2016-08-01

    Disrupted-In-Schizophrenia 1 (DISC1) is a genetic risk factor implicated in major mental disorders that involve disrupted neurodevelopment and synaptic signaling. Glial cells such as astrocytes can regulate neuronal and synaptic maturation. Although astrocytes express DISC1, the role of astrocyte DISC1 in synaptic regulation remains unknown. We expressed a pathogenic, dominant-negative form of DISC1, mutant DISC1, in astrocytes to elucidate the roles of astrocytic DISC1 in maturation of dendrites and excitatory and inhibitory synapses using a co-culture model. We found that wild-type primary neurons exhibited less elaborated dendritic arborization when co-cultured with astrocytes that express mutant DISC1, compared to control astrocytes. We observed significantly decreased density of excitatory but not inhibitory synapses on wild-type primary neurons that were co-cultured with astrocytes that express mutant DISC1, compared to control astrocytes. Treatment of co-cultures with D-serine restored dendritic development and density of excitatory synapses. Our findings show for the first time that mutant DISC1 diminished the capacity of astrocytes to support dendritic and synaptic maturation in co-cultured neurons, and that D-serine can restore the dendritic and synaptic abnormalities. The results provide a new insight into the mechanisms whereby genetic risk factors within astrocytes could contribute the pathogenesis of psychiatric disorders. Expression of mutant DISC1 (mDISC1) in astrocytes (A) decreases binding of endogenous DISC1 to serine racemase (SR) and production of D-serine (blue triangles) from L-serine (red triangles). As a result, neurons co-cultured with mutant DISC1 astrocytes exhibit diminished dendritic arborization (DIV10) and decreased linear density of VGLUT+(red)/PSD95 +  (green) excitatory synapses (DIV14). Filled circles with arrows denote membrane transporters for D-serine. Read the Editorial Highlight for this article on doi: 10.1111/jnc.13699

  14. Quinolinic acid induces disrupts cytoskeletal homeostasis in striatal neurons. Protective role of astrocyte-neuron interaction.

    PubMed

    Pierozan, Paula; Ferreira, Fernanda; de Lima, Bárbara Ortiz; Pessoa-Pureur, Regina

    2015-02-01

    Quinolinic acid (QUIN) is an endogenous metabolite of the kynurenine pathway involved in several neurological disorders. Among the several mechanisms involved in QUIN-mediated toxicity, disruption of the cytoskeleton has been demonstrated in striatally injected rats and in striatal slices. The present work searched for the actions of QUIN in primary striatal neurons. Neurons exposed to 10 µM QUIN presented hyperphosphorylated neurofilament (NF) subunits (NFL, NFM, and NFH). Hyperphosphorylation was abrogated in the presence of protein kinase A and protein kinase C inhibitors H89 (20 μM) and staurosporine (10 nM), respectively, as well as by specific antagonists to N-methyl-D-aspartate (50 µM DL-AP5) and metabotropic glutamate receptor 1 (100 µM MPEP). Also, intra- and extracellular Ca(2+) chelators (10 µM BAPTA-AM and 1 mM EGTA, respectively) and Ca(2+) influx through L-type voltage-dependent Ca(2+) channel (10 µM verapamil) are implicated in QUIN-mediated effects. Cells immunostained for the neuronal markers βIII-tubulin and microtubule-associated protein 2 showed altered neurite/neuron ratios and neurite outgrowth. NF hyperphosphorylation and morphological alterations were totally prevented by conditioned medium from QUIN-treated astrocytes. Cocultured astrocytes and neurons interacted with one another reciprocally, protecting them against QUIN injury. Cocultured cells preserved their cytoskeletal organization and cell morphology together with unaltered activity of the phosphorylating system associated with the cytoskeleton. This article describes cytoskeletal disruption as one of the most relevant actions of QUIN toxicity in striatal neurons in culture with soluble factors secreted by astrocytes, with neuron-astrocyte interaction playing a role in neuroprotection.

  15. Primary succession on slopes exposed to intense erosion: the case of Vesuvius Grand Cone

    NASA Astrophysics Data System (ADS)

    Stinca, Adriano; Battista Chirico, Giovanni; Bonanomi, Giuliano

    2014-05-01

    Mt. Vesuvius (1281 m a.s.l.) is an active volcano dominating the central part of the Campania Region coastline, with a distinctive barren crater summit, known as Grand Cone, formed during the eruption of AD 79. Local environmental factors hindered the colonization of the Vesuvius Grand Cone by vascular plants after the last eruptions of 1906 and 1944. The Grand Cone exhibits very steep planar slopes (33-35 degrees), covered by unconsolidated pyroclastic deposits, mainly formed by lapilli and gravels, characterized by an extremely low water holding capacity and very low organic matter and nitrogen contents, and exposed to intense water and wind erosion. In the last decade Genista aetnensis (Biv.) DC. (Fabaceae), has been expanding over the Grand Cone, facilitating the colonization by other species, especially herbaceous, with a dramatic change of the landscape appearance of the Vesuvius Grand Cone. G. aetnensis is a plant endemic of Mt. Etna and Eastern Sardinia and was firstly introduced at the base of Mt. Vesuvius within reforestation programs after the eruption of 1906. This plant is a nitrogen fixing species with a strong ability to colonize andosols, much more pronounced than the indigenous brooms (Cytisus scoparius and Spartium junceum). An intensive investigation has been conducted to explore the eco-hydrological processes driving the vegetation dynamics observed on the slopes of Grand Cone. Specific field surveys and laboratory experiments have been carried out to assess the effects of the G. aetnensis on soil physical and chemical properties, on the above- and below-ground microclimate, on the soil hydrological regime and on the distribution of coexisting species. The G. aetnensis triggers a pedogenetic process that contributes to a rapid increase of carbon and nitrogen stocks, available phosphorous, cation exchange capacity and a reduction of soil pH. The increase of carbon content also significantly improves the water retention properties in this coarse

  16. Transplantation of specific human astrocytes promotes functional recovery after spinal cord injury.

    PubMed

    Davies, Stephen J A; Shih, Chung-Hsuan; Noble, Mark; Mayer-Proschel, Margot; Davies, Jeannette E; Proschel, Christoph

    2011-03-02

    Repairing trauma to the central nervous system by replacement of glial support cells is an increasingly attractive therapeutic strategy. We have focused on the less-studied replacement of astrocytes, the major support cell in the central nervous system, by generating astrocytes from embryonic human glial precursor cells using two different astrocyte differentiation inducing factors. The resulting astrocytes differed in expression of multiple proteins thought to either promote or inhibit central nervous system homeostasis and regeneration. When transplanted into acute transection injuries of the adult rat spinal cord, astrocytes generated by exposing human glial precursor cells to bone morphogenetic protein promoted significant recovery of volitional foot placement, axonal growth and notably robust increases in neuronal survival in multiple spinal cord laminae. In marked contrast, human glial precursor cells and astrocytes generated from these cells by exposure to ciliary neurotrophic factor both failed to promote significant behavioral recovery or similarly robust neuronal survival and support of axon growth at sites of injury. Our studies thus demonstrate functional differences between human astrocyte populations and suggest that pre-differentiation of precursor cells into a specific astrocyte subtype is required to optimize astrocyte replacement therapies. To our knowledge, this study is the first to show functional differences in ability to promote repair of the injured adult central nervous system between two distinct subtypes of human astrocytes derived from a common fetal glial precursor population. These findings are consistent with our previous studies of transplanting specific subtypes of rodent glial precursor derived astrocytes into sites of spinal cord injury, and indicate a remarkable conservation from rat to human of functional differences between astrocyte subtypes. In addition, our studies provide a specific population of human astrocytes that

  17. Post-fire primary production and plant community dynamics in chaparral stands exposed to varying levels of nitrogen deposition.

    PubMed

    Pasquini, Sarah C; Vourlitis, George L

    2010-02-01

    High levels of atmospheric nitrogen (N) deposition to southern California chaparral shrublands may interact with fire to affect biomass production and plant species composition during secondary succession. To determine the potential interactions between post fire recovery and N deposition we compared rates of aboveground net primary production (ANPP), shrub growth, and the relative abundance of Adenostoma fasciculatum, other sub-dominant shrubs, and herbaceous species of three chaparral stands exposed to different levels of atmospheric N deposition over the first 3 years of post-fire succession. Our data suggest that rates of ANPP (gdw m(-2) month(-1)) and aboveground N storage (gN m(-2) month(-1)) for these chaparral stands were not related to N deposition even though sites exposed to high levels of N deposition had significantly higher rates of shrub growth (gdw plant(-1) month(-1)) and N uptake (gN plant(-1) month(-1)). However, high-N stands were composed of larger shrubs with a lower density, and this trade-off between shrub size and density may explain the low correlation between N deposition and post-fire ANPP. Differences in relative plant species abundance between sites were significantly correlated with N deposition exposure, where stands exposed to high N deposition had a lower relative abundance of A. fasciculatum and a higher relative abundance of other shrub and herbaceous species. While many factors can affect rates and patterns of post-fire recovery, these results suggest that chronic exposure to N deposition may significantly alter plant growth and species composition in successional chaparral stands.

  18. Post-fire primary production and plant community dynamics in chaparral stands exposed to varying levels of nitrogen deposition

    PubMed Central

    Pasquini, Sarah C.; Vourlitis, George L.

    2011-01-01

    High levels of atmospheric nitrogen (N) deposition to southern California chaparral shrublands may interact with fire to affect biomass production and plant species composition during secondary succession. To determine the potential interactions between post fire recovery and N deposition we compared rates of aboveground net primary production (ANPP), shrub growth, and the relative abundance of Adenostoma fasciculatum, other sub-dominant shrubs, and herbaceous species of three chaparral stands exposed to different levels of atmospheric N deposition over the first 3 years of post-fire succession. Our data suggest that rates of ANPP (gdw m−2 month−1) and aboveground N storage (gN m−2 month−1) for these chaparral stands were not related to N deposition even though sites exposed to high levels of N deposition had significantly higher rates of shrub growth (gdw plant−1 month−1) and N uptake (gN plant−1 month−1). However, high-N stands were composed of larger shrubs with a lower density, and this trade-off between shrub size and density may explain the low correlation between N deposition and post-fire ANPP. Differences in relative plant species abundance between sites were significantly correlated with N deposition exposure, where stands exposed to high N deposition had a lower relative abundance of A. fasciculatum and a higher relative abundance of other shrub and herbaceous species. While many factors can affect rates and patterns of post-fire recovery, these results suggest that chronic exposure to N deposition may significantly alter plant growth and species composition in successional chaparral stands. PMID:21731118

  19. Intermediate filaments are important for astrocyte response to oxidative stress induced by oxygen-glucose deprivation and reperfusion.

    PubMed

    de Pablo, Yolanda; Nilsson, Michael; Pekna, Marcela; Pekny, Milos

    2013-07-01

    As a response to central nervous system injury, astrocytes become reactive. Two cellular hallmarks of reactive gliosis are hypertrophy of astrocyte processes and upregulation of intermediate filament (nanofilament) proteins glial fibrillary acidic protein (GFAP), vimentin, nestin, and synemin. Astrocytes in mice devoid of GFAP and vimentin (GFAP (-/-) Vim (-/-)) do not form cytoplasmic intermediate filaments. GFAP (-/-) Vim (-/-) mice develop larger infarcts after ischemic stroke (Li et al. in J Cereb Blood Flow Metab 28(3):468-481, 2008). Here, we attempted to analyze the underlying mechanisms using oxygen-glucose deprivation (OGD), an in vitro ischemia model, examining a potential link between astrocyte intermediate filaments and reactive oxygen species (ROS). We observed a reorganization of the intermediate filament network in astrocytes exposed to OGD. ROS accumulation was higher in GFAP (-/-) Vim (-/-) than wild-type astrocytes when exposed to OGD followed by reperfusion or when exposed to hydrogen peroxide. These results indicate that the elimination of ROS is impaired in the absence of the intermediate filament system. Compared to wild-type astrocytes, GFAP (-/-) Vim (-/-) astrocytes exposed to OGD and reperfusion exhibited increased cell death and conferred lower degree of protection to cocultured neurons. We conclude that the astrocyte intermediate filament system is important for the cell response to oxidative stress induced by OGD followed by reperfusion.

  20. Temporal trends in aboveground net primary production of semi-arid shrublands exposed to experimental N deposition

    NASA Astrophysics Data System (ADS)

    Vourlitis, G. L.

    2011-12-01

    Southern Californian chaparral and coastal sage scrub (CSS) shrublands are exposed to high-levels of dry-atmospheric nitrogen (N) deposition. A field experiment was conducted over 8 years in a post-fire chaparral and a mature CSS stand to assess the effects of cumulative, dry-season N inputs on aboveground net primary production (NPP). We hypothesized that the NPP chaparral and CSS would significantly increase in response to N exposure because previous research indicated to the productivity of these semi-arid shrublands was limited by N. Our results indicate that N addition eventually increased the NPP of both shrublands; however, trends in NPP varied over time and were affected by interannual variations in rainfall. For example, added N in post-fire chaparral initially inhibited NPP, but over time NPP increased consistently in plots exposed to added N. For CSS, temporal trends in NPP were independent of cumulative N exposure; however, a pattern emerged where the effect of N exposure was significantly related to annual rainfall. NPP increased significantly to N exposure when rainfall exceeded approximately 400 mm year-1 but declined with N exposure during dry years. These results support our hypothesis that N enrichment will increase the NPP of these semi-arid shrublands; however, temporal patterns may take years to emerge (chaparral) or be modified by annual variations in rainfall (CSS).

  1. Chronic lithium treatment increased intracellular S100ß levels in rat primary neuronal culture.

    PubMed

    Emamghoreishi, Masoumeh; Keshavarz, Mojtaba; Nekooeian, Ali Akbar

    2015-01-01

    S100ß a neurotrophic factor mainly released by astrocytes, has been implicated in the pathogenesis of bipolar disorder. Thus, lithium may exert its neuroprotective effects to some extent through S100ß. Furthermore, the possible effects of lithium on astrocytes as well as on interactions between neurons and astrocytes as a part of its mechanisms of actions are unknown. This study was undertaken to determine the effect of lithium on S100β in neurons, astrocytes and a mixture of neurons and astrocytes. Rat primary astrocyte, neuronal and mixed neuro-astroglia cultures were prepared from cortices of 18-day's embryos. Cell cultures were exposed to lithium (1mM) or vehicle for 1day (acute) or 7 days (chronic). RT-PCR and ELISA determined S100β mRNA and intra- and extracellular protein levels. Chronic lithium treatment significantly increased intracellular S100β in neuronal and neuro-astroglia cultures in comparison to control cultures (P<0.05). Acute and chronic lithium treatments exerted no significant effects on intracellular S100β protein levels in astrocytes, and extracellular S100β protein levels in three studied cultures as compared to control cultures. Acute and chronic lithium treatments did not significantly alter S100β mRNA levels in three studied cultures, compared to control cultures. Chronic lithium treatment increased intracellular S100ß protein levels in a cell-type specific manner which may favor its neuroprotective action. The findings of this study suggest that lithium may exert its neuroprotective action, at least partly, by increasing neuronal S100ß level, with no effect on astrocytes or interaction between neurons and astrocytes.

  2. Low energy laser light (632.8 nm) suppresses amyloid-β peptide-induced oxidative and inflammatory responses in astrocytes.

    PubMed

    Yang, X; Askarova, S; Sheng, W; Chen, J K; Sun, A Y; Sun, G Y; Yao, G; Lee, J C-M

    2010-12-15

    Oxidative stress and inflammation are important processes in the progression of Alzheimer's disease (AD). Recent studies have implicated the role of amyloid β-peptides (Aβ) in mediating these processes. In astrocytes, oligomeric Aβ induces the assembly of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complexes resulting in its activation to produce anionic superoxide. Aβ also promotes production of pro-inflammatory factors in astrocytes. Since low energy laser has previously been reported to attenuate oxidative stress and inflammation in biological systems, the objective of this study was to examine whether this type of laser light was able to abrogate the oxidative and inflammatory responses induced by Aβ. Primary rat astrocytes were exposed to Helium-Neon laser (λ=632.8 nm), followed by the treatment with oligomeric Aβ. Primary rat astrocytes were used to measure Aβ-induced production of superoxide anions using fluorescence microscopy of dihydroethidium (DHE), assembly of NADPH oxidase subunits by the colocalization between the cytosolic p47(phox) subunit and the membrane gp91(phox) subunit using fluorescent confocal microscopy, phosphorylation of cytosolic phospholipase A(2) cPLA(2) and expressions of pro-inflammatory factors including interleukin-1β (IL-1β) and inducible nitric-oxide synthase (iNOS) using Western blot Analysis. Our data showed that laser light at 632.8 nm suppressed Aβ-induced superoxide production, colocalization between NADPH oxidase gp91(phox) and p47(phox) subunits, phosphorylation of cPLA(2,) and the expressions of IL-1β and iNOS in primary astrocytes. We demonstrated for the first time that 632.8 nm laser was capable of suppressing cellular pathways of oxidative stress and inflammatory responses critical in the pathogenesis in AD. This study should prove to provide the groundwork for further investigations for the potential use of laser therapy as a treatment for AD. PMID:20884337

  3. Cell-type-specific and differentiation-status-dependent variations in cytotoxicity of tributyltin in cultured rat cerebral neurons and astrocytes.

    PubMed

    Oyanagi, Koshi; Tashiro, Tomoko; Negishi, Takayuki

    2015-08-01

    Tributyltin (TBT) is an organotin used as an anti-fouling agent for fishing nets and ships and it is a widespread environmental contaminant at present. There is an increasing concern about imperceptible but serious adverse effect(s) of exposure to chemicals existing in the environment on various organs and their physiological functions, e.g. brain and mental function. Here, so as to contribute to improvement of and/or advances in in vitro cell-based assay systems for evaluating brain-targeted adverse effect of chemicals, we tried to evaluate cell-type-specific and differentiation-status-dependent variations in the cytotoxicity of TBT towards neurons and astrocytes using the four culture systems differing in the relative abundance of these two types of cells; primary neuron culture (> 95% neurons), primary neuron-astrocyte (2 : 1) mix culture, primary astrocyte culture (> 95% astrocytes), and passaged astrocyte culture (100% proliferative astrocytes). Cell viability was measured at 48 hr after exposure to TBT in serum-free medium. IC50's of TBT were 198 nM in primary neuron culture, 288 nM in primary neuron-astrocyte mix culture, 2001 nM in primary astrocyte culture, and 1989 nM in passaged astrocyte culture. Furthermore, in primary neuron-astrocyte mix culture, vulnerability of neurons cultured along with astrocytes to TBT toxicity was lower than that of neurons cultured purely in primary neuron culture. On the other hand, astrocytes in primary neuron-astrocyte mix culture were considered to be more vulnerable to TBT than those in primary or passaged astrocyte culture. The present study demonstrated variable cytotoxicity of TBT in neural cells depending on the culture condition.

  4. Connexin 43 stabilizes astrocytes in a stroke-like milieu to facilitate neuronal recovery

    PubMed Central

    Wu, Le-yu; Yu, Xue-li; Feng, Lin-yin

    2015-01-01

    Aim: Connexin 43 (Cx43) is a member of connexin family mainly expressed in astrocytes, which forms gap junctions and hemichannels and maintains the normal shape and function of astrocytes. In this study we investigated the role of Cx43 in astrocytes in facilitating neuronal recovery during ischemic stroke. Methods: Primary culture of astrocytes or a mixed culture of astrocytes and cortical neurons was subjected to oxygen glucose deprivation and reperfusion (OGD/R). The expression of Cx43 and Ephrin-A4 in astrocytes was detected using immunocytochemical staining and Western blot assays. Intercellular Ca2+ concentration was determined with Fluo-4 AM fluorescent staining. Middle cerebral artery occlusion (MCAO) model rats were used for in vivo studies. Results: OGD/R treatment of cultured astrocytes caused a decrement of Cx43 expression and translocation of Cx43 from cell membrane to cytoplasm, accompanied by cell retraction. Furthermore, OGD/R increased intracellular Ca2+ concentration, activated CaMKII/CREB pathways and upregulated expression of Ephrin-A4 in the astrocytes. All these changes in OGD/R-treated astrocytes were alleviated by overexpression of Cx43. In the cortical neurons cultured with astrocytes, OGD/R inhibited the neurite growth, whereas overexpression of Cx43 or knockdown of Ephrin-A4 in astrocytes restored the neurite growth. In MCAO model rats, neuronal recovery was found to be correlated with the recuperation of Cx43 and Ephrin-A4 in astrocytes. Conclusion: Cx43 can stabilize astrocytes and facilitate the resistance to the deleterious effects of a stroke-like milieu and promote neuronal recovery. PMID:26095039

  5. Modifications in astrocyte morphology and calcium signaling induced by a brain capillary endothelial cell line.

    PubMed

    Yoder, Elizabeth J

    2002-04-15

    Astrocytes extend specialized endfoot processes to perisynaptic and perivascular regions, and thus are positioned to mediate the bidirectional flow of metabolic, ionic, and other transmissive substances between neurons and the blood stream. While mutual structural and functional interactions between neurons and astrocytes have been documented, less is known about the interactions between astrocytes and cerebrovascular cells. For example, although the ability of astrocytes to induce structural and functional changes in endothelial cells is established, the reciprocity of brain endothelial cells to induce changes in astrocytes is undetermined. This issue is addressed in the present study. Changes in primary cultures of neonatal mouse cortical astrocytes were investigated following their coculture with mouse brain capillary endothelial (bEnd3) cells. The presence of bEnd3 cells altered the morphology of astrocytes by transforming them from confluent monolayers into networks of elongated multicellular columns. These columns did not occur when either bEnd3 cells or astrocytes were cocultured with other cell types, suggesting that astrocytes undergo specific morphological consequences when placed in close proximity to brain endothelial cells. In addition to these structural changes, the pharmacological profile of astrocytes was modified by coculture with bEnd3 cells. Astrocytes in the cocultures showed an increased Ca2+ responsiveness to bradykinin and glutamate, but no change in responsiveness to ATP, as compared to controls. Coculturing the astrocytes with a neuronal cell line resulted in increased responsiveness of the glial responses to glutamate but not to bradykinin. These studies indicate that brain endothelial cells induce changes in astrocyte morphology and pharmacology.

  6. Modifications in astrocyte morphology and calcium signaling induced by a brain capillary endothelial cell line.

    PubMed

    Yoder, Elizabeth J

    2002-04-15

    Astrocytes extend specialized endfoot processes to perisynaptic and perivascular regions, and thus are positioned to mediate the bidirectional flow of metabolic, ionic, and other transmissive substances between neurons and the blood stream. While mutual structural and functional interactions between neurons and astrocytes have been documented, less is known about the interactions between astrocytes and cerebrovascular cells. For example, although the ability of astrocytes to induce structural and functional changes in endothelial cells is established, the reciprocity of brain endothelial cells to induce changes in astrocytes is undetermined. This issue is addressed in the present study. Changes in primary cultures of neonatal mouse cortical astrocytes were investigated following their coculture with mouse brain capillary endothelial (bEnd3) cells. The presence of bEnd3 cells altered the morphology of astrocytes by transforming them from confluent monolayers into networks of elongated multicellular columns. These columns did not occur when either bEnd3 cells or astrocytes were cocultured with other cell types, suggesting that astrocytes undergo specific morphological consequences when placed in close proximity to brain endothelial cells. In addition to these structural changes, the pharmacological profile of astrocytes was modified by coculture with bEnd3 cells. Astrocytes in the cocultures showed an increased Ca2+ responsiveness to bradykinin and glutamate, but no change in responsiveness to ATP, as compared to controls. Coculturing the astrocytes with a neuronal cell line resulted in increased responsiveness of the glial responses to glutamate but not to bradykinin. These studies indicate that brain endothelial cells induce changes in astrocyte morphology and pharmacology. PMID:11948807

  7. Pituitary Adenylate cyclase-activating polypeptide orchestrates neuronal regulation of the astrocytic glutamate-releasing mechanism system xc (.).

    PubMed

    Kong, Linghai; Albano, Rebecca; Madayag, Aric; Raddatz, Nicholas; Mantsch, John R; Choi, SuJean; Lobner, Doug; Baker, David A

    2016-05-01

    Glutamate signaling is achieved by an elaborate network involving neurons and astrocytes. Hence, it is critical to better understand how neurons and astrocytes interact to coordinate the cellular regulation of glutamate signaling. In these studies, we used rat cortical cell cultures to examine whether neurons or releasable neuronal factors were capable of regulating system xc (-) (Sxc), a glutamate-releasing mechanism that is expressed primarily by astrocytes and has been shown to regulate synaptic transmission. We found that astrocytes cultured with neurons or exposed to neuronal-conditioned media displayed significantly higher levels of Sxc activity. Next, we demonstrated that the pituitary adenylate cyclase-activating polypeptide (PACAP) may be a neuronal factor capable of regulating astrocytes. In support, we found that PACAP expression was restricted to neurons, and that PACAP receptors were expressed in astrocytes. Interestingly, blockade of PACAP receptors in cultures comprised of astrocytes and neurons significantly decreased Sxc activity to the level observed in purified astrocytes, whereas application of PACAP to purified astrocytes increased Sxc activity to the level observed in cultures comprised of neurons and astrocytes. Collectively, these data reveal that neurons coordinate the actions of glutamate-related mechanisms expressed by astrocytes, such as Sxc, a process that likely involves PACAP. A critical gap in modeling excitatory signaling is how distinct components of the glutamate system expressed by neurons and astrocytes are coordinated. In these studies, we found that system xc (-) (Sxc), a glutamate release mechanism expressed by astrocytes, is regulated by releasable neuronal factors including PACAP. This represents a novel form of neuron-astrocyte communication, and highlights the possibility that pathological changes involving astrocytic Sxc may stem from altered neuronal activity.

  8. PUMA is invovled in ischemia/reperfusion-induced apoptosis of mouse cerebral astrocytes.

    PubMed

    Chen, H; Tian, M; Jin, L; Jia, H; Jin, Y

    2015-01-22

    PUMA (p53-upregulated modulator of apoptosis), a BH3-only member of the Bcl-2 protein family, is required for p53-dependent and p53-independent forms of apoptosis. PUMA has been invovled in the onset and progress of several diseases, including cancer, acquired immunodeficiency syndrome, and ischemic brain disease. Although many studies have shown that ischemia and reperfusion (I/R) can induce the apoptosis of astrocytes, the role of PUMA in I/R-mediated apoptosis of cerebral astrocyte apoptosis remains unclear. To mimic in vivo I/R conditions, primary mouse cerebral astrocytes were incubated in a combinational cultural condition of oxygen, glucose, and serum deprivation (OSGD) for 1 h followed by reperfusion (OSGD/R). Cell death determination assays and cell viability assays indicated that OSGD and OSGD/R induce the apoptosis of primary cerebral astrocytes. The expression of PUMA was significantly elevated in primary cerebral astrocytes during OSGD/R. Moreover, targeted down-regulation of PUMA by siRNA transfection significantly decreased the OSGD/R-induced apoptosis of primary cerebral astrocytes. We also found that OSGD and OSGD/R triggered the release of cytochrome c in astrocytes, indicating the dependence on a mitochondrial apoptotic pathway. Reactive oxygen species (ROS) was extremely generated during OSGD and OSGD/R, and the elimination of ROS by treated with N-acetyl-L-cysteine (NAC) remarkably inhibited the expression of PUMA and the apoptosis of primary cerebral astrocytes. The activation of Caspase 3 and Caspase 9 was extremely elevated in primary cerebral astrocytes during OSGD. In addition, we found that knockdown of PUMA led to the depressed expression of Bax, cleaved caspase-9 and caspase-3 during OSGD/R. These results indicate that PUMA is invovled in the apoptosis of cerebral astrocytes upon I/R injury.

  9. Astrocyte-mediated ischemic tolerance.

    PubMed

    Hirayama, Yuri; Ikeda-Matsuo, Yuri; Notomi, Shoji; Enaida, Hiroshi; Kinouchi, Hiroyuki; Koizumi, Schuichi

    2015-03-01

    Preconditioning (PC) using a preceding sublethal ischemic insult is an attractive strategy for protecting neurons by inducing ischemic tolerance in the brain. Although the underlying molecular mechanisms have been extensively studied, almost all studies have focused on neurons. Here, using a middle cerebral artery occlusion model in mice, we show that astrocytes play an essential role in the induction of brain ischemic tolerance. PC caused activation of glial cells without producing any noticeable brain damage. The spatiotemporal pattern of astrocytic, but not microglial, activation correlated well with that of ischemic tolerance. Interestingly, such activation in astrocytes lasted at least 8 weeks. Importantly, inhibiting astrocytes with fluorocitrate abolished the induction of ischemic tolerance. To investigate the underlying mechanisms, we focused on the P2X7 receptor as a key molecule in astrocyte-mediated ischemic tolerance. P2X7 receptors were dramatically upregulated in activated astrocytes. PC-induced ischemic tolerance was abolished in P2X7 receptor knock-out mice. Moreover, our results suggest that hypoxia-inducible factor-1α, a well known mediator of ischemic tolerance, is involved in P2X7 receptor-mediated ischemic tolerance. Unlike previous reports focusing on neuron-based mechanisms, our results show that astrocytes play indispensable roles in inducing ischemic tolerance, and that upregulation of P2X7 receptors in astrocytes is essential. PMID:25740510

  10. Role of Astrocytes in Epilepsy

    PubMed Central

    Coulter, Douglas A.; Steinhäuser, Christian

    2016-01-01

    Astrocytes express ion channels, transmitter receptors, and transporters and, thus, are endowed with the machinery to sense and respond to neuronal activity. Recent studies have implicated that astrocytes play important roles in physiology, but these cells also emerge as crucial actors in epilepsy. Astrocytes are abundantly coupled through gap junctions allowing them to redistribute elevated K+ and transmitter concentrations from sites of enhanced neuronal activity. Investigation of specimens from patients with pharmacoresistant temporal lobe epilepsy and epilepsy models revealed alterations in expression, localization, and function of astroglial K+ and water channels. In addition, malfunction of glutamate transporters and the astrocytic glutamate-converting enzyme, glutamine synthetase, has been observed in epileptic tissue. These findings suggest that dysfunctional astrocytes are crucial players in epilepsy and should be considered as promising targets for new therapeutic strategies. PMID:25732035

  11. Astrocytes: Key Regulators of Neuroinflammation.

    PubMed

    Colombo, Emanuela; Farina, Cinthia

    2016-09-01

    Astrocytes are crucial regulators of innate and adaptive immune responses in the injured central nervous system. Depending on timing and context, astrocyte activity may exacerbate inflammatory reactions and tissue damage, or promote immunosuppression and tissue repair. Recent literature has unveiled key factors and intracellular signaling pathways that govern astrocyte behavior during neuroinflammation. Here we have re-visited in vivo studies on astrocyte signaling in neuroinflammatory models focusing on evidences obtained from the analysis of transgenic mice where distinct genes involved in ligand binding, transcriptional regulation and cell communication have been manipulated in astrocytes. The integration of in vivo observations with in vitro data clarifies precise signaling steps, highlights the crosstalk among pathways and identifies shared effector mechanisms in neuroinflammation.

  12. Are astrocytes executive cells within the central nervous system?

    PubMed

    Sica, Roberto E; Caccuri, Roberto; Quarracino, Cecilia; Capani, Francisco

    2016-08-01

    Experimental evidence suggests that astrocytes play a crucial role in the physiology of the central nervous system (CNS) by modulating synaptic activity and plasticity. Based on what is currently known we postulate that astrocytes are fundamental, along with neurons, for the information processing that takes place within the CNS. On the other hand, experimental findings and human observations signal that some of the primary degenerative diseases of the CNS, like frontotemporal dementia, Parkinson's disease, Alzheimer's dementia, Huntington's dementia, primary cerebellar ataxias and amyotrophic lateral sclerosis, all of which affect the human species exclusively, may be due to astroglial dysfunction. This hypothesis is supported by observations that demonstrated that the killing of neurons by non-neural cells plays a major role in the pathogenesis of those diseases, at both their onset and their progression. Furthermore, recent findings suggest that astrocytes might be involved in the pathogenesis of some psychiatric disorders as well. PMID:27556379

  13. Differences in vulnerability of neurons and astrocytes to heme oxygenase-1 modulation: Implications for mitochondrial ferritin

    PubMed Central

    Yu, Xiaojun; Song, Ning; Guo, Xinli; Jiang, Hong; Zhang, Haoyun; Xie, Junxia

    2016-01-01

    Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) was observed in both astrocytes and neurons in the substantia nigra of patients with Parkinson’s disease (PD). In the current study, we investigated whether HO-1 behaves differently between neurons and astrocytes under the condition of neurotoxicity related to PD. The results showed a time-dependent HO-1 upregulation in primary cultured ventral mesencephalon neurons and astrocytes treated with the mitochondria complex I inhibitor 1-methyl-4-phenylpyridinium (MPP+) or recombinant α-synuclein. However, HO-1 upregulation appeared much later in neurons than in astrocytes. The HO-1 inhibitor zinc protoporphyrin (ZnPP) aggravated MPP+- or α-synuclein-induced oxidative damage in both astrocytes and neurons, indicating that this HO-1 response was cytoprotective. For neurons, the HO-1 activator cobalt protoporphyrin IX (CoPPIX) exerted protective effects against MPP+ or α-synuclein during moderate HO-1 upregulation, but it aggravated damage at the peak of the HO-1 response. For astrocytes, CoPPIXalways showed protective effects. Higher basal and CoPPIX-induced mitochondrial ferritin (MtFt) levels were detected in astrocytes. Lentivirus-mediated MtFt overexpression rescued the neuronal damage induced by CoPPIX, indicating that large MtFt buffering capacity contributes to pronounced HO-1 tolerance in astrocytes. Such findings suggest that astrocyte-targeted HO-1 interventions and MtFt modulations have potential as novel pharmacological strategies in PD. PMID:27097841

  14. Differences in vulnerability of neurons and astrocytes to heme oxygenase-1 modulation: Implications for mitochondrial ferritin.

    PubMed

    Yu, Xiaojun; Song, Ning; Guo, Xinli; Jiang, Hong; Zhang, Haoyun; Xie, Junxia

    2016-01-01

    Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) was observed in both astrocytes and neurons in the substantia nigra of patients with Parkinson's disease (PD). In the current study, we investigated whether HO-1 behaves differently between neurons and astrocytes under the condition of neurotoxicity related to PD. The results showed a time-dependent HO-1 upregulation in primary cultured ventral mesencephalon neurons and astrocytes treated with the mitochondria complex I inhibitor 1-methyl-4-phenylpyridinium (MPP(+)) or recombinant α-synuclein. However, HO-1 upregulation appeared much later in neurons than in astrocytes. The HO-1 inhibitor zinc protoporphyrin (ZnPP) aggravated MPP(+)- or α-synuclein-induced oxidative damage in both astrocytes and neurons, indicating that this HO-1 response was cytoprotective. For neurons, the HO-1 activator cobalt protoporphyrin IX (CoPPIX) exerted protective effects against MPP(+) or α-synuclein during moderate HO-1 upregulation, but it aggravated damage at the peak of the HO-1 response. For astrocytes, CoPPIXalways showed protective effects. Higher basal and CoPPIX-induced mitochondrial ferritin (MtFt) levels were detected in astrocytes. Lentivirus-mediated MtFt overexpression rescued the neuronal damage induced by CoPPIX, indicating that large MtFt buffering capacity contributes to pronounced HO-1 tolerance in astrocytes. Such findings suggest that astrocyte-targeted HO-1 interventions and MtFt modulations have potential as novel pharmacological strategies in PD. PMID:27097841

  15. Trophic and tropic effects of striatal astrocytes on cografted mesencephalic dopamine neurons and their axons.

    PubMed

    Pierret, P; Quenneville, N; Vandaele, S; Abbaszadeh, R; Lanctôt, C; Crine, P; Doucet, G

    1998-01-01

    Astrocytes from the ventral mesencephalon and from the striatum respectively promote the dendritic and axonal arborization of dopamine (DA) neurons in vitro. To test this response in vivo, astrocytes in primary cultures from the neonatal cerebral cortex, ventral mesencephalon, or striatum were coimplanted with fetal ventral mesencephalic tissue into the intact or DA-denervated striatum of adult rats and these cografts examined after 3-6 months by tyrosine hydroxylase (TH) immunohistochemistry (intact recipients) or after 5-6 months by in vitro [3H]DA-uptake autoradiography (DA-denervated recipients). In contrast with single ventral mesencephalic grafts, all types of cograft displayed a rather uniform distribution of TH-immunoreactive perikarya. The average size of TH-immunoreactive cell bodies was not significantly different in cografts containing cortical or mesencephalic astrocytes and in single ventral mesencephalic grafts, but it was significantly larger in cografts containing striatal astrocytes. Nevertheless, the number of [3H]DA-labeled terminals in the DA-lesioned host striatum was clearly smaller with cografts of striatal astrocytes than with single mesencephalic grafts or with cografts containing cortical astrocytes. On the other hand, cografts of striatal astrocytes contained much higher numbers of [3H]DA-labeled terminals than the other types of graft or cograft. Thus, while cografted astrocytes in general influence the distribution of DA neurons within the graft, astrocytes from the neonatal striatum have a trophic effect on DA perikarya and a tropic effect on DA axons, keeping the latter within the graft.

  16. Morphine Protects Spinal Cord Astrocytes from Glutamate-Induced Apoptosis via Reducing Endoplasmic Reticulum Stress

    PubMed Central

    Zhang, Chao; Wang, Chendan; Ren, Jianbo; Guo, Xiangjie; Yun, Keming

    2016-01-01

    Glutamate is not only a neurotransmitter but also an important neurotoxin in central nervous system (CNS). Chronic elevation of glutamate induces both neuronal and glial cell apoptosis. However, its effect on astrocytes is complex and still remains unclear. In this study, we investigated whether morphine, a common opioid ligand, could affect glutamate-induced apoptosis in astrocytes. Primary cultured astrocytes were incubated with glutamate in the presence/absence of morphine. It was found that morphine could reduce glutamate-induced apoptosis of astrocytes. Furthermore, glutamate activated Ca2+ release, thereby inducing endoplasmic reticulum (ER) stress in astrocytes, while morphine attenuated this deleterious effect. Using siRNA to reduce the expression of κ-opioid receptor, morphine could not effectively inhibit glutamate-stimulated Ca2+ release in astrocytes, the protective effect of morphine on glutamate-injured astrocytes was also suppressed. These results suggested that morphine could protect astrocytes from glutamate-induced apoptosis via reducing Ca2+ overload and ER stress pathways. In conclusion, this study indicated that excitotoxicity participated in the glutamate mediated apoptosis in astrocytes, while morphine attenuated this deleterious effect via regulating Ca2+ release and ER stress. PMID:27783050

  17. [Novel function of astrocytes revealed by optogenetics].

    PubMed

    Beppu, Kaoru; Matsui, Ko

    2014-12-01

    Astrocytes respond to neuronal activity. However, whether astrocytic activity has any significance in brain function is unknown. Signaling pathway leading from astrocytes to neurons would be required for astrocytes to participate in neuronal functions and, here, we investigated the presence of such pathway. Optogenetics was used to manipulate astrocytic activity. A light-sensitive protein, channelrhodopsin-2 (ChR2), was selectively expressed in astrocytes. Photostimulation of these astrocytes induced glutamate release which modulated neuronal activity and animal behavior. Such glutamate release was triggered by intracellular acidification produced by ChR2 photoactivation. Astrocytic acidification occurs upon brain ischemia, and we found that another optogenetic tool, archaerhodopsin (ArchT), could counter the acidification and suppress astrocytic glutamate release. Controlling of astrocytic pH may become a therapeutic strategy upon ischemia.

  18. Volume transmission signalling via astrocytes

    PubMed Central

    Hirase, Hajime; Iwai, Youichi; Takata, Norio; Shinohara, Yoshiaki; Mishima, Tsuneko

    2014-01-01

    The influence of astrocytes on synaptic function has been increasingly studied, owing to the discovery of both gliotransmission and morphological ensheathment of synapses. While astrocytes exhibit at best modest membrane potential fluctuations, activation of G-protein coupled receptors (GPCRs) leads to a prominent elevation of intracellular calcium which has been reported to correlate with gliotransmission. In this review, the possible role of astrocytic GPCR activation is discussed as a trigger to promote synaptic plasticity, by affecting synaptic receptors through gliotransmitters. Moreover, we suggest that volume transmission of neuromodulators could be a biological mechanism to activate astrocytic GPCRs and thereby to switch synaptic networks to the plastic mode during states of attention in cerebral cortical structures. PMID:25225097

  19. INTEGRATED DISINFECTION BY-PRODUCTS (DBP) MIXTURES RESEARCH: GENE EXPRESSION ALTERATIONS IN PRIMARY RAT HEPATOCYTE CULTURES EXPOSED TO DBP MIXTURES FORMED BY CHLORINATION AND OZONATION/POSTCHLORINATION

    EPA Science Inventory

    What is the study?
    This study was designed to provide data on the in vitro toxicity of water concentrates containing complex mixtures of DBPs. Rat hepatocytes in primary culture were exposed for 24 hr to full strength, 1:10 or 1:20 dilutions of chlorination or ozonation/chl...

  20. Determine the yield of micronucleated cells in primary human fibroblasts exposed to focused soft X-rays.

    SciTech Connect

    Kevin M. Prise

    2007-01-02

    This project was a small part of a larger collaborative study headed by Dr Aloke Chatterjee, (Lawrence Berkeley National Laboratory) and including Drs Les Braby, John Ford (Texas A&M) and Kathy Held (MGH Boston), which was developing an integrated theoretical and experimental model of the radiation-induced bystander response. Our part of the study has been to determine the effectiveness of soft X-rays at inducing chromosomal damage under conditions of direct and bystander exposure. The aim was to compare this with the effectiveness of the low energy 60 kV electron microbeam available at Texas A&M. Previous studies have been performed with primary human fibroblasts measuring micronuclei formation to determine the relative yields of direct versus bystander mediated micronuclei formation after cells were individually irradiated utilizing our novel focused soft X-ray microprobe, which is capable of producing localized submicron beams of carbon-K (278 eV) X-rays. Only a brief overview is given here as the study has been published in several papers. Our original hypothesis was to study yields of bystander-induced micronucleated cells in both wild-type and mutant fibroblast from mouse embryo fibroblasts. Difficulties with the level of background micronuclei in the MEFs prevented systematic studies of bystander responses in the laboratories involved in the collaboration. We then performed these studies with AG1522 primary human fibroblast cells using a siRNA approach developed by John Ford at Texas A&M to knock down DNA PKcs in the first instance. Our soft X-ray source has been in routine use for carbon-K X-rays and is now available with Aluminium-K (1.49 keV) and titanium-K (4.5 keV), although the dose-rate from titanium is still too low at present for most experiments, where large numbers of cells need to be exposed. A separately funded project developed a new soft X-ray microprobe which will give much greater flexibility for changing energies and giving high dose

  1. Hydrolysis of polyphosphoinositides in astrocytes by platelet-activating factor.

    PubMed

    Murphy, S; Welk, G

    1990-06-12

    In primary astrocyte cultures, picomolar concentrations of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) evoked the formation of inositol phosphates (InsP), including inositol trisphosphate. This effect was not observed with the biologically inert lyso-PAF, nor in cells pretreated with phorbol myristate acetate to downregulate receptors. PAF at concentrations greater than or equal to 10(-9) M did not elevate InsP, suggesting some form of uncoupling of the receptor from phospholipase C. The responsiveness of astrocytes to PAF is further evidence for the role of these cells in the central nervous system response to trauma. PMID:2164941

  2. Rat nucleus accumbens core astrocytes modulate reward and the motivation to self-administer ethanol after abstinence.

    PubMed

    Bull, Cecilia; Freitas, Kelen C C; Zou, Shiping; Poland, Ryan S; Syed, Wahab A; Urban, Daniel J; Minter, Sabrina C; Shelton, Keith L; Hauser, Kurt F; Negus, S Stevens; Knapp, Pamela E; Bowers, M Scott

    2014-11-01

    Our understanding of the active role that astrocytes play in modulating neuronal function and behavior is rapidly expanding, but little is known about the role that astrocytes may play in drug-seeking behavior for commonly abused substances. Given that the nucleus accumbens is critically involved in substance abuse and motivation, we sought to determine whether nucleus accumbens astrocytes influence the motivation to self-administer ethanol following abstinence. We found that the packing density of astrocytes that were expressing glial fibrillary acidic protein increased in the nucleus accumbens core (NAcore) during abstinence from EtOH self-administration. No change was observed in the nucleus accumbens shell. This increased NAcore astrocyte density positively correlated with the motivation for ethanol. Astrocytes can communicate with one another and influence neuronal activity through gap-junction hemichannels. Because of this, the effect of blocking gap-junction hemichannels on the motivation for ethanol was examined. The motivation to self-administer ethanol after 3 weeks abstinence was increased following microinjection of gap-junction hemichannel blockers into the NAcore at doses that block both neuronal and astrocytic channels. In contrast, no effect was observed following microinjection of doses that are not thought to block astrocytic channels or following microinjection of either dose into the nucleus accumbens shell. Additionally, the motivation for sucrose after 3 weeks abstinence was unaffected by NAcore gap-junction hemichannel blockers. Next, Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) were selectively expressed in NAcore astrocytes to test the effect of astrocyte stimulation. DREADD activation increased cytosolic calcium in primary astrocytes, facilitated responding for rewarding brain stimulation, and reduced the motivation for ethanol after 3 weeks abstinence. This is the first work to modulate drug-seeking behavior with

  3. Astrocytes expressing mutant SOD1 and TDP43 trigger motoneuron death that is mediated via sodium channels and nitroxidative stress.

    PubMed

    Rojas, Fabiola; Cortes, Nicole; Abarzua, Sebastian; Dyrda, Agnieszka; van Zundert, Brigitte

    2014-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal paralytic disorder caused by dysfunction and degeneration of motor neurons. Multiple disease-causing mutations, including in the genes for SOD1 and TDP-43, have been identified in ALS. Astrocytes expressing mutant SOD1 are strongly implicated in the pathogenesis of ALS: we have shown that media conditioned by astrocytes carrying mutant SOD1(G93A) contains toxic factor(s) that kill motoneurons by activating voltage-sensitive sodium (Na v ) channels. In contrast, a recent study suggests that astrocytes expressing mutated TDP43 contribute to ALS pathology, but do so via cell-autonomous processes and lack non-cell-autonomous toxicity. Here we investigate whether astrocytes that express diverse ALS-causing mutations release toxic factor(s) that induce motoneuron death, and if so, whether they do so via a common pathogenic pathway. We exposed primary cultures of wild-type spinal cord cells to conditioned medium derived from astrocytes (ACM) that express SOD1 (ACM-SOD1(G93A) and ACM-SOD1(G86R)) or TDP43 (ACM-TDP43(A315T)) mutants; we show that such exposure rapidly (within 30-60 min) increases dichlorofluorescein (DCF) fluorescence (indicative of nitroxidative stress) and leads to extensive motoneuron-specific death within a few days. Co-application of the diverse ACMs with anti-oxidants Trolox or esculetin (but not with resveratrol) strongly improves motoneuron survival. We also find that co-incubation of the cultures in the ACMs with Na v channel blockers (including mexiletine, spermidine, or riluzole) prevents both intracellular nitroxidative stress and motoneuron death. Together, our data document that two completely unrelated ALS models lead to the death of motoneuron via non-cell-autonomous processes, and show that astrocytes expressing mutations in SOD1 and TDP43 trigger such cell death through a common pathogenic pathway that involves nitroxidative stress, induced at least in part by Na v channel activity. PMID

  4. Active sulforhodamine 101 uptake into hippocampal astrocytes.

    PubMed

    Schnell, Christian; Hagos, Yohannes; Hülsmann, Swen

    2012-01-01

    Sulforhodamine 101 (SR101) is widely used as a marker of astrocytes. In this study we investigated labeling of astrocytes by SR101 in acute slices from the ventrolateral medulla and the hippocampus of transgenic mice expressing EGFP under the control of the astrocyte-specific human GFAP promoter. While SR101 efficiently and specifically labeled EGFP-expressing astrocytes in hippocampus, we found that the same staining procedure failed to label astrocytes efficiently in the ventrolateral medulla. Although carbenoxolone is able to decrease the SR101-labeling of astrocytes in the hippocampus, it is unlikely that SR101 is taken up via gap-junction hemichannels because mefloquine, a blocker for pannexin and connexin hemichannels, was unable to prevent SR101-labeling of hippocampal astrocytes. However, SR101-labeling of the hippocampal astrocytes was significantly reduced by substrates of organic anion transport polypeptides, including estron-3-sulfate and dehydroepiandrosterone sulfate, suggesting that SR101 is actively transported into hippocampal astrocytes.

  5. Parkinsonian toxin-induced oxidative stress inhibits basal autophagy in astrocytes via NQO2/quinone oxidoreductase 2: Implications for neuroprotection

    PubMed Central

    Janda, Elzbieta; Lascala, Antonella; Carresi, Cristina; Parafati, Maddalena; Aprigliano, Serafina; Russo, Vanessa; Savoia, Claudia; Ziviani, Elena; Musolino, Vincenzo; Morani, Federica; Isidoro, Ciro; Mollace, Vincenzo

    2015-01-01

    Oxidative stress (OS) stimulates autophagy in different cellular systems, but it remains controversial if this rule can be generalized. We have analyzed the effect of chronic OS induced by the parkinsonian toxin paraquat (PQ) on autophagy in astrocytoma cells and primary astrocytes, which represent the first cellular target of neurotoxins in the brain. PQ decreased the basal levels of LC3-II and LC3-positive vesicles, and its colocalization with lysosomal markers, both in the absence and presence of chloroquine. This was paralleled by increased number and size of SQSTM1/p62 aggregates. Downregulation of autophagy was also observed in cells chronically exposed to hydrogen peroxide or nonlethal concentrations of PQ, and it was associated with a reduced astrocyte capability to protect dopaminergic cells from OS in co-cultures. Surprisingly, PQ treatment led to inhibition of MTOR, activation of MAPK8/JNK1 and MAPK1/ERK2-MAPK3/ERK1 and upregulation of BECN1/Beclin 1 expression, all signals typically correlating with induction of autophagy. Reduction of OS by NMDPEF, a specific NQO2 inhibitor, but not by N-acetylcysteine, abrogated the inhibitory effect of PQ and restored autophagic flux. Activation of NQO2 by PQ or menadione and genetic manipulation of its expression confirmed the role of this enzyme in the inhibitory action of PQ on autophagy. PQ did not induce NFE2L2/NRF2, but when it was co-administered with NMDPEF NFE2L2 activity was enhanced in a SQSTM1-independent fashion. Thus, a prolonged OS in astrocytes inhibits LC3 lipidation and impairs autophagosome formation and autophagic flux, in spite of concomitant activation of several pro-autophagic signals. These findings outline an unanticipated neuroprotective role of astrocyte autophagy and identify in NQO2 a novel pharmacological target for its positive modulation. PMID:26046590

  6. Galunisertib inhibits glioma vasculogenic mimicry formation induced by astrocytes.

    PubMed

    Zhang, Chao; Chen, Wenliang; Zhang, Xin; Huang, Bin; Chen, Aanjing; He, Ying; Wang, Jian; Li, Xingang

    2016-03-15

    Gliomas are among the most lethal primary brain tumors found in humans. In high-grade gliomas, vasculogenic mimicry is often detected and has been correlated with prognosis, thus suggesting its potential as a therapeutic target. Vasculogenic mimicry mainly forms vascular-like channels independent of endothelial cells; however, little is known about the relationship between astrocytes and vasculogenic mimicry. In our study, we demonstrated that the presence of astrocytes promoted vasculogenic mimicry. With suspension microarray technology and in vitro tube formation assays, we identified that astrocytes relied on TGF-β1 to enhance vasculogenic mimicry. We also found that vasculogenic mimicry was inhibited by galunisertib, a promising TGF-β1 inhibitor currently being studied in an ongoing trial in glioma patients. The inhibition was partially attributed to a decrease in autophagy after galunisertib treatment. Moreover, we observed a decrease in VE-cadherin and smooth muscle actin-α expression, as well as down-regulation of Akt and Flk phosphorylation in galunisertib-treated glioma cells. By comparing tumor weight and volume in a xenograft model, we acquired promising results to support our theory. This study expands our understanding of the role of astrocytes in gliomas and demonstrates that galunisertib inhibits glioma vasculogenic mimicry induced by astrocytes.

  7. Persistent oxygen-glucose deprivation induces astrocytic death through two different pathways and calpain-mediated proteolysis of cytoskeletal proteins during astrocytic oncosis.

    PubMed

    Cao, Xu; Zhang, Ying; Zou, Liangyu; Xiao, Haibing; Chu, Yinghao; Chu, Xiaofan

    2010-07-26

    Astrocytes are thought to play a role in the maintenance of homeostasis and the provision of metabolic substrates for neurons as well as the coupling of cerebral blood flow to neuronal activity. Accordingly, astrocytic death due to various types of injury can critically influence neuronal survival. The exact pathway of cell death after brain ischemia is under debate. In the present study, we used astrocytes from rat primary culture treated with persistent oxygen-glucose-deprivation (OGD) as a model of ischemia to examine the pathway of cell death and the relevant mechanisms. We observed changes in the cellular morphology, the energy metabolism of astrocytes, and the percentage of apoptosis or oncosis of the astrocytes induced by OGD. Electron microscopy revealed the co-existence of ultrastructural features in both apoptosis and oncosis in individual cells. The cellular ATP content was gradually decreased and the percentages of apoptotic and oncotic cells were increased during OGD. After 4h of OGD, ATP depletion to less than 35% of the control was observed, and oncosis became the primary pathway for astrocytic death. Increased plasma membrane permeability due to oncosis was associated with increased calpain-mediated degradation of several cytoskeletal proteins, including paxillin, vinculin, vimentin and GFAP. Pre-treatment with the calpain inhibitor 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) could delay the OGD-induced astrocytic oncosis. These results suggest that there is a narrow range of ATP that determines astrocytic oncotic death induced by persistent OGD and that calpain-mediated hydrolysis of the cytoskeletal-associated proteins may contribute to astrocytes oncosis. PMID:20493926

  8. α1-Adrenergic receptors mediate coordinated Ca2+ signaling of cortical astrocytes in awake, behaving mice.

    PubMed

    Ding, Fengfei; O'Donnell, John; Thrane, Alexander S; Zeppenfeld, Douglas; Kang, Hongyi; Xie, Lulu; Wang, Fushun; Nedergaard, Maiken

    2013-12-01

    Astrocyte Ca2+ signals in awake behaving mice are widespread, coordinated and differ fundamentally from the locally restricted Ca2+ transients observed ex vivo and in anesthetized animals. Here we show that the synchronized release of norepinephrine (NE) from locus coeruleus (LC) projections throughout the cerebral cortex mediate long-ranging Ca2+ signals by activation of astrocytic α1-adrenergic receptors. When LC output was triggered by either physiological sensory (whisker) stimulation or an air-puff startle response, astrocytes responded with fast Ca2+ transients that encompassed the entire imaged field (positioned over either frontal or parietal cortex). The application of adrenergic inhibitors, including α1-adrenergic antagonist prazosin, potently suppressed both evoked, as well as the frequently observed spontaneous astroglial Ca2+ signals. The LC-specific neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), which reduced cortical NE content by >90%, prevented nearly all astrocytic Ca2+ signals in awake mice. The observations indicate that in adult, unanesthetized mice, astrocytes do not respond directly to glutamatergic signaling evoked by sensory stimulation. Instead astrocytes appear to be the primary target for NE, with astrocytic Ca2+ signaling being triggered by the α1-adrenergic receptor. In turn, astrocytes may coordinate the broad effects of neuromodulators on neuronal activity.

  9. Mutant Disrupted-In-Schizophrenia 1 in astrocytes: focus on glutamate metabolism

    PubMed Central

    Abazyan, Sofya; Yang, Eun Ju; Abazyan, Bagrat; Xia, Meng; Yang, Chunxia; Rojas, Camilo; Slusher, Barbara; Sattler, Rita; Pletnikov, Mikhail

    2014-01-01

    Disrupted-In-Schizophrenia 1 (DISC1) is a genetic risk factor that has been implicated in major mental disorders. DISC1 binds to and stabilizes serine racemase (SR) to regulate production of D-serine by astrocytes, contributing to glutamate (GLU) neurotransmission. However, the possible involvement of astrocytic DISC1 in synthesis, metabolism, re-uptake or secretion of GLU remains unexplored. Thus, we studied the effects of dominant-negative mutant DISC1 on various aspects of GLU metabolism using primary astrocyte cultures and the hippocampal tissue from transgenic mice with astrocyte-restricted expression of mutant DISC1. While mutant DISC1 had no significant effects on astrocyte proliferation, GLU re-uptake, Glutaminase or Glutamate carboxypeptidase II activity, expression of mutant DISC1 was associated with increased levels of alanine-serine-cysteine transporter 2, vesicular glutamate transporters 1 and 3 in primary astrocytes and in the hippocampus as well as elevated expression of the NR1 subunit and diminished expression of the NR2A subunit of NMDA receptors in the hippocampus at postnatal day 21. Our findings indicate that decreased D-serine production by astrocytic mutant DISC1 may lead to compensatory changes in levels of the amino acid transporters and NMDA receptors in the context of tripartite synapse. PMID:25131692

  10. Glutamate Mediated Astrocytic Filtering of Neuronal Activity

    PubMed Central

    Herzog, Nitzan; De Pittà, Maurizio; Jacob, Eshel Ben; Berry, Hugues; Hanein, Yael

    2014-01-01

    Neuron-astrocyte communication is an important regulatory mechanism in various brain functions but its complexity and role are yet to be fully understood. In particular, the temporal pattern of astrocyte response to neuronal firing has not been fully characterized. Here, we used neuron-astrocyte cultures on multi-electrode arrays coupled to Ca2+ imaging and explored the range of neuronal stimulation frequencies while keeping constant the amount of stimulation. Our results reveal that astrocytes specifically respond to the frequency of neuronal stimulation by intracellular Ca2+ transients, with a clear onset of astrocytic activation at neuron firing rates around 3-5 Hz. The cell-to-cell heterogeneity of the astrocyte Ca2+ response was however large and increasing with stimulation frequency. Astrocytic activation by neurons was abolished with antagonists of type I metabotropic glutamate receptor, validating the glutamate-dependence of this neuron-to-astrocyte pathway. Using a realistic biophysical model of glutamate-based intracellular calcium signaling in astrocytes, we suggest that the stepwise response is due to the supralinear dynamics of intracellular IP3 and that the heterogeneity of the responses may be due to the heterogeneity of the astrocyte-to-astrocyte couplings via gap junction channels. Therefore our results present astrocyte intracellular Ca2+ activity as a nonlinear integrator of glutamate-dependent neuronal activity. PMID:25521344

  11. Protective Effects of Gastrodin Against Autophagy-Mediated Astrocyte Death.

    PubMed

    Wang, Xin-shang; Tian, Zhen; Zhang, Nan; Han, Jing; Guo, Hong-liang; Zhao, Ming-gao; Liu, Shui-bing

    2016-03-01

    Gastrodin is an active ingredient derived from the rhizome of Gastrodia elata. This compound is usually used to treat convulsive illness, dizziness, vertigo, and headache. This study aimed to investigate the effect of gastrodin on the autophagy of glial cells exposed to lipopolysaccharides (LPS, 1 µg/mL). Autophagy is a form of programmed cell death, although it also promotes cell survival. In cultured astrocytes, LPS exposure induced excessive autophagy and apoptosis, which were significantly prevented by the pretreatment cells with gastrodin (10 μM). The protective effects of gastrodin via autophagy inhibition were verified by the decreased levels of LC3-II, P62, and Beclin-1, which are classical markers for autophagy. Furthermore, gastrodin protected astrocytes from apoptosis through Bcl-2 and Bax signaling pathway. The treatment of astrocytes with rapamycin (500 nM), wortmannin (100 nM), and LY294002 (10 μM), which are inhibitors of mTOR and PI3K, respectively, eliminated the known effects of gastrodin on the inhibited Beclin-1 expression. Furthermore, gastrodin blocked the down-regulation of glutamine synthetase induced by LPS exposure in astrocytes. Our results suggest that gastrodin can be used as a preventive agent for the excessive autophagy induced by LPS. PMID:26643508

  12. Functional Oxygen Sensitivity of Astrocytes.

    PubMed

    Angelova, Plamena R; Kasymov, Vitaliy; Christie, Isabel; Sheikhbahaei, Shahriar; Turovsky, Egor; Marina, Nephtali; Korsak, Alla; Zwicker, Jennifer; Teschemacher, Anja G; Ackland, Gareth L; Funk, Gregory D; Kasparov, Sergey; Abramov, Andrey Y; Gourine, Alexander V

    2015-07-22

    In terrestrial mammals, the oxygen storage capacity of the CNS is limited, and neuronal function is rapidly impaired if oxygen supply is interrupted even for a short period of time. However, oxygen tension monitored by the peripheral (arterial) chemoreceptors is not sensitive to regional CNS differences in partial pressure of oxygen (PO2 ) that reflect variable levels of neuronal activity or local tissue hypoxia, pointing to the necessity of a functional brain oxygen sensor. This experimental animal (rats and mice) study shows that astrocytes, the most numerous brain glial cells, are sensitive to physiological changes in PO2 . Astrocytes respond to decreases in PO2 a few millimeters of mercury below normal brain oxygenation with elevations in intracellular calcium ([Ca(2+)]i). The hypoxia sensor of astrocytes resides in the mitochondria in which oxygen is consumed. Physiological decrease in PO2 inhibits astroglial mitochondrial respiration, leading to mitochondrial depolarization, production of free radicals, lipid peroxidation, activation of phospholipase C, IP3 receptors, and release of Ca(2+) from the intracellular stores. Hypoxia-induced [Ca(2+)]i increases in astrocytes trigger fusion of vesicular compartments containing ATP. Blockade of astrocytic signaling by overexpression of ATP-degrading enzymes or targeted astrocyte-specific expression of tetanus toxin light chain (to interfere with vesicular release mechanisms) within the brainstem respiratory rhythm-generating circuits reveals the fundamental physiological role of astroglial oxygen sensitivity; in low-oxygen conditions (environmental hypoxia), this mechanism increases breathing activity even in the absence of peripheral chemoreceptor oxygen sensing. These results demonstrate that astrocytes are functionally specialized CNS oxygen sensors tuned for rapid detection of physiological changes in brain oxygenation. Significance statement: Most, if not all, animal cells possess mechanisms that allow them to

  13. Increased proliferation and gliogenesis of cultured rat neural progenitor cells by lipopolysaccharide-stimulated astrocytes.

    PubMed

    Go, Hyo Sang; Shin, Chan Young; Lee, Sung Hoon; Jeon, Se-Jin; Kim, Ki Chan; Choi, Chang Soon; Ko, Kwang Ho

    2009-01-01

    Neural progenitor cells (NPC) are self-renewing multipotent cells that generate neurons and glial cells in the brain. NPCs generate neurons and glia not only during development but also after neural injury. Recent studies have shown that endogenous NPCs are activated after brain injury and migrate toward damaged areas where astrocyte activation occurs. Considering the massive proliferation of astrocytes as well as the production of several kinds of cytoactive molecules after brain injury, such as NO, growth factors and cytokines, it is tempting to think that cytoactive molecules released by activated glial cells regulate neural progenitor differentiation and proliferation through inflammatory mediators. To test this hypothesis, we stimulated rat primary astrocytes with lipopolysaccharide (LPS) to induce the activation of astrocytes. After addition of the conditioned media from LPS-stimulated astrocytes to NPC culture, proliferation was examined by MTT assay and bromodeoxyuridine (BrdU) incorporation. The differentiation of NPC into neurons and astrocytes was examined by Western blot, ELISA and immunocytochemical staining with cell-type-specific markers. Conditioned media from LPS-stimulated astrocytes increased NPC proliferation as well as gliogenesis as compared with control conditioned media from astrocytes without LPS stimulation. In contrast, neurogenesis was decreased by LPS-conditioned media. To investigate the molecular mechanism mediating glial differentiation and proliferation of NPC by reactive astrocytes, we added inhibitors of the Erk and JNK pathways during LPS stimulation. These inhibitors - except for a p38 inhibitor - decreased NPC proliferation and glial differentiation. These results suggest that LPS stimulated astrocytes generate factors regulating NPC proliferation and gliogenesis via the Erk and JNK pathways. PMID:19609085

  14. Acute and chronic mu opioids differentially regulate thrombospondins 1 and 2 isoforms in astrocytes.

    PubMed

    Phamduong, Ellen; Rathore, Maanjot K; Crews, Nicholas R; D'Angelo, Alexander S; Leinweber, Andrew L; Kappera, Pranay; Krenning, Thomas M; Rendell, Victoria R; Belcheva, Mariana M; Coscia, Carmine J

    2014-02-19

    Chronic opioids induce synaptic plasticity, a major neuronal adaptation. Astrocyte activation in synaptogenesis may play a critical role in opioid tolerance, withdrawal, and dependence. Thrombospondins 1 and 2 (TSP1/2) are astrocyte-secreted matricellular glycoproteins that promote neurite outgrowth as well as dendritic spine and synapse formation, all of which are inhibited by chronic μ opioids. In prior studies, we discovered that the mechanism of TSP1 regulation by μ opioids in astrocytes involves crosstalk between three different classes of receptors, μ opioid receptor, EGFR and TGFβR. Moreover, TGFβ1 stimulated TSP1 expression via EGFR and ERK/MAPK activation, indicating that EGFR is a signaling hub for opioid and TGFβ1 actions. Using various selective antagonists, and inhibitors, here we compared the mechanisms of chronic opioid regulation of TSP1/2 isoform expression in vivo and in immortalized rat cortical astrocytes. TSP1/2 release from astrocytes was also monitored. Acute and chronic μ opioids, morphine, and the prototypic μ ligand, DAMGO, modulated TSP2 protein levels. TSP2 but not TSP1 protein content was up-regulated by acute (3 h) morphine or DAMGO by an ERK/MAPK dependent mechanism. Paradoxically, TSP2 protein levels were altered neither by TGFβ1 nor by astrocytic neurotrophic factors, EGF, CNTF, and BMP4. TSP1/2 immunofluorescence was increased in astrocytes subjected to scratch-wounding, suggesting TSPs may be useful markers for the "reactive" state of these cells and potentially for different types of injury. Previously, we determined that chronic morphine attenuated both neurite outgrowth and synapse formation in cocultures of primary astrocytes and neurons under similar temporal conditions that μ opioids reduced TSP1 protein levels in astrocytes. Here we found that, after the same 8 day treatment, morphine or DAMGO diminished TSP2 protein levels in astrocytes. Therefore, μ opioids may deter synaptogenesis via both TSP1/2 isoforms, but

  15. Induction of anchorage-independent growth in primary human cells exposed to protons or HZE ions separately or in dual exposures.

    PubMed

    Sutherland, B M; Cuomo, N C; Bennett, P V

    2005-10-01

    Travelers on space missions will be exposed to a complex radiation environment that includes protons and heavy charged particles. Since protons are present at much higher levels than are heavy ions, the most likely scenario for cellular radiation exposure will be proton exposure followed by a hit by a heavy ion. Although the effects of individual ion species on human cells are being investigated extensively, little is known about the effects of exposure to both radiation types. One useful measure of mammalian cell damage is induction of the ability to grow in a semi-solid agar medium highly inhibitory to the growth of normal human cells, termed neoplastic transformation. Using primary human cells, we evaluated induction of soft-agar growth and survival of cells exposed to protons only or to heavy charged particles (600 MeV/nucleon silicon) only as well as of cells exposed to protons followed after a 4-day interval by silicon ions. Both ions alone efficiently transformed the human cells to anchorage-independent growth. Initial experiments indicate that the dose responses for neoplastic transformation of cells exposed to protons and then after 4 days to silicon ions appear similar to that of cells exposed to silicon ions alone.

  16. Induction of anchorage-independent growth in primary human cells exposed to protons or HZE ions separately or in dual exposures.

    PubMed

    Sutherland, B M; Cuomo, N C; Bennett, P V

    2005-10-01

    Travelers on space missions will be exposed to a complex radiation environment that includes protons and heavy charged particles. Since protons are present at much higher levels than are heavy ions, the most likely scenario for cellular radiation exposure will be proton exposure followed by a hit by a heavy ion. Although the effects of individual ion species on human cells are being investigated extensively, little is known about the effects of exposure to both radiation types. One useful measure of mammalian cell damage is induction of the ability to grow in a semi-solid agar medium highly inhibitory to the growth of normal human cells, termed neoplastic transformation. Using primary human cells, we evaluated induction of soft-agar growth and survival of cells exposed to protons only or to heavy charged particles (600 MeV/nucleon silicon) only as well as of cells exposed to protons followed after a 4-day interval by silicon ions. Both ions alone efficiently transformed the human cells to anchorage-independent growth. Initial experiments indicate that the dose responses for neoplastic transformation of cells exposed to protons and then after 4 days to silicon ions appear similar to that of cells exposed to silicon ions alone. PMID:16187755

  17. Cleavable ester linked magnetic nanoparticles for labeling of solvent exposed primary amine groups of peptides/proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to study the solvent exposed lysine residues of peptides/proteins, we previously reported disulfide linked N-hydrosuccinimide ester modified silica coated iron oxide magnetic nanoparticles (NHS-SS-SiO2@Fe3O4 MNPs). The presence of a disulfide bond in the linker limits the use of disulfide r...

  18. Gene expression as a sensitive endpoint to evaluate cell differentiation and maturation of the developing central nervous system in primary cultures of rat cerebellar granule cells (CGCs) exposed to pesticides

    SciTech Connect

    Hogberg, Helena T.; Kinsner-Ovaskainen, Agnieszka; Hartung, Thomas; Coecke, Sandra; Bal-Price, Anna K.

    2009-03-15

    The major advantage of primary neuronal cultures for developmental neurotoxicity (DNT) testing is their ability to replicate the crucial stages of neurodevelopment. In our studies using primary culture of cerebellar granule cells (CGCs) we have evaluated whether the gene expression relevant to the most critical developmental processes such as neuronal differentiation (NF-68 and NF-200) and functional maturation (NMDA and GABA{sub A} receptors), proliferation and differentiation of astrocytes (GFAP and S100{beta}) as well as the presence of neural precursor cells (nestin and Sox10) could be used as an endpoint for in vitro DNT. The expression of these genes was assessed after exposure to various pesticides (paraquat parathion, dichlorvos, pentachlorophenol and cycloheximide) that could induce developmental neurotoxicity through different mechanisms. All studied pesticides significantly modified the expression of selected genes, related to the different stages of neuronal and/or glial cell development and maturation. The most significant changes were observed after exposure to paraquat and parathion (i.e. down-regulation of mRNA expression of NF-68 and NF-200, NMDA and GABA{sub A} receptors). Similarly, dichlorvos affected mainly neurons (decreased mRNA expression of NF-68 and GABA{sub A} receptors) whereas cycloheximide had an effect on neurons and astrocytes, as significant decreases in the mRNA expression of both neurofilaments (NF-68 and NF-200) and the astrocyte marker (S100{beta}) were observed. Our results suggest that toxicity induced by pesticides that target multiple pathways of neurodevelopment can be identified by studying expression of genes that are involved in different stages of cell development and maturation, and that gene expression could be used as a sensitive endpoint for initial screening to identify the compounds with the potential to cause developmental neurotoxicity.

  19. [Glial fibrillary acidic protein: the component of intermediate filaments in the vertebrate brain astrocytes].

    PubMed

    Sukhorukova, E G; Kruzhevskiĭ, D É; Alekseeva, O S

    2015-01-01

    Glial fibrillary acidic protein (GFAP) refers to the type III intermediate filament proteins and is the essential component of the cytoskeleton in astrocytes of all vertebrates. This review presents current data on the molecular organization of GFAP in a comparative aspect. The results of most relevant studies using immunocytochemical labeling of the protein are summarized. The data on the changes in expression of GFAP in Alexander disease caused by the primary pathology of astrocytes are presented.

  20. Endocytosis-mediated HIV-1 entry and its significance in the elusive behavior of the virus in astrocytes.

    PubMed

    Chauhan, Ashok; Mehla, Rajeev; Vijayakumar, Theophilus Sunder; Handy, Indhira

    2014-05-01

    Astrocytes protect neurons but also evoke a proinflammatory response to injury and viral infections including HIV. We investigated the mechanism of HIV-1 infection in primary astrocytes, which showed minimal but productive viral infection independent of CXCR4. As with ectopic-CD4-expressing astrocytes, lysosomotropic agents led to increased HIV-1 infection in wild-type but not Rabs 5, 7, and 11-ablated astrocytes. Instead, HIV-1 infection was decreased in Rab-depleted astrocytes, corroborating viral entry by endocytosis. HIV-1 produced persistent infection in astrocytes (160 days); no evidence of latent infection was seen. Notably, one caveat is that endosomal modifiers enhanced wild-type HIV-1 infection (M- and T-tropic) in astrocytes, suggesting endocytic entry of the virus. Impeding endocytosis by inhibition of Rab 5, 7 or 11 will inhibit HIV infection in astrocytes. Although the contribution of such low-level infection in astrocytes to neurological complications is unclear, it may serve as an elusive viral reservoir in the central nervous system.

  1. Astrocytes Release Polyunsaturated Fatty Acids by Lipopolysaccharide Stimuli.

    PubMed

    Aizawa, Fuka; Nishinaka, Takashi; Yamashita, Takuya; Nakamoto, Kazuo; Koyama, Yutaka; Kasuya, Fumiyo; Tokuyama, Shogo

    2016-01-01

    We previously reported that levels of long-chain fatty acids (FAs) including docosahexaenoic acids (DHA) increase in the hypothalamus of inflammatory pain model mice. However, the precise mechanisms underlying the increment of free fatty acids (FFAs) in the brain during inflammation remains unknown. In this study, we characterized FFAs released by inflammatory stimulation in rat primary cultured astrocytes, and tested the involvement of phospholipase A2 (PLA2) on these mechanisms. Lipopolysaccharide (LPS) stimulation significantly increased the levels of several FAs in the astrocytes. Under these conditions, mRNA expression of cytosolic PLA2 (cPLA2) and calcium-independent PLA2 (iPLA2) in LPS-treated group increased compared with the control group. Furthermore, in the culture media, the levels of DHA and arachidonic acid (ARA) significantly increased by LPS stimuli compared with those of a vehicle-treated control group whereas the levels of saturated FAs (SFAs), namely palmitic acid (PAM) and stearic acid (STA), did not change. In summary, our findings suggest that astrocytes specifically release DHA and ARA by inflammatory conditions. Therefore astrocytes might function as a regulatory factor of DHA and ARA in the brain. PMID:27374285

  2. Central Role of Maladapted Astrocytic Plasticity in Ischemic Brain Edema Formation.

    PubMed

    Wang, Yu-Feng; Parpura, Vladimir

    2016-01-01

    Brain edema formation and the ensuing brain damages are the major cause of high mortality and long term disability following the occurrence of ischemic stroke. In this process, oxygen and glucose deprivation and the resulting reperfusion injury play primary roles. In response to the ischemic insult, the neurovascular unit experiences both intracellular and extracellular edemas, associated with maladapted astrocytic plasticity. The astrocytic plasticity includes both morphological and functional plasticity. The former involves a reactive gliosis and the subsequent glial retraction. It relates to the capacity of astrocytes to buffer changes in extracellular chemical levels, particularly K(+) and glutamate, as well as the integrity of the blood-brain barrier (BBB). The latter involves the expression and activity of a series of ion and water transport proteins. These molecules are grouped together around glial fibrillary acidic protein (GFAP) and water channel protein aquaporin 4 (AQP4) to form functional networks, regulate hydromineral balance across cell membranes and maintain the integrity of the BBB. Intense ischemic challenges can disrupt these capacities of astrocytes and result in their maladaptation. The maladapted astrocytic plasticity in ischemic stroke cannot only disrupt the hydromineral homeostasis across astrocyte membrane and the BBB, but also leads to disorders of the whole neurovascular unit. This review focuses on how the maladapted astrocytic plasticity in ischemic stroke plays the central role in the brain edema formation. PMID:27242440

  3. α7 Nicotinic Receptor Promotes the Neuroprotective Functions of Astrocytes against Oxaliplatin Neurotoxicity

    PubMed Central

    Di Cesare Mannelli, Lorenzo; Tenci, Barbara; Zanardelli, Matteo; Failli, Paola; Ghelardini, Carla

    2015-01-01

    Neuropathies are characterized by a complex response of the central nervous system to injuries. Glial cells are recruited to maintain neuronal homeostasis but dysregulated activation leads to pain signaling amplification and reduces the glial neuroprotective power. Recently, we highlighted the property of α7 nicotinic-acetylcholine-receptor (nAChR) agonists to relieve pain and induce neuroprotection simultaneously with a strong increase in astrocyte density. Aimed to study the role of α7 nAChR in the neuron-glia cross-talk, we treated primary rat neurons and astrocytes with the neurotoxic anticancer drug oxaliplatin evaluating the effect of the α7 nAChR agonist PNU-282987 (PNU). Oxaliplatin (1 μM, 48 h) reduced cell viability and increased caspase-3 activity of neuron monocultures without damaging astrocytes. In cocultures, astrocytes were not able to protect neurons by oxaliplatin even if glial cell metabolism was stimulated (pyruvate increase). On the contrary, the coculture incubation with 10 μM PNU improved neuron viability and inhibited apoptosis. In the absence of astrocytes, the protection disappeared. Furthermore, PNU promoted the release of the anti-inflammatory cytokine TGF-β1 and the expression of the glutamate-detoxifying enzyme glutamine synthetase. The α7 nAChR stimulation protects neurons from oxaliplatin toxicity through an astrocyte-mediated mechanism. α7 nAChR is suggested for recovering the homeostatic role of astrocytes. PMID:26146570

  4. Central Role of Maladapted Astrocytic Plasticity in Ischemic Brain Edema Formation

    PubMed Central

    Wang, Yu-Feng; Parpura, Vladimir

    2016-01-01

    Brain edema formation and the ensuing brain damages are the major cause of high mortality and long term disability following the occurrence of ischemic stroke. In this process, oxygen and glucose deprivation and the resulting reperfusion injury play primary roles. In response to the ischemic insult, the neurovascular unit experiences both intracellular and extracellular edemas, associated with maladapted astrocytic plasticity. The astrocytic plasticity includes both morphological and functional plasticity. The former involves a reactive gliosis and the subsequent glial retraction. It relates to the capacity of astrocytes to buffer changes in extracellular chemical levels, particularly K+ and glutamate, as well as the integrity of the blood-brain barrier (BBB). The latter involves the expression and activity of a series of ion and water transport proteins. These molecules are grouped together around glial fibrillary acidic protein (GFAP) and water channel protein aquaporin 4 (AQP4) to form functional networks, regulate hydromineral balance across cell membranes and maintain the integrity of the BBB. Intense ischemic challenges can disrupt these capacities of astrocytes and result in their maladaptation. The maladapted astrocytic plasticity in ischemic stroke cannot only disrupt the hydromineral homeostasis across astrocyte membrane and the BBB, but also leads to disorders of the whole neurovascular unit. This review focuses on how the maladapted astrocytic plasticity in ischemic stroke plays the central role in the brain edema formation. PMID:27242440

  5. Characterization of Amino Acid Profile and Enzymatic Activity in Adult Rat Astrocyte Cultures.

    PubMed

    Souza, Débora Guerini; Bellaver, Bruna; Hansel, Gisele; Arús, Bernardo Assein; Bellaver, Gabriela; Longoni, Aline; Kolling, Janaina; Wyse, Angela T S; Souza, Diogo Onofre; Quincozes-Santos, André

    2016-07-01

    Astrocytes are multitasking players in brain complexity, possessing several receptors and mechanisms to detect, participate and modulate neuronal communication. The functionality of astrocytes has been mainly unraveled through the study of primary astrocyte cultures, and recently our research group characterized a model of astrocyte cultures derived from adult Wistar rats. We, herein, aim to characterize other basal functions of these cells to explore the potential of this model for studying the adult brain. To characterize the astrocytic phenotype, we determined the presence of GFAP, GLAST and GLT 1 proteins in cells by immunofluorescence. Next, we determined the concentrations of thirteen amino acids, ATP, ADP, adenosine and calcium in astrocyte cultures, as well as the activities of Na(+)/K(+)-ATPase and acetylcholine esterase. Furthermore, we assessed the presence of the GABA transporter 1 (GAT 1) and cannabinoid receptor 1 (CB 1) in the astrocytes. Cells demonstrated the presence of glutamine, consistent with their role in the glutamate-glutamine cycle, as well as glutamate and D-serine, amino acids classically known to act as gliotransmitters. ATP was produced and released by the cells and ADP was consumed. Calcium levels were in agreement with those reported in the literature, as were the enzymatic activities measured. The presence of GAT 1 was detected, but the presence of CB 1 was not, suggesting a decreased neuroprotective capacity in adult astrocytes under in vitro conditions. Taken together, our results show cellular functionality regarding the astrocytic role in gliotransmission and neurotransmitter management since they are able to produce and release gliotransmitters and to modulate the cholinergic and GABAergic systems. PMID:26915106

  6. NMO sera down-regulate AQP4 in human astrocyte and induce cytotoxicity independent of complement.

    PubMed

    Haruki, Hiroyo; Sano, Yasuteru; Shimizu, Fumitaka; Omoto, Masatoshi; Tasaki, Ayako; Oishi, Mariko; Koga, Michiaki; Saito, Kazuyuki; Takahashi, Toshiyuki; Nakada, Tsutomu; Kanda, Takashi

    2013-08-15

    Autoantibodies against astrocyte water channel aquaporin-4 (AQP4) are highly specific for neuromyelitis optica (NMO). However, the molecular mechanism of NMO still remains unclear. The purpose of this study was to identify the possible humoral mechanisms responsible for the occurrence of astrocytic damage. Human primary astrocytes (AST) were immortalized by retroviral vectors harboring temperature-sensitive SV40 T antigen gene and AQP4 cDNA (M23), designated as hAST-AQP4. The effects of NMO sera on the content and localization of AQP4, including cytotoxicity and astrocytic morphology, were evaluated. In addition, this study examined whether the amount and localization of AQP4 protein in astrocytes were influenced by direct contact with the immortalized human brain microvascular endothelial cell line, TY09. NMO sera alone induced cytotoxicity and addition of complement had a more harmful effect on hAST-AQP4. NMO sera also decreased AQP4 mRNA and protein. NMO sera alone up-regulated TNFα and IL-6 in astrocytes and co-incubation with anti-TNFα and anti-IL-6 neutralizing antibodies blocked both the cytotoxicity and reduction of AQP4 in astrocytes. In the experiment using the in vitro BBB models, AQP4 protein mainly localized at the astrocytic membrane after co-culture with TY09, in contact with TY09. The future elucidation of factors that up-regulate AQP4 in astrocytes presumably released by blood brain barrier forming endothelial cells and that block the production of inflammatory cytokines may therefore lead to the development of a novel therapeutic strategy.

  7. Methamphetamine Inhibits the Glucose Uptake by Human Neurons and Astrocytes: Stabilization by Acetyl-L-Carnitine

    PubMed Central

    Szlachetka, Adam M.; Haorah, James

    2011-01-01

    Methamphetamine (METH), an addictive psycho-stimulant drug exerts euphoric effects on users and abusers. It is also known to cause cognitive impairment and neurotoxicity. Here, we hypothesized that METH exposure impairs the glucose uptake and metabolism in human neurons and astrocytes. Deprivation of glucose is expected to cause neurotoxicity and neuronal degeneration due to depletion of energy. We found that METH exposure inhibited the glucose uptake by neurons and astrocytes, in which neurons were more sensitive to METH than astrocytes in primary culture. Adaptability of these cells to fatty acid oxidation as an alternative source of energy during glucose limitation appeared to regulate this differential sensitivity. Decrease in neuronal glucose uptake by METH was associated with reduction of glucose transporter protein-3 (GLUT3). Surprisingly, METH exposure showed biphasic effects on astrocytic glucose uptake, in which 20 µM increased the uptake while 200 µM inhibited glucose uptake. Dual effects of METH on glucose uptake were paralleled to changes in the expression of astrocytic glucose transporter protein-1 (GLUT1). The adaptive nature of astrocyte to mitochondrial β-oxidation of fatty acid appeared to contribute the survival of astrocytes during METH-induced glucose deprivation. This differential adaptive nature of neurons and astrocytes also governed the differential sensitivity to the toxicity of METH in these brain cells. The effect of acetyl-L-carnitine for enhanced production of ATP from fatty oxidation in glucose-free culture condition validated the adaptive nature of neurons and astrocytes. These findings suggest that deprivation of glucose-derived energy may contribute to neurotoxicity of METH abusers. PMID:21556365

  8. Astrocytic mitochondrial membrane hyperpolarization following extended oxygen and glucose deprivation.

    PubMed

    Korenić, Andrej; Boltze, Johannes; Deten, Alexander; Peters, Myriam; Andjus, Pavle; Radenović, Lidija

    2014-01-01

    Astrocytes can tolerate longer periods of oxygen and glucose deprivation (OGD) as compared to neurons. The reasons for this reduced vulnerability are not well understood. Particularly, changes in mitochondrial membrane potential (Δψ(m)) in astrocytes, an indicator of the cellular redox state, have not been investigated during reperfusion after extended OGD exposure. Here, we subjected primary mouse astrocytes to glucose deprivation (GD), OGD and combinations of both conditions varying in duration and sequence. Changes in Δψ(m), visualized by change in the fluorescence of JC-1, were investigated within one hour after reconstitution of oxygen and glucose supply, intended to model in vivo reperfusion. In all experiments, astrocytes showed resilience to extended periods of OGD, which had little effect on Δψ(m) during reperfusion, whereas GD caused a robust Δψ(m) negativation. In case no Δψ(m) negativation was observed after OGD, subsequent chemical oxygen deprivation (OD) induced by sodium azide caused depolarization, which, however, was significantly delayed as compared to normoxic group. When GD preceded OD for 12 h, Δψ(m) hyperpolarization was induced by both GD and subsequent OD, but significant interaction between these conditions was not detected. However, when GD was extended to 48 h preceding OGD, hyperpolarization enhanced during reperfusion. This implicates synergistic effects of both conditions in that sequence. These findings provide novel information regarding the role of the two main substrates of electron transport chain (glucose and oxygen) and their hyperpolarizing effect on Δψ(m) during substrate deprivation, thus shedding new light on mechanisms of astrocyte resilience to prolonged ischemic injury. PMID:24587410

  9. Synaptopathy in the noise-exposed and aging cochlea: Primary neural degeneration in acquired sensorineural hearing loss.

    PubMed

    Kujawa, Sharon G; Liberman, M Charles

    2015-12-01

    The classic view of sensorineural hearing loss (SNHL) is that the "primary" targets are hair cells, and that cochlear-nerve loss is "secondary" to hair cell degeneration. Our recent work in mouse and guinea pig has challenged that view. In noise-induced hearing loss, exposures causing only reversible threshold shifts (and no hair cell loss) nevertheless cause permanent loss of >50% of cochlear-nerve/hair-cell synapses. Similarly, in age-related hearing loss, degeneration of cochlear synapses precedes both hair cell loss and threshold elevation. This primary neural degeneration has remained hidden for three reasons: 1) the spiral ganglion cells, the cochlear neural elements commonly assessed in studies of SNHL, survive for years despite loss of synaptic connection with hair cells, 2) the synaptic terminals of cochlear nerve fibers are unmyelinated and difficult to see in the light microscope, and 3) the degeneration is selective for cochlear-nerve fibers with high thresholds. Although not required for threshold detection in quiet (e.g. threshold audiometry or auditory brainstem response threshold), these high-threshold fibers are critical for hearing in noisy environments. Our research suggests that 1) primary neural degeneration is an important contributor to the perceptual handicap in SNHL, and 2) in cases where the hair cells survive, neurotrophin therapies can elicit neurite outgrowth from spiral ganglion neurons and re-establishment of their peripheral synapses. This article is part of a Special Issue entitled .

  10. Ferrous Iron Induces Nrf2 Expression in Mouse Brain Astrocytes to Prevent Neurotoxicity.

    PubMed

    Cui, Zhenwen; Zhong, Zhihong; Yang, Yong; Wang, Baofeng; Sun, Yuhao; Sun, Qingfang; Yang, Guo-Yuan; Bian, Liuguan

    2016-08-01

    Free radical damage caused by ferrous iron is involved in the pathogenesis of secondary brain injury after intracerebral hemorrhage (ICH). NF-E2-related factor 2 (Nrf2), a major phase II gene regulator that binds to antioxidant response element, represents an important cellular cytoprotective mechanism against oxidative damage. We hypothesized that Nrf2 might protect astrocytes from damage by Fe(2+) . Therefore, we examined cytotoxicity in primary astrocytes induced by iron overload and evaluated the effects of Fe(2+) on Nrf2 expression. The results demonstrated that 24-h Fe(2+) exposure exerted time- and concentration-dependent cytotoxicity in astrocytes. Furthermore, Fe(2+) exposure in astrocytes resulted in time- and concentration-dependent increases in Nrf2 expression, which preceded Fe(2+) toxicity. Nrf2-specific siRNA further knocked down Nrf2 levels, resulting in greater Fe(2+) -induced astrocyte cytotoxicity. These data indicate that induction of Nrf2 expression could serve as an adaptive self-defense mechanism, although it is insufficient to completely protect primary astrocytes from Fe(2+) -induced neurotoxicity. PMID:27037625

  11. Diphenylarsinic Acid Induced Activation of Cultured Rat Cerebellar Astrocytes: Phosphorylation of Mitogen-Activated Protein Kinases, Upregulation of Transcription Factors, and Release of Brain-Active Cytokines.

    PubMed

    Negishi, Takayuki; Matsumoto, Mami; Kojima, Mikiya; Asai, Ryota; Kanehira, Tomoko; Sakaguchi, Fumika; Takahata, Kazuaki; Arakaki, Rina; Aoyama, Yohei; Yoshida, Hikari; Yoshida, Kenji; Yukawa, Kazunori; Tashiro, Tomoko; Hirano, Seishiro

    2016-03-01

    Diphenylarsinic acid (DPAA) was detected as the primary compound responsible for the arsenic poisoning that occurred in Kamisu, Ibaraki, Japan, where people using water from a well that was contaminated with a high level of arsenic developed neurological (mostly cerebellar) symptoms and dysregulation of regional cerebral blood flow. To understand the underlying molecular mechanism of DPAA-induced cerebellar symptoms, we focused on astrocytes, which have a brain-protective function. Incubation with 10 µM DPAA for 96 h promoted cell proliferation, increased the expression of antioxidative stress proteins (heme oxygenase-1 and heat shock protein 70), and induced the release of cytokines (MCP-1, adrenomedullin, FGF2, CXCL1, and IL-6). Furthermore, DPAA overpoweringly increased the phosphorylation of three major mitogen-activated protein kinases (MAPKs) (ERK1/2, p38MAPK, and SAPK/JNK), which indicated MAPK activation, and subsequently induced expression and/or phosphorylation of transcription factors (Nrf2, CREB, c-Jun, and c-Fos) in cultured rat cerebellar astrocytes. Structure-activity relationship analyses of DPAA and other related pentavalent organic arsenicals revealed that DPAA at 10 µM activated astrocytes most effective among organic arsenicals tested at the same dose. These results suggest that in a cerebellum exposed to DPAA, abnormal activation of the MAPK-transcription factor pathway and irregular secretion of these neuroactive, glioactive, and/or vasoactive cytokines in astrocytes can be the direct/indirect cause of functional abnormalities in surrounding neurons, glial cells, and vascular cells: This in turn might lead to the onset of cerebellar symptoms and disruption of cerebral blood flow. PMID:26645585

  12. Astrocyte calcium signaling: the third wave.

    PubMed

    Bazargani, Narges; Attwell, David

    2016-02-01

    The discovery that transient elevations of calcium concentration occur in astrocytes, and release 'gliotransmitters' which act on neurons and vascular smooth muscle, led to the idea that astrocytes are powerful regulators of neuronal spiking, synaptic plasticity and brain blood flow. These findings were challenged by a second wave of reports that astrocyte calcium transients did not mediate functions attributed to gliotransmitters and were too slow to generate blood flow increases. Remarkably, the tide has now turned again: the most important calcium transients occur in fine astrocyte processes not resolved in earlier studies, and new mechanisms have been discovered by which astrocyte [Ca(2+)]i is raised and exerts its effects. Here we review how this third wave of discoveries has changed our understanding of astrocyte calcium signaling and its consequences for neuronal function.

  13. Astrocytes Control Synapse Formation, Function, and Elimination

    PubMed Central

    Chung, Won-Suk; Allen, Nicola J.; Eroglu, Cagla

    2015-01-01

    Astrocytes, through their close associations with synapses, can monitor and alter synaptic function, thus actively controlling synaptic transmission in the adult brain. Besides their important role at adult synapses, in the last three decades a number of critical findings have highlighted the importance of astrocytes in the establishment of synaptic connectivity in the developing brain. In this article, we will review the key findings on astrocytic control of synapse formation, function, and elimination. First, we will summarize our current structural and functional understanding of astrocytes at the synapse. Then, we will discuss the cellular and molecular mechanisms through which developing and mature astrocytes instruct the formation, maturation, and refinement of synapses. Our aim is to provide an overview of astrocytes as important players in the establishment of a functional nervous system. PMID:25663667

  14. Astrocytes in the tempest of multiple sclerosis.

    PubMed

    Miljković, Djordje; Timotijević, Gordana; Mostarica Stojković, Marija

    2011-12-01

    Astrocytes are the most abundant cell population within the CNS of mammals. Their glial role is perfectly performed in the healthy CNS as they support functions of neurons. The omnipresence of astrocytes throughout the white and grey matter and their intimate relation with blood vessels of the CNS, as well as numerous immunity-related actions that these cells are capable of, imply that astrocytes should have a prominent role in neuroinflammatory disorders, such as multiple sclerosis (MS). The role of astrocytes in MS is rather ambiguous, as they have the capacity to both stimulate and restrain neuroinflammation and tissue destruction. In this paper we present some of the proved and the proposed functions of astrocytes in neuroinflammation and discuss the effect of MS therapeutics on astrocytes. PMID:21443873

  15. Involvement of astrocytes in neurovascular communication.

    PubMed

    Nuriya, M; Hirase, H

    2016-01-01

    The vascular interface of the brain is distinct from that of the peripheral tissue in that astrocytes, the most numerous glial cell type in the gray matter, cover the vasculature with their endfeet. This morphological feature of the gliovascular junction has prompted neuroscientists to suggest possible functional roles of astrocytes including astrocytic modulation of the vasculature. Additionally, astrocytes develop an intricate morphology that intimately apposes neuronal synapses, making them an ideal cellular mediator of neurovascular coupling. In this article, we first introduce the classical anatomical and physiological findings that led to the proposal of various gliovascular interaction models. Next, we touch on the technological advances in the past few decades that enabled investigations and evaluations of neuro-glio-vascular interactions in situ. We then review recent experimental findings on the roles of astrocytes in neurovascular coupling from the viewpoints of intra- and intercellular signalings in astrocytes. PMID:27130410

  16. Astrocytic glycogenolysis: mechanisms and functions.

    PubMed

    Hertz, Leif; Xu, Junnan; Song, Dan; Du, Ting; Li, Baoman; Yan, Enzhi; Peng, Liang

    2015-02-01

    Until the demonstration little more than 20 years ago that glycogenolysis occurs during normal whisker stimulation glycogenolysis was regarded as a relatively uninteresting emergency procedure. Since then, a series of important astrocytic functions has been shown to be critically dependent on glycogenolytic activity to support the signaling mechanisms necessary for these functions to operate. This applies to glutamate formation and uptake and to release of ATP as a transmitter, stimulated by other transmitters or elevated K(+) concentrations and affecting not only other astrocytes but also most other brain cells. It is also relevant for astrocytic K(+) uptake both during the period when the extracellular K(+) concentration is still elevated after neuronal excitation, and capable of stimulating glycogenolytic activity, and during the subsequent undershoot after intense neuronal activity, when glycogenolysis may be stimulated by noradrenaline. Both elevated K(+) concentrations and several transmitters, including the β-adrenergic agonist isoproterenol and vasopressin increase free cytosolic Ca(2+) concentration in astrocytes, which stimulates phosphorylase kinase so that it activates the transformation of the inactive glycogen phosphorylase a to the active phosphorylase b. Contrary to common belief cyclic AMP plays at most a facilitatory role, and only when free cytosolic Ca(2+) concentration is also increased. Cyclic AMP is not increased during activation of glycogenolysis by either elevated K(+) concentrations or the stimulation of the serotonergic 5-HT(2B) receptor. Not all agents that stimulate glycogenolysis do so by directly activating phophorylase kinase--some do so by activating processes requiring glycogenolysis, e.g. for synthesis of glutamate. PMID:24744118

  17. Different responses of astrocytes and neurons to nitric oxide: The role of glycolytically generated ATP in astrocyte protection

    PubMed Central

    Almeida, Angeles; Almeida, Julia; Bolaños, Juan P.; Moncada, Salvador

    2001-01-01

    It was recently proposed that in Jurkat cells, after inhibition of respiration by NO, glycolytically generated ATP plays a critical role in preventing the collapse of mitochondrial membrane potential (Δψm) and thus apoptotic cell death. We have investigated this observation further in primary cultures of rat cortical neurons and astrocytes—cell types that differ greatly in their glycolytic capacity. Continuous and significant (≈85%) inhibition of respiration by NO (1.4 μM at 175 μM O2) generated by [(z)-1-[2-aminoethyl]-N-[2-ammonioethyl]amino]diazen-1-ium-1,2 diolate (DETA-NO) initially (10 min) depleted ATP concentrations by ≈25% in both cell types and increased the rate of glycolysis in astrocytes but not in neurons. Activation of glycolysis in astrocytes, as judged by lactate production, prevented further ATP depletion, whereas in neurons, which do not invoke this mechanism, there was a progressive decrease in ATP concentrations over the next 60 min. During this time, there was a persistent mitochondrial hyperpolarization and absence of apoptotic cell death in astrocytes, whereas in the neurons there was a progressive fall in Δψm and increased apoptosis. After glucose deprivation or treatment with inhibitors of the F1F0-ATPase and adenine nucleotide translocase, astrocytes responded to NO with a fall in Δψm and apoptotic cell death similar to the response in neurons. Finally, although treatment of astrocytes with NO partially prevented staurosporin-induced collapse in Δψm and cell death, NO and staurosporin synergized in decreasing Δψm and inducing apoptosis in neurons. These results demonstrate that although inhibition of cellular respiration by NO leads to neurotoxicity, it may also result in initial neuroprotection, depending on the glycolytic capacity of the particular cell. PMID:11742096

  18. Targeting astrocytes in bipolar disorder.

    PubMed

    Peng, Liang; Li, Baoman; Verkhratsky, Alexei

    2016-06-01

    Astrocytes are homeostatic cells of the central nervous system, which are critical for development and maintenance of synaptic transmission and hence of synaptically connected neuronal ensembles. Astrocytic densities are reduced in bipolar disorder, and therefore deficient astroglial function may contribute to overall disbalance in neurotransmission and to pathological evolution. Classical anti-bipolar drugs (lithium salts, valproic acid and carbamazepine) affect expression of astroglial genes and modify astroglial signalling and homeostatic cascades. Many effects of both antidepressant and anti-bipolar drugs are exerted through regulation of glutamate homeostasis and glutamatergic transmission, through K(+) buffering, through regulation of calcium-dependent phospholipase A2 (that controls metabolism of arachidonic acid) or through Ca(2+) homeostatic and signalling pathways. Sometimes anti-depressant and anti-bipolar drugs exert opposite effects, and some effects on gene expression in drug treated animals are opposite in neurones vs. astrocytes. Changes in the intracellular pH induced by anti-bipolar drugs affect uptake of myo-inositol and thereby signalling via inositoltrisphosphate (InsP3), this being in accord with one of the main theories of mechanism of action for these drugs. PMID:27015045

  19. Astrocytes: Everything but the glue

    PubMed Central

    Gonzalez-Perez, Oscar; Lopez-Virgen, Veronica; Quiñones-Hinojosa, Alfredo

    2015-01-01

    The current knowledge in neuroscience indicates that neural tissue has two major cell populations: neurons and glia (term derived from the Greek word for glue). Neuronal population is characterized by the capacity to produce action potentials, whereas glial cells are typically identified as the subordinate cell population of neurons. To date, this point of view has changed dramatically and growing evidence indicates that glial cells play a crucial role in normal mental functions and the pathogenesis of neurological diseases. Classically, glial cells include four major populations clearly discernible in the adult brain: astrocytes, oligodendrocytes, microglia cells and NG2 glia. Astrocytes, also referred as to astroglia, are by far the most abundant cell lineage in the adult brain. These cells are in close contact with several tissue components of the brain parenchyma including neurons, vasculature, extracellular matrix and other glial populations. Hence, the number and strategic position of astrocytes provide them with exceptional capacity for modulating multiple functions in the neural tissue. PMID:25938129

  20. Disentangling calcium-driven astrocyte physiology.

    PubMed

    Rusakov, Dmitri A

    2015-04-01

    Astrocytes seem to rely on relatively sluggish and spatially blurred Ca(2+) waves to communicate with fast and point-precise neural circuits. This apparent discrepancy could, however, reflect our current inability to understand the microscopic mechanisms involved. Difficulties in detecting and interpreting astrocyte Ca(2+) signals may have led to some prominent controversies in the field. Here, we argue that a deeper understanding of astrocyte physiology requires a qualitative leap in our experimental and analytical strategies.

  1. The effect of resilience on posttraumatic stress disorder in trauma-exposed inner-city primary care patients.

    PubMed

    Wrenn, Glenda L; Wingo, Aliza P; Moore, Renee; Pelletier, Tiffany; Gutman, Alisa R; Bradley, Bekh; Ressler, Kerry J

    2011-07-01

    Posttraumatic stress disorder (PTSD) has previously been associated with increased risk for a variety of chronic medical conditions and it is often underdiagnosed in minority civilian populations. The current study examined the effects of resilience on the likelihood of having a diagnosis of PTSD in an inner-city sample of primary care patients (n=767). We measured resilience with the Connor-Davidson Resilience Scale, trauma with the Childhood Trauma Questionnaire and Trauma Events Inventory, and assessed for PTSD with the modified PTSD symptom scale. Multiple logistic regression model with presence/absence of PTSD as the outcome yielded 3 significant factors: childhood abuse, nonchild abuse trauma, and resilience. One type of childhood abuse in moderate to severe range (OR, 2.01; p = .0001), 2 or more types of childhood abuse in moderate to severe range (OR, 4.00; p < or = .0001), and 2 or more types of nonchildhood abuse trauma exposure (OR, 3.33; p < or = .0001), were significantly associated with an increased likelihood of PTSD, while resilience was robustly and significantly associated with a decreased likelihood of PTSD (OR, 0.93; p < or = .0001). By understanding the role of resilience in recovery from adverse experiences, improved treatment and interventional methods may be developed. Furthermore, these results suggest a role for assessing resilience in highly traumatized primary care populations as a way to better characterize risk for PTSD and direct screening/psychiatric referral efforts.

  2. Proteomic changes in chicken primary hepatocytes exposed to T-2 toxin are associated with oxidative stress and mitochondrial enhancement.

    PubMed

    Mu, Peiqiang; Xu, Ming; Zhang, Lei; Wu, Kaixin; Wu, Jun; Jiang, Jun; Chen, Qingmei; Wang, Lijuan; Tang, Xianqing; Deng, Yiqun

    2013-11-01

    T-2 toxin is a mycotoxin that is toxic to plants, animals, and humans. However, its molecular mechanism remains unclear, especially in chickens. In this study, using 2D electrophoresis with MALDI-TOF/TOF-MS, 53 proteins were identified as up- or downregulated by T-2 toxin in chicken primary hepatocytes. Functional network analysis by ingenuity pathway analysis showed that the top network altered by T-2 toxin is associated with neurological disease, cancer, organismal injury, and abnormalities. Most of the identified proteins were associated with one of eight functional classes, including cell redox homeostasis, transcriptional or translational regulation, cell cycle or cell proliferation, stress response, lipid metabolism, transport, carbohydrate metabolism, and protein degradation. Subcellular location categorization showed that the identified proteins were predominantly located in the mitochondrion (34%) and interestingly, the expression of all the identified mitochondrial proteins was increased. Further cellular analysis showed that T-2 toxin was able to induce the ROS accumulation and could lead to an increase in mitochondrial mass and adenosine 5'-triphosphate content, which indicated that oxidative stress and mitochondrial enhancement occurred in T-2 toxin-treated cells. Overall, these results characterize the global proteomic response of chicken primary hepatocytes to T-2 toxin, which may lead to a better understanding of the molecular mechanisms underlying its toxicity.

  3. Astrocytic Actions on Extrasynaptic Neuronal Currents

    PubMed Central

    Pál, Balázs

    2015-01-01

    In the last few decades, knowledge about astrocytic functions has significantly increased. It was demonstrated that astrocytes are not passive elements of the central nervous system (CNS), but active partners of neurons. There is a growing body of knowledge about the calcium excitability of astrocytes, the actions of different gliotransmitters and their release mechanisms, as well as the participation of astrocytes in the regulation of synaptic functions and their contribution to synaptic plasticity. However, astrocytic functions are even more complex than being a partner of the “tripartite synapse,” as they can influence extrasynaptic neuronal currents either by releasing substances or regulating ambient neurotransmitter levels. Several types of currents or changes of membrane potential with different kinetics and via different mechanisms can be elicited by astrocytic activity. Astrocyte-dependent phasic or tonic, inward or outward currents were described in several brain areas. Such currents, together with the synaptic actions of astrocytes, can contribute to neuromodulatory mechanisms, neurosensory and -secretory processes, cortical oscillatory activity, memory, and learning or overall neuronal excitability. This mini-review is an attempt to give a brief summary of astrocyte-dependent extrasynaptic neuronal currents and their possible functional significance. PMID:26696832

  4. Thiopental sodium preserves the responsiveness to glutamate but not acetylcholine in rat primary cultured neurons exposed to hypoxia.

    PubMed

    Morita, Tomotaka; Shibuta, Satoshi; Kosaka, Jun; Fujino, Yuji

    2016-06-15

    Although many in vitro studies demonstrated that thiopental sodium (TPS) is a promising neuroprotective agent, clinical attempts to use TPS showed mainly unsatisfactory results. We investigated the neuroprotective effects of TPS against hypoxic insults (HI), and the responses of the neurons to l-glutamate and acetylcholine application. Neurons prepared from E17 Wistar rats were used after 2weeks in culture. The neurons were exposed to 12-h HI with or without TPS. HI-induced neurotoxicity was evaluated morphologically. Moreover, we investigated the dynamics of the free intracellular calcium ([Ca(2+)]i) in the surviving neurons after HI with or without TPS pretreatment following the application of neurotransmitters. TPS was neuroprotective against HI according to the morphological examinations (0.73±0.06 vs. 0.52±0.07, P=0.04). While the response to l-glutamate was maintained (0.89±0.08 vs. 1.02±0.09, P=0.60), the [Ca(2+)]i response to acetylcholine was notably impaired (0.59±0.02 vs. 0.94±0.04, P<0.01). Though TPS to cortical cultures was neuroprotective against HI morphologically, the [Ca(2+)]i response not to l-glutamate but to acetylcholine was impaired. This may partially explain the inconsistent results regarding the neuroprotective effects of TPS between experimental studies and clinical settings. PMID:27206889

  5. Microglia trigger astrocyte-mediated neuroprotection via purinergic gliotransmission

    NASA Astrophysics Data System (ADS)

    Shinozaki, Youichi; Nomura, Masatoshi; Iwatsuki, Ken; Moriyama, Yoshinori; Gachet, Christian; Koizumi, Schuichi

    2014-03-01

    Microglia are highly sensitive to even small changes in the brain environment, such as invasion of non-hazardous toxicants or the presymptomatic state of diseases. However, the physiological or pathophysiological consequences of their responses remain unknown. Here, we report that cultured microglia sense low concentrations of the neurotoxicant methylmercury (MeHglow) and provide neuroprotection against MeHg, for which astrocytes are also required. When exposed to MeHglow, microglia exocytosed ATP via p38 MAPK- and vesicular nucleotide transporter (VNUT)-dependent mechanisms. Astrocytes responded to the microglia-derived ATP via P2Y1 receptors and released interleukin-6 (IL-6), thereby protecting neurons against MeHglow. These neuroprotective actions were also observed in organotypic hippocampal slices from wild-type mice, but not in slices prepared from VNUT knockout or P2Y1 receptor knockout mice. These findings suggest that microglia sense and respond to even non-hazardous toxicants such as MeHglow and change their phenotype into a neuroprotective one, for which astrocytic support is required.

  6. Astrocytic TIMP-1 Promotes Oligodendrocyte Differentiation and Enhances CNS Myelination

    PubMed Central

    Moore, Craig S.; Milner, Richard; Nishiyama, Akiko; Frausto, Ricardo F.; Serwanski, David R.; Pagarigan, Roberto R.; Whitton, J. Lindsay; Miller, Robert H.; Crocker, Stephen J.

    2011-01-01

    Tissue inhibitor of metalloproteinase-1 (TIMP-1) is an extracellular protein and endogenous regulator of matrix metalloproteinases (MMPs) secreted by astrocytes in response to CNS myelin injury. We have previously reported that adult TIMP-1KO mice exhibit poor myelin repair following demyelinating injury. This observation led us to hypothesize a role for TIMP-1 in oligodendrogenesis and CNS myelination. Herein, we demonstrate that compact myelin formation is significantly delayed in TIMP-1KO mice which coincided with dramatically reduced numbers of white matter astrocytes in the developing CNS. Analysis of differentiation in CNS progenitor cells (neurosphere) cultures from TIMP-1KO mice revealed a specific deficit of NG2+ oligodendrocyte progenitor cells. Application of rmTIMP-1 to TIMP-1KO neurosphere cultures evoked a dose-dependent increase in NG2+ cell numbers, while treatment with GM6001, a potent broad spectrum MMP inhibitor did not. Similarly, administration of recombinant murine TIMP-1 (rmTIMP-1) to A2B5+ immunopanned oligodendrocyte progenitors significantly increased the number of differentiated O1+ oligodendrocytes, while antisera to TIMP-1 reduced oligodendrocyte numbers. We also determined that A2B5+ oligodendrocyte progenitors grown in conditioned media derived from TIMP-1KO primary glial cultures resulted in reduced differentiation of mature O1+ oligodendrocytes. Finally, we report that addition of rmTIMP-1 to primary glial cultures resulted in a dose-dependent proliferative response of astrocytes. Together, these findings describe a previously uncharacterized role for TIMP-1 in the regulation of oligodendrocytes and astrocytes during development and provide a novel function for TIMP-1 on myelination in the developing CNS. PMID:21508247

  7. Diphenylarsinic acid increased the synthesis and release of neuroactive and vasoactive peptides in rat cerebellar astrocytes.

    PubMed

    Negishi, Takayuki; Takahashi, Masaki; Matsunaga, Yuki; Hirano, Seishiro; Tashiro, Tomoko

    2012-06-01

    An incident of poisoning occurred in Japan in 2003 when high-level contamination with arsenic, mainly diphenylarsinic acid (DPAA), was found in well water. People using this water particularly experienced cerebellar symptoms. In the present study, we investigated the adverse effects of DPAA on the cerebellum in vitro and in vivo to understand the biological mechanisms that cause cerebellar symptoms. Comprehensive gene expression analyses in primary cultured ratcerebellar cells exposed to 10 μM DPAA for 24 hours indicated significant alterations in the mRNA expression of genes encoding antioxidative stress proteins (heme oxigenase 1 and heat shock protein72) and neuroactive and vasoactive peptides (neuropeptide Y, adrenomedullin, monocyte chemoattractant protein 1, and fibroblast growth factor 2). Further analyses of proteins revealed that cultured cerebellar astrocytes expressed these antioxidative stress proteins and peptides in response to exposure to DPAA. In addition, these adverseeffects were also observed in the cerebellum exposed in vivo to DPAA (100 mg/L) for 21 days. These results suggested that cerebellarastrocytes irregularly secrete neuroactive and vasoactive peptidesagainst DPAA-induced oxidative stress, which leads to abnormal neural functions and disrupted cerebellar autoregulation dynamics and results in the onset of cerebellar symptoms.

  8. Astrocyte-to-neuron intercellular prion transfer is mediated by cell-cell contact

    PubMed Central

    Victoria, Guiliana Soraya; Arkhipenko, Alexander; Zhu, Seng; Syan, Sylvie; Zurzolo, Chiara

    2016-01-01

    Prion diseases are caused by misfolding of the cellular protein PrPC to an infectious conformer, PrPSc. Intercellular PrPSc transfer propagates conversion and allows infectivity to move from the periphery to the brain. However, how prions spread between cells of the central nervous system is unclear. Astrocytes are specialized non-neuronal cells within the brain that have a number of functions indispensable for brain homeostasis. Interestingly, they are one of the earliest sites of prion accumulation in the brain. A fundamental question arising from this observation is whether these cells are involved in intercellular prion transfer and thereby disease propagation. Using co-culture systems between primary infected astrocytes and granule neurons or neuronal cell lines, we provide direct evidence that prion-infected astrocytes can disseminate prion to neurons. Though astrocytes are capable of secreting PrP, this is an inefficient method of transferring prion infectivity. Efficient transfer required co-culturing and direct cell contact. Astrocytes form numerous intercellular connections including tunneling nanotubes, containing PrPSc, often colocalized with endolysosomal vesicles, which may constitute the major mechanism of transfer. Because of their role in intercellular transfer of prions astrocytes may influence progression of the disease. PMID:26857744

  9. STIM1 and Orai1 mediate thrombin-induced Ca(2+) influx in rat cortical astrocytes.

    PubMed

    Moreno, Claudia; Sampieri, Alicia; Vivas, Oscar; Peña-Segura, Claudia; Vaca, Luis

    2012-12-01

    In astrocytes, thrombin leads to cytoplasmic Ca(2+) elevations modulating a variety of cytoprotective and cytotoxic responses. Astrocytes respond to thrombin stimulation with a biphasic Ca(2+) increase generated by an interplay between ER-Ca(2+) release and store-operated Ca(2+) entry (SOCE). In many cell types, STIM1 and Orai1 have been demonstrated to be central components of SOCE. STIM1 senses the ER-Ca(2+) depletion and binds Orai1 to activate Ca(2+) influx. Here we used immunocytochemistry, overexpression and siRNA assays to investigate the role of STIM1 and Orai1 in the thrombin-induced Ca(2+) response in primary cultures of rat cortical astrocytes. We found that STIM1 and Orai1 are endogenously expressed in cortical astrocytes and distribute accordingly with other mammalian cells. Importantly, native and overexpressed STIM1 reorganized in puncta under thrombin stimulation and this reorganization was reversible. In addition, the overexpression of STIM1 and Orai1 increased by twofold the Ca(2+) influx evoked by thrombin, while knockdown of endogenous STIM1 and Orai1 significantly decreased this Ca(2+) influx. These results indicate that STIM1 and Orai1 underlie an important fraction of the Ca(2+) response that astrocytes exhibit in the presence of thrombin. Thrombin stimulation in astrocytes leads to ER-Ca(2+) release which causes STIM1 reorganization allowing the activation of Orai1 and the subsequent Ca(2+) influx.

  10. Differential Acute and Chronic Effects of Leptin on Hypothalamic Astrocyte Morphology and Synaptic Protein Levels

    PubMed Central

    García-Cáceres, Cristina; Fuente-Martín, Esther; Burgos-Ramos, Emma; Granado, Miriam; Frago, Laura M.; Barrios, Vicente; Horvath, Tamas

    2011-01-01

    Astrocytes participate in neuroendocrine functions partially through modulation of synaptic input density in the hypothalamus. Indeed, glial ensheathing of neurons is modified by specific hormones, thus determining the availability of neuronal membrane space for synaptic inputs, with the loss of this plasticity possibly being involved in pathological processes. Leptin modulates synaptic inputs in the hypothalamus, but whether astrocytes participate in this action is unknown. Here we report that astrocyte structural proteins, such as glial fibrillary acidic protein (GFAP) and vimentin, are induced and astrocyte morphology modified by chronic leptin administration (intracerebroventricular, 2 wk), with these changes being inversely related to modifications in synaptic protein densities. Similar changes in glial structural proteins were observed in adult male rats that had increased body weight and circulating leptin levels due to neonatal overnutrition (overnutrition: four pups/litter vs. control: 12 pups/litter). However, acute leptin treatment reduced hypothalamic GFAP levels and induced synaptic protein levels 1 h after administration, with no effect on vimentin. In primary hypothalamic astrocyte cultures leptin also reduced GFAP levels at 1 h, with an induction at 24 h, indicating a possible direct effect of leptin. Hence, one mechanism by which leptin may affect metabolism is by modifying hypothalamic astrocyte morphology, which in turn could alter synaptic inputs to hypothalamic neurons. Furthermore, the responses to acute and chronic leptin exposure are inverse, raising the possibility that increased glial activation in response to chronic leptin exposure could be involved in central leptin resistance. PMID:21343257

  11. Dynamics of β-adrenergic/cAMP signaling and morphological changes in cultured astrocytes.

    PubMed

    Vardjan, Nina; Kreft, Marko; Zorec, Robert

    2014-04-01

    The morphology of astrocytes, likely regulated by cAMP, determines the structural association between astrocytes and the synapse, consequently modulating synaptic function. β-Adrenergic receptors (β-AR), which increase cytosolic cAMP concentration ([cAMP]i ), may affect cell morphology. However, the real-time dynamics of β-AR-mediated cAMP signaling in single live astrocytes and its effect on cell morphology have not been studied. We used the fluorescence resonance energy transfer (FRET)-based cAMP biosensor Epac1-camps to study time-dependent changes in [cAMP]i ; morphological changes in primary rat astrocytes were monitored by real-time confocal microscopy. Stimulation of β-AR by adrenaline, noradrenaline, and isoprenaline, a specific agonist of β-AR, rapidly increased [cAMP]i (∼15 s). The FRET signal response, mediated via β-AR, was faster than in the presence of forskolin (twofold) and dibutyryl-cAMP (>35-fold), which directly activate adenylyl cyclase and Epac1-camps, respectively, likely due to slow entry of these agents into the cytosol. Oscillations in [cAMP]i have not been recorded, indicating that cAMP-dependent processes operate in a slow time domain. Most Epac1-camps expressing astrocytes revealed a morphological change upon β-AR activation and attained a stellate morphology within 1 h. The morphological changes exhibited a bell-shaped dependency on [cAMP]i . The 5-10% decrease in cell cross-sectional area and the 30-50% increase in cell perimeter are likely due to withdrawal of the cytoplasm to the perinuclear region and the appearance of protrusions on the surface of astrocytes. Because astrocyte processes ensheath neurons, β-AR/cAMP-mediated morphological changes can modify the geometry of the extracellular space, affecting synaptic, neuronal, and astrocyte functions in health and disease. PMID:24464905

  12. Globular adiponectin induces a pro-inflammatory response in human astrocytic cells

    SciTech Connect

    Wan, Zhongxiao; Mah, Dorrian; Simtchouk, Svetlana; Klegeris, Andis; Little, Jonathan P.

    2014-03-28

    Highlights: • Adiponectin receptors are expressed in human astrocytes. • Globular adiponectin induces secretion of IL-6 and MCP-1 from cultured astrocytes. • Adiponectin may play a pro-inflammatory role in astrocytes. - Abstract: Neuroinflammation, mediated in part by activated brain astrocytes, plays a critical role in the development of neurodegenerative disorders, including Alzheimer’s disease (AD). Adiponectin is the most abundant adipokine secreted from adipose tissue and has been reported to exert both anti- and pro-inflammatory effects in peripheral tissues; however, the effects of adiponectin on astrocytes remain unknown. Shifts in peripheral concentrations of adipokines, including adiponectin, could contribute to the observed link between midlife adiposity and increased AD risk. The aim of the present study was to characterize the effects of globular adiponectin (gAd) on pro-inflammatory cytokine mRNA expression and secretion in human U373 MG astrocytic cells and to explore the potential involvement of nuclear factor (NF)-κB, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and phosphatidylinositide 3-kinases (PI3 K) signaling pathways in these processes. We demonstrated expression of adiponectin receptor 1 (adipoR1) and adipoR2 in U373 MG cells and primary human astrocytes. gAd induced secretion of interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1, and gene expression of IL-6, MCP-1, IL-1β and IL-8 in U373 MG cells. Using specific inhibitors, we found that NF-κB, p38MAPK and ERK1/2 pathways are involved in gAd-induced induction of cytokines with ERK1/2 contributing the most. These findings provide evidence that gAd may induce a pro-inflammatory phenotype in human astrocytes.

  13. The NRTIs Lamivudine, Stavudine and Zidovudine Have Reduced HIV-1 Inhibitory Activity in Astrocytes

    PubMed Central

    Gray, Lachlan R.; Tachedjian, Gilda; Ellett, Anne M.; Roche, Michael J.; Cheng, Wan-Jung; Guillemin, Gilles J.; Brew, Bruce J.; Turville, Stuart G.; Wesselingh, Steve L.; Gorry, Paul R.; Churchill, Melissa J.

    2013-01-01

    HIV-1 establishes infection in astrocytes and macroage-lineage cells of the central nervous system (CNS). Certain antiretroviral drugs (ARVs) can penetrate the CNS, and are therefore often used in neurologically active combined antiretroviral therapy (Neuro-cART) regimens, but their relative activity in the different susceptible CNS cell populations is unknown. Here, we determined the HIV-1 inhibitory activity of CNS-penetrating ARVs in astrocytes and macrophage-lineage cells. Primary human fetal astrocytes (PFA) and the SVG human astrocyte cell line were used as in vitro models for astrocyte infection, and monocyte-derived macrophages (MDM) were used as an in vitro model for infection of macrophage-lineage cells. The CNS-penetrating ARVs tested were the nucleoside reverse transcriptase inhibitors (NRTIs) abacavir (ABC), lamivudine (3TC), stavudine (d4T) and zidovudine (ZDV), the non-NRTIs efavirenz (EFV), etravirine (ETR) and nevirapine (NVP), and the integrase inhibitor raltegravir (RAL). Drug inhibition assays were performed using single-round HIV-1 entry assays with luciferase viruses pseudotyped with HIV-1 YU-2 envelope or vesicular stomatitis virus G protein (VSV-G). All the ARVs tested could effectively inhibit HIV-1 infection in macrophages, with EC90s below concentrations known to be achievable in the cerebral spinal fluid (CSF). Most of the ARVs had similar potency in astrocytes, however the NRTIs 3TC, d4T and ZDV had insufficient HIV-1 inhibitory activity in astrocytes, with EC90s 12-, 187- and 110-fold greater than achievable CSF concentrations, respectively. Our data suggest that 3TC, d4T and ZDV may not adequately target astrocyte infection in vivo, which has potential implications for their inclusion in Neuro-cART regimens. PMID:23614033

  14. Protein interacting with C kinase 1 suppresses invasion and anchorage-independent growth of astrocytic tumor cells

    PubMed Central

    Cockbill, Louisa M. R.; Murk, Kai; Love, Seth; Hanley, Jonathan G.

    2015-01-01

    Astrocytic tumors are the most common form of primary brain tumor. Astrocytic tumor cells infiltrate the surrounding CNS tissue, allowing them to evade removal upon surgical resection of the primary tumor. Dynamic changes to the actin cytoskeleton are crucial to cancer cell invasion, but the specific mechanisms that underlie the particularly invasive phenotype of astrocytic tumor cells are unclear. Protein interacting with C kinase 1 (PICK1) is a PDZ and BAR domain–containing protein that inhibits actin-related protein 2/3 (Arp2/3)-dependent actin polymerization and is involved in regulating the trafficking of a number of cell-surface receptors. Here we report that, in contrast to other cancers, PICK1 expression is down-regulated in grade IV astrocytic tumor cell lines and also in clinical cases of the disease in which grade IV tumors have progressed from lower-grade tumors. Exogenous expression of PICK1 in the grade IV astrocytic cell line U251 reduces their capacity for anchorage-independent growth, two-dimensional migration, and invasion through a three-dimensional matrix, strongly suggesting that low PICK1 expression plays an important role in astrocytic tumorigenesis. We propose that PICK1 negatively regulates neoplastic infiltration of astrocytic tumors and that manipulation of PICK1 is an attractive possibility for therapeutic intervention. PMID:26466675

  15. Astrocytes and Developmental White Matter Disorders

    ERIC Educational Resources Information Center

    Sen, Ellora; Levison, Steven W.

    2006-01-01

    There is an increasing awareness that the astrocytes in the immature periventricular white matter are vulnerable to ischemia and respond to inflammation. Here we provide a synopsis of the articles that have evaluated the causes and consequences of developmental brain injuries to white matter astrocytes as well as the consequences of several…

  16. Nitric Oxide in Astrocyte-Neuron Signaling

    SciTech Connect

    Nianzhen Li

    2002-06-27

    Astrocytes, a subtype of glial cell, have recently been shown to exhibit Ca{sup 2+} elevations in response to neurotransmitters. A Ca{sup 2+} elevation can propagate to adjacent astrocytes as a Ca{sup 2+} wave, which allows an astrocyte to communicate with its neighbors. Additionally, glutamate can be released from astrocytes via a Ca{sup 2+}-dependent mechanism, thus modulating neuronal activity and synaptic transmission. In this dissertation, the author investigated the roles of another endogenous signal, nitric oxide (NO), in astrocyte-neuron signaling. First the author tested if NO is generated during astrocytic Ca{sup 2+} signaling by imaging NO in purified murine cortical astrocyte cultures. Physiological concentrations of a natural messenger, ATP, caused a Ca{sup 2+}-dependent NO production. To test the roles of NO in astrocytic Ca{sup 2+} signaling, the author applied NO to astrocyte cultures via addition of a NO donor, S-nitrosol-N-acetylpenicillamine (SNAP). NO induced an influx of external Ca{sup 2+}, possibly through store-operated Ca{sup 2+} channels. The NO-induced Ca{sup 2+} signaling is cGMP-independent since 8-Br-cGMP, an agonistic analog of cGMP, did not induce a detectable Ca{sup 2+} change. The consequence of this NO-induced Ca{sup 2+} influx was assessed by simultaneously monitoring of cytosolic and internal store Ca{sup 2+} using fluorescent Ca{sup 2+} indicators x-rhod-1 and mag-fluo-4. Blockage of NO signaling with the NO scavenger PTIO significantly reduced the refilling percentage of internal stores following ATP-induced Ca{sup 2+} release, suggesting that NO modulates internal store refilling. Furthermore, locally photo-release of NO to a single astrocyte led to a Ca{sup 2+} elevation in the stimulated astrocyte and a subsequent Ca{sup 2+} wave to neighbors. Finally, the author tested the role of NO inglutamate-mediated astrocyte-neuron signaling by recording the astrocyte-evoked glutamate-dependent neuronal slow inward current (SIC

  17. Knockdown of apoptosis signal-regulating kinase 1 affects ischaemia-induced astrocyte activation and glial scar formation.

    PubMed

    Cheon, So Yeong; Cho, Kyoung Joo; Song, Juhyun; Kim, Gyung Whan

    2016-04-01

    Reactive astrocytes play an essential role in determining the tissue response to ischaemia. Formation of a glial scar can block the neuronal outgrowth that is required for restoration of damaged tissue. Therefore, regulation of astrocyte activation is important; however, the mediator of this process has not been fully elucidated. Apoptosis signal-regulating kinase 1 (ASK1) is an early responder to oxidative stress, and plays a pivotal role in the intracellular signalling pathway of apoptosis, inflammation, and differentiation. To confirm whether ASK1 mediates astrocyte activation and leads to glial scar formation after cerebral ischaemia, we conducted in vivo and in vitro experiments. C57BL/6 mice were subjected to occlusion of the middle cerebral artery, and astrocyte cultures were exposed to oxygen-glucose deprivation. After silencing of ASK1 , astrocyte-associated genes were downregulated, as seen with the use of microarrays. The glial fibrillary acidic protein (GFAP) level was decreased, and correlated with the reduction in the ASK1 level. In astrocytes, reduction in the ASK1 level decreased the activity of the p38 pathway, and the levels of transcription factors for GFAP and GFAP transcripts after hypoxia. In the chronic phase, ASK1 depletion reduced glial scar formation and conserved neuronal structure, which may lead to better functional recovery. These data suggest that ASK1 may be an important mediator of ischaemia-induced astrocyte activation and scar formation, and could provide a potential therapeutic target for treatment after ischaemic stroke. PMID:26797817

  18. Exposure to high glutamate concentration activates aerobic glycolysis but inhibits ATP-linked respiration in cultured cortical astrocytes.

    PubMed

    Shen, Yao; Tian, Yueyang; Shi, Xiaojie; Yang, Jianbo; Ouyang, Li; Gao, Jieqiong; Lu, Jianxin

    2014-08-01

    Astrocytes play a key role in removing the synaptically released glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. However, high concentration of glutamate leads to toxicity in astrocytes, and the underlying mechanisms are unclear. The purpose of this study was to investigate whether energy metabolism disorder, especially impairment of mitochondrial respiration, is involved in the glutamate-induced gliotoxicity. Exposure to 10-mM glutamate for 48 h stimulated glycolysis and respiration in astrocytes. However, the increased oxygen consumption was used for proton leak and non-mitochondrial respiration, but not for oxidative phosphorylation and ATP generation. When the exposure time extended to 72 h, glycolysis was still activated for ATP generation, but the mitochondrial ATP-linked respiration of astrocytes was reduced. The glutamate-induced astrocyte damage can be mimicked by the non-metabolized substrate d-aspartate but reversed by the non-selective glutamate transporter inhibitor TBOA. In addition, the glutamate toxicity can be partially reversed by vitamin E. These findings demonstrate that changes of bioenergetic profile occur in cultured cortical astrocytes exposed to high concentration of glutamate and highlight the role of mitochondria respiration in glutamate-induced gliotoxicity in cortical astrocytes.

  19. Large-scale recording of astrocyte activity

    PubMed Central

    Nimmerjahn, Axel; Bergles, Dwight E.

    2015-01-01

    Astrocytes are highly ramified glial cells found throughout the central nervous system (CNS). They express a variety of neurotransmitter receptors that can induce widespread chemical excitation, placing these cells in an optimal position to exert global effects on brain physiology. However, the activity patterns of only a small fraction of astrocytes have been examined and techniques to manipulate their behavior are limited. As a result, little is known about how astrocytes modulate CNS function on synaptic, microcircuit, or systems levels. Here, we review current and emerging approaches for visualizing and manipulating astrocyte activity in vivo. Deciphering how astrocyte network activity is controlled in different physiological and pathological contexts is critical for defining their roles in the healthy and diseased CNS. PMID:25665733

  20. Probing the Complexities of Astrocyte Calcium Signaling.

    PubMed

    Shigetomi, Eiji; Patel, Sandip; Khakh, Baljit S

    2016-04-01

    Astrocytes are abundant glial cells that tile the entire central nervous system and mediate well-established functions for neurons, blood vessels, and other glia. These ubiquitous cells display intracellular Ca(2+) signals, which have been intensely studied for 25 years. Recently, the use of improved methods has unearthed the panoply of astrocyte Ca(2+) signals and a variable landscape of basal Ca(2+) levels. In vivo studies have started to reveal the settings under which astrocytes display behaviorally relevant Ca(2+) signaling. Studies in mice have emphasized how astrocyte Ca(2+) signaling is altered in distinct neurodegenerative diseases. Progress in the past few years, fueled by methodological advances, has thus reignited interest in astrocyte Ca(2+) signaling for brain function and dysfunction. PMID:26896246

  1. Three-Dimensional Environment Sustains Morphological Heterogeneity and Promotes Phenotypic Progression During Astrocyte Development.

    PubMed

    Balasubramanian, Swarnalatha; Packard, John A; Leach, Jennie B; Powell, Elizabeth M

    2016-06-01

    Astrocytes are critical for coordinating normal brain function by regulating brain metabolic homeostasis, synaptogenesis and neurotransmission, and blood-brain barrier permeability and maintenance. Dysregulation of normal astrocyte ontogeny contributes to neurodevelopmental and neurodegenerative disorders, epilepsies, and adverse responses to injury. To achieve these multiple essential roles, astrocyte phenotypes are regionally, morphologically, and functionally heterogeneous. Therefore, the best regenerative medicine strategies may require selective production of distinct astrocyte subpopulations at defined maturation levels. However, little is known about the mechanisms that direct astrocyte diversity or whether heterogeneity is represented in biomaterials. In vitro studies report lack of normal morphologies and overrepresentation of the glial scar type of reactive astrocyte morphology and expression of markers, questioning how well the in vitro astrocytes represent glia in vivo and whether in vitro tissue engineering methods are suitable for regenerative medicine applications. Our previous work with neurons suggests that the three-dimensional (3D) environment, when compared with standard two-dimensional (2D) substrate, yields cellular and molecular behaviors that more closely approximately normal ontogeny. To specifically study the effects of dimensionality, we used purified glial fibrillary acidic protein (GFAP)-expressing primary cerebral cortical astrocyte cultures from single pups and characterized the cellular maturation profiles in 2D and 3D milieu. We identified four morphological groups in vitro: round, bipolar, stellate, and putative perivascular. In the 3D hydrogel culture environment, postnatal astrocytes transitioned from a population of nearly all round cells and very few bipolar cells toward a population with significant fractions of round, stellate, and putative perivascular cells within a few days, following the in vivo ontogeny. In 2D, however

  2. Acamprosate {monocalcium bis(3-acetamidopropane-1-sulfonate)} reduces ethanol-drinking behavior in rats and glutamate-induced toxicity in ethanol-exposed primary rat cortical neuronal cultures.

    PubMed

    Oka, Michiko; Hirouchi, Masaaki; Tamura, Masaru; Sugahara, Seishi; Oyama, Tatsuya

    2013-10-15

    Acamprosate, the calcium salt of bis(3-acetamidopropane-1-sulfonate), contributes to the maintenance of abstinence in alcohol-dependent patients, but its mechanism of action in the central nervous system is unclear. Here, we report the effect of acamprosate on ethanol-drinking behavior in standard laboratory Wistar rats, including voluntary ethanol consumption and the ethanol-deprivation effect. After forced ethanol consumption arranged by the provision of only one drinking bottle containing 10% ethanol, the rats were given a choice between two drinking bottles, one containing water and the other containing 10% ethanol. In rats selected for high ethanol preference, repeated oral administration of acamprosate diminished voluntary ethanol drinking. After three months of continuous access to two bottles, rats were deprived of ethanol for three days and then presented with two bottles again. After ethanol deprivation, ethanol preference was increased, and the increase was largely abolished by acamprosate. After exposure of primary neuronal cultures of rat cerebral cortex to ethanol for four days, neurotoxicity, as measured by the extracellular leakage of lactate dehydrogenase (LDH), was induced by incubation with glutamate for 1h followed by incubation in the absence of ethanol for 24h. The N-methyl-D-aspartate receptor blocker 5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine, the metabotropic glutamate receptor subtype 5 antagonist 6-methyl-2-(phenylethynyl)pyridine and the voltage-gated calcium-channel blocker nifedipine all inhibited glutamate-induced LDH leakage from ethanol-exposed neurons. Acamprosate inhibited the glutamate-induced LDH leakage from ethanol-exposed neurons more strongly than that from intact neurons. In conclusion, acamprosate showed effective reduction of drinking behavior in rats and protected ethanol-exposed neurons by multiple blocking of glutamate signaling.

  3. Astrocytic αVβ3 Integrin Inhibits Neurite Outgrowth and Promotes Retraction of Neuronal Processes by Clustering Thy-1

    PubMed Central

    Herrera-Molina, Rodrigo; Frischknecht, Renato; Maldonado, Horacio; Seidenbecher, Constanze I.; Gundelfinger, Eckart D.; Hetz, Claudio; Aylwin, María de la Luz; Schneider, Pascal; Quest, Andrew F. G.; Leyton, Lisette

    2012-01-01

    Thy-1 is a membrane glycoprotein suggested to stabilize or inhibit growth of neuronal processes. However, its precise function has remained obscure, because its endogenous ligand is unknown. We previously showed that Thy-1 binds directly to αVβ3 integrin in trans eliciting responses in astrocytes. Nonetheless, whether αVβ3 integrin might also serve as a Thy-1-ligand triggering a neuronal response has not been explored. Thus, utilizing primary neurons and a neuron-derived cell line CAD, Thy-1-mediated effects of αVβ3 integrin on growth and retraction of neuronal processes were tested. In astrocyte-neuron co-cultures, endogenous αVβ3 integrin restricted neurite outgrowth. Likewise, αVβ3-Fc was sufficient to suppress neurite extension in Thy-1(+), but not in Thy-1(−) CAD cells. In differentiating primary neurons exposed to αVβ3-Fc, fewer and shorter dendrites were detected. This effect was abolished by cleavage of Thy-1 from the neuronal surface using phosphoinositide-specific phospholipase C (PI-PLC). Moreover, αVβ3-Fc also induced retraction of already extended Thy-1(+)-axon-like neurites in differentiated CAD cells as well as of axonal terminals in differentiated primary neurons. Axonal retraction occurred when redistribution and clustering of Thy-1 molecules in the plasma membrane was induced by αVβ3 integrin. Binding of αVβ3-Fc was detected in Thy-1 clusters during axon retraction of primary neurons. Moreover, αVβ3-Fc-induced Thy-1 clustering correlated in time and space with redistribution and inactivation of Src kinase. Thus, our data indicates that αVβ3 integrin is a ligand for Thy-1 that upon binding not only restricts the growth of neurites, but also induces retraction of already existing processes by inducing Thy-1 clustering. We propose that these events participate in bi-directional astrocyte-neuron communication relevant to axonal repair after neuronal damage. PMID:22479590

  4. Macrophages and CD4+ T lymphocytes from two multiply exposed, uninfected individuals resist infection with primary non-syncytium-inducing isolates of human immunodeficiency virus type 1.

    PubMed Central

    Connor, R I; Paxton, W A; Sheridan, K E; Koup, R A

    1996-01-01

    Despite multiple, high-risk sexual exposures, some individuals remain uninfected with human immunodeficiency virus type 1 (HIV-1). CD4+ lymphocytes from these individuals are less susceptible to infection in vitro with some strains of HIV-1, suggesting that the phenotype of the virus may influence its ability to interact with certain CD4+ cells. In the present study, we examined the susceptibility of CD4+ T lymphocytes and macrophages from two exposed uninfected individuals (EU2 and EU3) to infection with a panel of biologically cloned isolates of HIV-1 having either a non-syncytium-inducing (NSI) or a syncytium-inducing (SI) phenotype. Our results indicate that CD4+ T lymphocytes from EU2 and EU3 are resistant to infection with NSI isolates of HIV-1 but are susceptible to infection with primary SI isolates. In addition, we found that macrophages from EU2 and EU3 are resistant to infection with both NSI and SI isolates. The latter finding was confirmed by using several uncloned NSI and SI isolates obtained from patients during acute HIV-1 infection. In further experiments, env clones encoding glycoproteins characteristic of NSI or SI viruses were used in single-cycle infectivity assays to evaluate infection of CD4+ lymphocytes and macrophages from EU2 and EU3. Consistent with our previous results, we found that macrophages from these individuals are resistant to infection with NSI and SI env-pseudotyped viruses, while CD4+ T lymphocytes are resistant to NSI, but not SI, pseudotyped viruses. Overall, our results demonstrate that CD4+ cells from two exposed uninfected individuals resist infection in vitro with primary, macrophage-tropic, NSI isolates of HIV-1, which is the predominant viral phenotype found following HIV-1 transmission. Furthermore, infection with NSI isolates was blocked in both CD4+ T lymphocytes and macrophages from these individuals, suggesting that there may be a common mechanism for resistance in both cell types. PMID:8971004

  5. Astrocytes as new targets to improve cognitive functions.

    PubMed

    Dallérac, Glenn; Rouach, Nathalie

    2016-09-01

    Astrocytes are now viewed as key elements of brain wiring as well as neuronal communication. Indeed, they not only bridge the gap between metabolic supplies by blood vessels and neurons, but also allow fine control of neurotransmission by providing appropriate signaling molecules and insulation through a tight enwrapping of synapses. Recognition that astroglia is essential to neuronal communication is nevertheless fairly recent and the large body of evidence dissecting such role has focused on the synaptic level by identifying neuro- and gliotransmitters uptaken and released at synaptic or extrasynaptic sites. Yet, more integrated research deciphering the impact of astroglial functions on neuronal network activity have led to the reasonable assumption that the role of astrocytes in supervising synaptic activity translates in influencing neuronal processing and cognitive functions. Several investigations using recent genetic tools now support this notion by showing that inactivating or boosting astroglial function directly affects cognitive abilities. Accordingly, brain diseases resulting in impaired cognitive functions have seen their physiopathological mechanisms revisited in light of this primary protagonist of brain processing. We here provide a review of the current knowledge on the role of astrocytes in cognition and in several brain diseases including neurodegenerative disorders, psychiatric illnesses, as well as other conditions such as epilepsy. Potential astroglial therapeutic targets are also discussed. PMID:26969413

  6. The Role of TLR4 and Fyn Interaction on Lipopolysaccharide-Stimulated PAI-1 Expression in Astrocytes.

    PubMed

    Ko, Hyun Myung; Lee, Sung Hoon; Kim, Ki Chan; Joo, So Hyun; Choi, Wahn Soo; Shin, Chan Young

    2015-08-01

    Plasminogen activator inhibitor-1 (PAI-1) is an endogenous inhibitor of tissue plasminogen activator (tPA) that acts as a neuromodulator in various neurophysiological and pathological conditions. Several researchers including us reported the induction of PAI-1 during inflammatory condition; however, the mechanism regulating PAI-1 induction is not yet clear. In this study, we investigated the role of non-receptor tyrosine kinase Fyn in the regulation of lipopolysaccharide (LPS)-induced upregulation of PAI-1 in rat primary astrocyte. The activation of toll-like receptor 4 (TLR4) signaling, induced by its ligand LPS, stimulated a physical interaction between TLR4 and Fyn along with phosphorylation of tyrosine residue in both molecules as determined by co-immunoprecipitation experiments. Immunofluorescence staining also showed increased co-localization of TLR4-Fyn on cultured rat primary astrocytes after LPS treatment. The increased TRLR4-Fyn interaction induced expression of PAI-1 through the activation of PI3k/Akt/NFĸB pathway. Treatment with Src kinase inhibitor (PP2) or transfection of Fyn small interfering RNA (siRNA) into cultured rat primary astrocytes inhibited phosphorylation of tyrosine residue of TLR4 and blocked the interaction between TLR4 and Fyn resulting to the inhibition of LPS-induced expression of PAI-1. The activation of PI3K/Akt/NFĸB signaling cascades was also inhibited by Fyn knockdown in rat primary astrocytes. The induction of PAI-1 in rat primary astrocytes, which resulted in downregulation of tPA activity in culture supernatants, inhibited neurite outgrowth in cultured rat primary cortical neuron. The inhibition of neurite extension was prevented by PP2 or Fyn siRNA treatment in rat primary astrocytes. These results suggest the critical physiological role of TRL4-Fyn interaction in the modulation of PAI-1-tPA axis in astrocytes during neuroinflammatory responses such as ischemia/reperfusion injuries.

  7. Involvement of connexin43 in the infrasonic noise-induced glutamate release by cultured astrocytes.

    PubMed

    Jiang, Shan; Wang, Yong-Qiang; Xu, Cheng-Feng; Li, Ya-Na; Guo, Rong; Li, Ling

    2014-05-01

    Infrasonic noise/infrasound is a type of environmental noise that threatens public health as a nonspecific biological stressor. Glutamate-related excitotoxicity is thought to be responsible for infrasound-induced impairment of learning and memory. In addition to neurons, astrocytes are also capable of releasing glutamate. In the present study, to identify the effect of infrasound on astroglial glutamate release, cultured astrocytes were exposed to infrasound at 16 Hz, 130 dB for different times. We found that infrasound exposure caused a significant increase in glutamate levels in the extracellular fluid. Moreover, blocking the connexin43 (Cx43) hemichannel or gap junction, decreasing the probability of Cx43 being open or inhibiting of Cx43 expression blocked this increase. The results suggest that glutamate release by Cx43 hemichannels/gap junctions is involved in the response of cultured astrocytes to infrasound. PMID:24634254

  8. The pathophysiological role of astrocytic endothelin-1.

    PubMed

    Hostenbach, Stéphanie; D'haeseleer, Miguel; Kooijman, Ron; De Keyser, Jacques

    2016-09-01

    In the normal central nervous system, endothelin-1 (ET-1) is found in some types of neurons, epithelial cells of the choroid plexus, and endothelial cells of microvessels, but it is usually not detectable in glial cells. However, in different pathological conditions, astrocytes adapting a reactive phenotype express high levels of ET-1 and its receptors, mainly the ETB receptor. ET-1 released by reactive astrocytes appears mainly to have neurodeleterious effects by mechanisms that include constriction of cerebral arterioles leading to impairment of the cerebral microcirculation, increase of blood brain barrier permeability, inflammation, excitotoxicity, impairment of fast axonal transport, and astrogliosis. A few studies in rodents found that ET-1 increased the astrocytic expression of brain-derived neurotrophic factor, glial cell-line derived neurotrophic factor and neurotropin-3, and the production of endocannabinoids. However, whether this occurs in physiological or pathological conditions is unclear. This review summarizes current knowledge about the role of the astrocytic ET-1 system in acute and chronic neurological conditions, including multiple sclerosis, ischemic stroke and hypoxic/ischemic brain injury, traumatic brain injury, subarachnoid hemorrhage, Alzheimer's disease, Binswanger's disease and post-stroke dementia, amyotrophic lateral sclerosis, and CNS infections. Counteracting the harmful effects of astrocytic ET-1 may represent a promising therapeutic target for mitigating secondary brain damage in a variety of neurological diseases. We also briefly address the role of astrocytic ET-1 in astrocytic tumors and pain. PMID:27132521

  9. Astrocytes regulate cortical state switching in vivo.

    PubMed

    Poskanzer, Kira E; Yuste, Rafael

    2016-05-10

    The role of astrocytes in neuronal function has received increasing recognition, but disagreement remains about their function at the circuit level. Here we use in vivo two-photon calcium imaging of neocortical astrocytes while monitoring the activity state of the local neuronal circuit electrophysiologically and optically. We find that astrocytic calcium activity precedes spontaneous circuit shifts to the slow-oscillation-dominated state, a neocortical rhythm characterized by synchronized neuronal firing and important for sleep and memory. Further, we show that optogenetic activation of astrocytes switches the local neuronal circuit to this slow-oscillation state. Finally, using two-photon imaging of extracellular glutamate, we find that astrocytic transients in glutamate co-occur with shifts to the synchronized state and that optogenetically activated astrocytes can generate these glutamate transients. We conclude that astrocytes can indeed trigger the low-frequency state of a cortical circuit by altering extracellular glutamate, and therefore play a causal role in the control of cortical synchronizations. PMID:27122314

  10. Astrocytes regulate cortical state switching in vivo

    PubMed Central

    Poskanzer, Kira E.; Yuste, Rafael

    2016-01-01

    The role of astrocytes in neuronal function has received increasing recognition, but disagreement remains about their function at the circuit level. Here we use in vivo two-photon calcium imaging of neocortical astrocytes while monitoring the activity state of the local neuronal circuit electrophysiologically and optically. We find that astrocytic calcium activity precedes spontaneous circuit shifts to the slow-oscillation–dominated state, a neocortical rhythm characterized by synchronized neuronal firing and important for sleep and memory. Further, we show that optogenetic activation of astrocytes switches the local neuronal circuit to this slow-oscillation state. Finally, using two-photon imaging of extracellular glutamate, we find that astrocytic transients in glutamate co-occur with shifts to the synchronized state and that optogenetically activated astrocytes can generate these glutamate transients. We conclude that astrocytes can indeed trigger the low-frequency state of a cortical circuit by altering extracellular glutamate, and therefore play a causal role in the control of cortical synchronizations. PMID:27122314

  11. Artificial astrocytes improve neural network performance.

    PubMed

    Porto-Pazos, Ana B; Veiguela, Noha; Mesejo, Pablo; Navarrete, Marta; Alvarellos, Alberto; Ibáñez, Oscar; Pazos, Alejandro; Araque, Alfonso

    2011-01-01

    Compelling evidence indicates the existence of bidirectional communication between astrocytes and neurons. Astrocytes, a type of glial cells classically considered to be passive supportive cells, have been recently demonstrated to be actively involved in the processing and regulation of synaptic information, suggesting that brain function arises from the activity of neuron-glia networks. However, the actual impact of astrocytes in neural network function is largely unknown and its application in artificial intelligence remains untested. We have investigated the consequences of including artificial astrocytes, which present the biologically defined properties involved in astrocyte-neuron communication, on artificial neural network performance. Using connectionist systems and evolutionary algorithms, we have compared the performance of artificial neural networks (NN) and artificial neuron-glia networks (NGN) to solve classification problems. We show that the degree of success of NGN is superior to NN. Analysis of performances of NN with different number of neurons or different architectures indicate that the effects of NGN cannot be accounted for an increased number of network elements, but rather they are specifically due to astrocytes. Furthermore, the relative efficacy of NGN vs. NN increases as the complexity of the network increases. These results indicate that artificial astrocytes improve neural network performance, and established the concept of Artificial Neuron-Glia Networks, which represents a novel concept in Artificial Intelligence with implications in computational science as well as in the understanding of brain function. PMID:21526157

  12. Artificial Astrocytes Improve Neural Network Performance

    PubMed Central

    Porto-Pazos, Ana B.; Veiguela, Noha; Mesejo, Pablo; Navarrete, Marta; Alvarellos, Alberto; Ibáñez, Oscar; Pazos, Alejandro; Araque, Alfonso

    2011-01-01

    Compelling evidence indicates the existence of bidirectional communication between astrocytes and neurons. Astrocytes, a type of glial cells classically considered to be passive supportive cells, have been recently demonstrated to be actively involved in the processing and regulation of synaptic information, suggesting that brain function arises from the activity of neuron-glia networks. However, the actual impact of astrocytes in neural network function is largely unknown and its application in artificial intelligence remains untested. We have investigated the consequences of including artificial astrocytes, which present the biologically defined properties involved in astrocyte-neuron communication, on artificial neural network performance. Using connectionist systems and evolutionary algorithms, we have compared the performance of artificial neural networks (NN) and artificial neuron-glia networks (NGN) to solve classification problems. We show that the degree of success of NGN is superior to NN. Analysis of performances of NN with different number of neurons or different architectures indicate that the effects of NGN cannot be accounted for an increased number of network elements, but rather they are specifically due to astrocytes. Furthermore, the relative efficacy of NGN vs. NN increases as the complexity of the network increases. These results indicate that artificial astrocytes improve neural network performance, and established the concept of Artificial Neuron-Glia Networks, which represents a novel concept in Artificial Intelligence with implications in computational science as well as in the understanding of brain function. PMID:21526157

  13. 1-BP inhibits NF-kappaB activity and Bcl-xL expression in astrocytes in vitro and reduces Bcl-xL expression in the brains of rats in vivo.

    PubMed

    Yoshida, Y; Liu, J Q; Nakano, Y; Ueno, S; Ohmori, S; Fueta, Y; Ishidao, T; Kunugita, N; Yamashita, U; Hori, H

    2007-03-01

    1-Bromopropane (1-BP) has been widely used as a substitute for chlorofluorocarbon that destroys the ozone layer. Although the central neurotoxicity of 1-BP has been recently reported, a molecular mechanism is not clear. In particular, the effects on cells in brain have not been fully analyzed. Here, we studied the effects of 1-BP on the activation of transcription factors involved in anti-apoptotic function or cell survival in astrocytes. Astrocytoma cell lines, U251, U373 and VM, or murine primary astrocytes were used for in vitro assay. DNA binding activities of NF-kappaB in these cells induced by interleukin (IL)-1 or LPS were inhibited by 1-BP. Consequently, the treatment of U251 cells with 1-BP resulted in suppression of NF-kappaB reporter activity. Furthermore, 1-BP blocked IkappaBalpha degradation, which is important for NF-kappaB activation. In addition, the level of Bcl-xL mRNA, which is known as an anti-apoptotic gene, were reduced in U251 treated with 1-BP or in the brain from rat exposed to 1-BP (400 ppm, 12 weeks). These results suggest that subchronic inhalation exposure to 1-BP vapor may affect the Bcl-xL expression in astrocytes.

  14. NH4+ triggers the release of astrocytic lactate via mitochondrial pyruvate shunting

    PubMed Central

    Lerchundi, Rodrigo; Fernández-Moncada, Ignacio; Contreras-Baeza, Yasna; Sotelo-Hitschfeld, Tamara; Mächler, Philipp; Wyss, Matthias T.; Stobart, Jillian; Baeza-Lehnert, Felipe; Alegría, Karin; Weber, Bruno; Barros, L. Felipe

    2015-01-01

    Neural activity is accompanied by a transient mismatch between local glucose and oxygen metabolism, a phenomenon of physiological and pathophysiological importance termed aerobic glycolysis. Previous studies have proposed glutamate and K+ as the neuronal signals that trigger aerobic glycolysis in astrocytes. Here we used a panel of genetically encoded FRET sensors in vitro and in vivo to investigate the participation of NH4+, a by-product of catabolism that is also released by active neurons. Astrocytes in mixed cortical cultures responded to physiological levels of NH4+ with an acute rise in cytosolic lactate followed by lactate release into the extracellular space, as detected by a lactate-sniffer. An acute increase in astrocytic lactate was also observed in acute hippocampal slices exposed to NH4+ and in the somatosensory cortex of anesthetized mice in response to i.v. NH4+. Unexpectedly, NH4+ had no effect on astrocytic glucose consumption. Parallel measurements showed simultaneous cytosolic pyruvate accumulation and NADH depletion, suggesting the involvement of mitochondria. An inhibitor-stop technique confirmed a strong inhibition of mitochondrial pyruvate uptake that can be explained by mitochondrial matrix acidification. These results show that physiological NH4+ diverts the flux of pyruvate from mitochondria to lactate production and release. Considering that NH4+ is produced stoichiometrically with glutamate during excitatory neurotransmission, we propose that NH4+ behaves as an intercellular signal and that pyruvate shunting contributes to aerobic lactate production by astrocytes. PMID:26286989

  15. Ischemic neurons activate astrocytes to disrupt endothelial barrier via increasing VEGF expression

    PubMed Central

    Li, Ying-Na; Pan, Rong; Qin, Xu-Jun; Yang, Wei-Lin; Qi, Zhifeng; Liu, Wenlan; Liu, Ke Jian

    2014-01-01

    Blood brain barrier (BBB) disruption occurring within the first few hours of ischemic stroke onset is closely associated with hemorrhagic transformation following thrombolytic therapy. However, the mechanism of this acute BBB disruption remains unclear. In the neurovascular unit, neurons do not have direct contact with the endothelial barrier, however they are highly sensitive and vulnerable to ischemic injury, and may act as the initiator for disrupting BBB when cerebral ischemia occurs. Herein we employed oxygen-glucose deprivation (OGD) and an in vitro BBB system consisting of brain microvascular cells and astrocytes to test this hypothesis. Neurons (CATH.a cells) were exposed to OGD for 3-hours before co-culturing with endothelial monolayer (bEnd 3 cells), or endothelial cells plus astrocytes (C8-D1A cells). Incubation of OGD-treated neurons with endothelial monolayer alone did not increase endothelial permeability. However, when astrocytes were present, the endothelial permeability was significantly increased, which was accompanied by loss of occludin and claudin-5 proteins as well as increased VEGF secretion into the conditioned medium. Importantly, all these changes were abolished when VEGF was knocked down in astrocytes by siRNA. Our findings suggest that ischemic neurons activate astrocytes to increase VEGF production, which in turn induces endothelial barrier disruption. PMID:24251624

  16. NH4(+) triggers the release of astrocytic lactate via mitochondrial pyruvate shunting.

    PubMed

    Lerchundi, Rodrigo; Fernández-Moncada, Ignacio; Contreras-Baeza, Yasna; Sotelo-Hitschfeld, Tamara; Mächler, Philipp; Wyss, Matthias T; Stobart, Jillian; Baeza-Lehnert, Felipe; Alegría, Karin; Weber, Bruno; Barros, L Felipe

    2015-09-01

    Neural activity is accompanied by a transient mismatch between local glucose and oxygen metabolism, a phenomenon of physiological and pathophysiological importance termed aerobic glycolysis. Previous studies have proposed glutamate and K(+) as the neuronal signals that trigger aerobic glycolysis in astrocytes. Here we used a panel of genetically encoded FRET sensors in vitro and in vivo to investigate the participation of NH4(+), a by-product of catabolism that is also released by active neurons. Astrocytes in mixed cortical cultures responded to physiological levels of NH4(+) with an acute rise in cytosolic lactate followed by lactate release into the extracellular space, as detected by a lactate-sniffer. An acute increase in astrocytic lactate was also observed in acute hippocampal slices exposed to NH4(+) and in the somatosensory cortex of anesthetized mice in response to i.v. NH4(+). Unexpectedly, NH4(+) had no effect on astrocytic glucose consumption. Parallel measurements showed simultaneous cytosolic pyruvate accumulation and NADH depletion, suggesting the involvement of mitochondria. An inhibitor-stop technique confirmed a strong inhibition of mitochondrial pyruvate uptake that can be explained by mitochondrial matrix acidification. These results show that physiological NH4(+) diverts the flux of pyruvate from mitochondria to lactate production and release. Considering that NH4(+) is produced stoichiometrically with glutamate during excitatory neurotransmission, we propose that NH4(+) behaves as an intercellular signal and that pyruvate shunting contributes to aerobic lactate production by astrocytes. PMID:26286989

  17. Characterization of Subcellular Responses Induced by Exposure of Microbubbles to Astrocytes.

    PubMed

    Sun, Shan; Kanagaraj, Johnwesly; Cho, Lindsey; Kang, Dongkoo; Xiao, Shu; Cho, Michael

    2015-10-01

    Blast traumatic brain injury (bTBI) has now been identified to associate with adverse health consequences among combat veterans. Post-traumatic stress disorder linked with explosive blasts, for example, may result from such brain injury. The fundamental questions about the nature, diagnosis, and long-term consequences of bTBI and causative relationship to post-traumatic stress disorder remain elusive, however. A better understanding of brain tissue injury requires elucidation of potential mechanisms. One such mechanism may be generation of microcavitation bubbles in the brain after an explosive blast and their subsequent interaction with brain cells. Using a controlled electrical discharge system, we have successfully generated shock waves (∼10 MPa) and microbubbles (20-30 μm) in the cell culture of mouse astrocytes. Detachment of astrocytes from the substrate after exposure to microbubbles was observed, and it depended on repetitive exposures. Of the cells that survived the initial assault, several subcellular changes were monitored and determined using fluorescent microscopy, including cell viability, cytoskeletal reorganization, changes in focal adhesion, membrane permeability, and potential onset of apoptosis. While the astrocytes impacted by the shock wave only demonstrated essentially unaltered cellular behavior, the astrocytes exposed to microbubbles exhibited significantly different responses, including production of reactive oxygen species by collapse of microbubbles. In the present study, we characterized and report for the first time the altered biophysical and subcellular properties in astrocytes in response to exposure to the combination of shock waves and microbubbles. PMID:25843567

  18. Astrocytes Protect Against Isoflurane Neurotoxicity by Buffering pro Brain-Derived Neurotrophic Factor

    PubMed Central

    Stary, Creed M.; Sun, Xiaoyun; Giffard, Rona G.

    2015-01-01

    Background Isoflurane induces cell death in neurons undergoing synaptogenesis via increased production of pro-brain derived neurotrophic factor (proBDNF) and activation of post-synaptic p75 neurotrophin receptor (p75NTR). Astrocytes express p75NTR but their role in neuronal p75NTR mediated cell death remains unclear. We 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 hours following 1 hour 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 6–8 replicate cultures/condition, and was repeated at least three times. Results Exposure to isoflurane significantly (p<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 elevated levels of proBDNF induced by isoflurane. These findings may hold clinical significance for the neonatal and injured brain where elevated levels of proBDNF impair neurogenesis. PMID:26270940

  19. Spatio-temporal characteristics of metabotropic glutamate receptor 5 traffic at or near the plasma membrane in astrocytes.

    PubMed

    Lee, William; Parpura, Vladimir

    2016-06-01

    Astrocytes can sense extracellular glutamate and respond to it by elevating their intracellular Ca(2+) levels via the activation of G-protein coupled receptors, such as metabotropic glutamate receptor 5 (mGluR5), which, during early postnatal development, is the primary receptor responsible for glutamatergic signaling in astrocytes. However, the detailed spatio-temporal characteristics of mGluR5 traffic at or near the plasma membrane of astrocytes are not well understood. To address this issue, we expressed recombinant fluorescent protein chimera of mGluR5 and used total internal reflection fluorescence microscopy on rat visual cortical astrocytes in culture. We used astrocytes lacking major processes, otherwise posing as a diffusion barrier, to infer into the general dynamics of this receptor. We found that plasmalemmal mGluR5 clusters in distinct areas, the size, and initial spatio-temporal level of occupancy of which dictated mGluR5 trafficking characteristics upon glutamate stimulation. These findings will be valuable in the interpretation of point-to-point information transfer and volume transmission between astrocytes and neurons, as well as that of paracrine signaling within astrocytic networks. PMID:27014856

  20. C/EBPβ regulates multiple IL-1β-induced human astrocyte inflammatory genes

    PubMed Central

    2012-01-01

    Background CCAAT enhancer-binding protein (C/EBP)β regulates gene expression in multiple organ systems and cell types, including astrocytes in the central nervous system (CNS). Inflammatory stimuli, interleukin (IL)-1β, tumor necrosis factor-α, human immunodeficiency virus (HIV)-1 and lipopolysaccharide induce astrocyte C/EBPβ expression. C/EBPβ is detectable in brains of Alzheimer’s disease (AD), Parkinson’s disease (PD) and HIV-1-associated dementia (HAD) patients, yet little is known about how C/EBPβ contributes to astrocyte gene regulation during neuroinflammation. Methods The expression of 92 human inflammation genes was compared between IL-1β-treated primary human astrocytes and astrocytes transfected with C/EBPβ-specific small interfering (si)RNA prior to IL-1β treatment for 12 h. Transcripts altered by > two-fold compared to control were subjected to one-way analysis of variance and Newman-Keuls post-test for multiple comparisons. Expression of two genes, cyclooxygenase-2 (COX-2) and bradykinin receptor B2 (BDKRB2) was further confirmed in additional human astrocyte donors. Astrocytes were treated with mitogen-activated protein kinase-selective inhibitors, then with IL-1β for 12 or 24 h followed by COX-2 and BDKRB2, expression analyses. Results IL-1β altered expression of 29 of 92 human inflammation genes by at least two-fold in primary human astrocytes in 12 h. C/EBPβ knockdown affected expression of 17 out of 29 IL-1β-regulated genes by > 25%. Two genes relevant to neuroinflammation, COX-2 and BDKRB2, were robustly decreased and increased, respectively, in response to C/EBPβ knockdown, and expression was confirmed in two additional donors. COX-2 and BDKRB2 mRNA remained altered in siRNA-transfected astrocytes at 12, 24 or 72 h. Inhibiting p38 kinase (p38K) activation blocked IL-1β-induced astrocyte COX-2 mRNA and protein expression, but not IL-1β-induced astrocyte BDKRB2 expression. Inhibiting extracellular

  1. RNA-binding protein QKI regulates Glial fibrillary acidic protein expression in human astrocytes.

    PubMed

    Radomska, Katarzyna J; Halvardson, Jonatan; Reinius, Björn; Lindholm Carlström, Eva; Emilsson, Lina; Feuk, Lars; Jazin, Elena

    2013-04-01

    Linkage, association and expression studies previously pointed to the human QKI, KH domain containing, RNA-binding (QKI) as a candidate gene for schizophrenia. Functional studies of the mouse orthologue Qk focused mainly on its role in oligodendrocyte development and myelination, while its function in astroglia remained unexplored. Here, we show that QKI is highly expressed in human primary astrocytes and that its splice forms encode proteins targeting different subcellular localizations. Uncovering the role of QKI in astrocytes is of interest in light of growing evidence implicating astrocyte dysfunction in the pathogenesis of several disorders of the central nervous system. We selectively silenced QKI splice variants in human primary astrocytes and used RNA sequencing to identify differential expression and splice variant composition at the genome-wide level. We found that an mRNA expression of Glial fibrillary acidic protein (GFAP), encoding a major component of astrocyte intermediate filaments, was down-regulated after QKI7 splice variant silencing. Moreover, we identified a potential QKI-binding site within the 3' untranslated region of human GFAP. This sequence was not conserved between mice and humans, raising the possibility that GFAP is a target for QKI in humans but not rodents. Haloperidol treatment of primary astrocytes resulted in coordinated increases in QKI7 and GFAP expression. Taken together, our results provide the first link between QKI and GFAP, two genes with alterations previously observed independently in schizophrenic patients. Our findings for QKI, together with its well-known role in myelination, suggest that QKI is a hub regulator of glia function in humans.

  2. Neuronal and astrocyte dysfunction diverges from embryonic fibroblasts in the Ndufs4fky/fky mouse

    PubMed Central

    Bird, Matthew J.; Wijeyeratne, Xiaonan W.; Komen, Jasper C.; Laskowski, Adrienne; Ryan, Michael T.; Thorburn, David R.; Frazier, Ann E.

    2014-01-01

    Mitochondrial dysfunction causes a range of early-onset neurological diseases and contributes to neurodegenerative conditions. The mechanisms of neurological damage however are poorly understood, as accessing relevant tissue from patients is difficult, and appropriate models are limited. Hence, we assessed mitochondrial function in neurologically relevant primary cell lines from a CI (complex I) deficient Ndufs4 KO (knockout) mouse (Ndufs4fky/fky) modelling aspects of the mitochondrial disease LS (Leigh syndrome), as well as MEFs (mouse embryonic fibroblasts). Although CI structure and function were compromised in all Ndufs4fky/fky cell types, the mitochondrial membrane potential was selectively impaired in the MEFs, correlating with decreased CI-dependent ATP synthesis. In addition, increased ROS (reactive oxygen species) generation and altered sensitivity to cell death were only observed in Ndufs4fky/fky primary MEFs. In contrast, Ndufs4fky/fky primary isocortical neurons and primary isocortical astrocytes displayed only impaired ATP generation without mitochondrial membrane potential changes. Therefore the neurological dysfunction in the Ndufs4fky/fky mouse may partly originate from a more severe ATP depletion in neurons and astrocytes, even at the expense of maintaining the mitochondrial membrane potential. This may provide protection from cell death, but would ultimately compromise cell functionality in neurons and astrocytes. Furthermore, RET (reverse electron transfer) from complex II to CI appears more prominent in neurons than MEFs or astrocytes, and is attenuated in Ndufs4fky/fky cells. PMID:25312000

  3. S100b counteracts effects of the neurotoxicant trimethyltin on astrocytes and microglia.

    PubMed

    Reali, Camilla; Scintu, Franca; Pillai, Rita; Donato, Rosario; Michetti, Fabrizio; Sogos, Valeria

    2005-09-01

    Central nervous system degenerative diseases are often characterized by an early, strong reaction of astrocytes and microglia. Both these cell types can play a double role, protecting neurons against degeneration through the synthesis and secretion of trophic factors or inducing degeneration through the secretion of toxic molecules. Therefore, we studied the effects of S100B and trimethyltin (TMT) on human astrocytes and microglia with two glial models, primary cultures of human fetal astrocytes and a microglia cell line. After treatment with 10(-5) M TMT, astrocytes showed morphological alterations associated with an increase in glial fibrillary acidic protein (GFAP) expression and changes in GFAP filament organization. Administration of S100B before TMT treatment prevented TMT-induced changes in morphology and GFAP expression. A decrease in inducible nitric oxide synthase expression was observed in astrocytes treated with TMT, whereas the same treatment induced iNOS expression in microglia. In both cases, S100B prevented TMT-induced changes. Tumor necrosis factor-alpha mRNA expression in astrocytes was not modified by TMT treatment, whereas it was increased in microglia cells. S100B pretreatment blocked the TMT-induced increase in TNF-alpha expression in microglia. To trace the mechanisms involved in S100B activity, the effect of BAY 11-7082, an inhibitor of nuclear factor-kappaB (NF-kappaB) activation, and of PD98059, an inhibitor of MEK-ERK1/2, were investigated. Results showed that the protective effects of S100B against TMT toxicity in astrocytes depend on NF-kappaB, but not on ERK1/2 activation. These results might help in understanding the role played by glial cells in brain injury after exposure to chemical neurotoxicants and support the view that S100B may protect brain cells in case of injury. (c) 2005 Wiley-Liss, Inc.

  4. Antagonistic interactions between dexamethasone and fluoxetine modulate morphodynamics and expression of cytokines in astrocytes.

    PubMed

    Henkel, A W; Alali, H; Devassy, A; Alawadi, M M; Redzic, Z B

    2014-11-01

    The "plasticity hypothesis" proposes that major depression is caused by morphological and biochemical modifications in neurons and astrocytes and those beneficial pharmacological effects of selective-serotonin-reuptake-inhibitors (SSRI) are at least partially associated with modifications of cellular communications between these cells. In this study we examined effects of the antidepressant fluoxetine on cultured astrocytes that were, in some cases, pretreated with dexamethasone, a cortisol analog known to trigger depressive disorder. Primary rat astrocytes were purified and treated with dexamethasone and the SSRI fluoxetine in physiological concentrations so that both drugs did not affect cell viability. Expression of interleukin-2 (IL-2) and glia-derived-neurotrophic-factor (GDNF) were analyzed and monitored and cell viability, apoptosis, cluster formation, particle-removing capacity and cell mobility were also monitored. Pre-studies without any drugs on mixed neuron-astrocyte co-cultures suggested that astrocytes interacted with neurons and other brain cells in vitro by actively assembling them into clusters. Treatment of purified astrocytes with dexamethasone significantly decreased their mobility compared to controls but had no effect on cluster formation. Dexamethasone-treated cells removed fewer extracellular particles derived from dead cells and cell debris. Both effects were abolished by simultaneous application of fluoxetine. Intracellular IL-2 increased, while GDNF amount expression was diminished following dexamethasone treatment. Simultaneous administration of fluoxetine reversed dexamethasone-triggered IL-2 elevation but had no effect on decreased GDNF concentration. These results suggest that mobility and growth factor equilibrium of astrocytes are affected by dexamethasone and by fluoxetine and that fluoxetine could reverse some changes induced by dexamethasone. PMID:25242644

  5. Astrocytes: The missing link in neurological disease?

    PubMed Central

    Lin, Chia-Ching John; Deneen, Benjamin

    2013-01-01

    The central nervous system (CNS) is comprised of numerous cell types that work in concert to facilitate proper function and homeostasis. Disruption of these carefully orchestrated networks results in neuronal dysfunction, manifesting itself in a variety of neurological disorders. While neuronal dysregulation is causative of symptoms manifest in the clinic, the etiology of these disorders is often more complex than simply a loss of neurons or intrinsic dysregulation of their function. In the adult brain, astrocytes comprise the most abundant cell type and play key roles in CNS physiology, therefore it stands to reason that dysregulation of normal astrocyte function contributes to the etiology and progression of varied neurological disorders. We review here some neurological disorders associated with an astrocyte factor and discuss how the related astrocyte dysfunction contributes to the etiology and/or progression of these disorders. PMID:24365571

  6. Proliferative effects of humoral factors derived from neuroblastoma cells on cultured astrocytes.

    PubMed

    Ohashi, M; Amano, S; Hazama, F; Handa, J

    1994-08-29

    The proliferative effects of humoral factors released from N18-RE105 neuroblastoma (NRE) cells on cultured astrocytes were assessed in separate co-culture and conditioned medium studies. In both experimental conditions, the humoral factors derived from neuroblastoma cells had growth-promoting effects on C6 glioma cells of astroglial lineage, but not on primary cultured astrocytes from new-born rat cerebral cortex. It is assumed that neuron-derived humoral factors include astroglial growth factors and that differences in responsiveness between two kinds of cells are probably related to the stages of astroglial maturation processes.

  7. Inflammatory mediators alter the astrocyte transcriptome and calcium signaling elicited by multiple G-protein-coupled receptors.

    PubMed

    Hamby, Mary E; Coppola, Giovanni; Ao, Yan; Geschwind, Daniel H; Khakh, Baljit S; Sofroniew, Michael V

    2012-10-17

    Inflammation features in CNS disorders such as stroke, trauma, neurodegeneration, infection, and autoimmunity in which astrocytes play critical roles. To elucidate how inflammatory mediators alter astrocyte functions, we examined effects of transforming growth factor-β1 (TGF-β1), lipopolysaccharide (LPS), and interferon-gamma (IFNγ), alone and in combination, on purified, mouse primary cortical astrocyte cultures. We used microarrays to conduct whole-genome expression profiling, and measured calcium signaling, which is implicated in mediating dynamic astrocyte functions. Combinatorial exposure to TGF-β1, LPS, and IFNγ significantly modulated astrocyte expression of >6800 gene probes, including >380 synergistic changes not predicted by summing individual treatment effects. Bioinformatic analyses revealed significantly and markedly upregulated molecular networks and pathways associated in particular with immune signaling and regulation of cell injury, death, growth, and proliferation. Highly regulated genes included chemokines, growth factors, enzymes, channels, transporters, and intercellular and intracellular signal transducers. Notably, numerous genes for G-protein-coupled receptors (GPCRs) and G-protein effectors involved in calcium signaling were significantly regulated, mostly down (for example, Cxcr4, Adra2a, Ednra, P2ry1, Gnao1, Gng7), but some up (for example, P2ry14, P2ry6, Ccrl2, Gnb4). We tested selected cases and found that changes in GPCR gene expression were accompanied by significant, parallel changes in astrocyte calcium signaling evoked by corresponding GPCR-specific ligands. These findings identify pronounced changes in the astrocyte transcriptome induced by TGF-β1, LPS, and IFNγ, and show that these inflammatory stimuli upregulate astrocyte molecular networks associated with immune- and injury-related functions and significantly alter astrocyte calcium signaling stimulated by multiple GPCRs.

  8. Galectin-9 Protein Is Up-regulated in Astrocytes by Tumor Necrosis Factor and Promotes Encephalitogenic T-cell Apoptosis*

    PubMed Central

    Steelman, Andrew J.; Smith, Roger; Welsh, C. Jane; Li, Jianrong

    2013-01-01

    Demyelination and axonal damage in multiple sclerosis (MS) are thought to be a consequence of inflammatory processes that are perpetuated by activated glia and infiltrating leukocytes. Galectin-9 is a β-galactoside binding lectin capable of modulating immune responses and appears to be up-regulated in MS. However, its role in the pathogenesis of MS has yet to be determined. Here, we report that proinflammatory cytokines induce galectin-9 (Gal-9) expression in primary astrocytes and the mechanism by which TNF up-regulates Gal-9. Astrocytes did not express Gal-9 under basal conditions nor did IL-6, IL-10, or IL-13 trigger Gal-9 expression. In contrast, IL-1β, IFN-γ, and particularly TNF up-regulated Gal-9 in astrocytes. TNF-induced Gal-9 expression was dependent on TNF receptor 1 (TNFR1) as TNF failed to induce Gal-9 in TNFR1−/− astrocytes. Blockade of the JNK MAP kinase pathway with the JNK inhibitor SP600125 abrogated TNF-induced Gal-9, whereas p38 and MEK inhibitors had minimal effects. Furthermore, specific knockdown of c-Jun via siRNA in astrocytes before TNF treatment greatly suppressed Gal-9 transcription, suggesting that TNF induces astroglial Gal-9 through the TNF/TNFR1/JNK/cJun signaling pathway. Finally, utilizing astrocytes from Lgals9 mutant (Gal-9−/−) mice as well as a myelin basic protein-specific Tim-3+ encephalitogenic T-cell clone (LCN-8), we found that conditioned medium from TNF-stimulated Gal-9+/+ but not Gal-9−/− astrocytes increased the percentage of apoptotic encephalitogenic T-cells. Together, our results suggest that Gal-9 is induced in astrocytes by TNF via the JNK/c-Jun pathway and that astrocyte-derived Gal-9 may function as an immunoregulatory protein in response to ongoing neuroinflammation. PMID:23836896

  9. Impaired Autophagy in APOE4 Astrocytes.

    PubMed

    Simonovitch, Shira; Schmukler, Eran; Bespalko, Alina; Iram, Tal; Frenkel, Dan; Holtzman, David M; Masliah, Eliezer; Michaelson, Danny M; Pinkas-Kramarski, Ronit

    2016-01-01

    Alzheimer's disease (AD) is the most prevalent form of dementia in elderly. Genetic studies revealed allelic segregation of the apolipoprotein E (ApoE) gene in sporadic AD and in families with higher risk of AD. The mechanisms underlying the pathological effects of ApoE4 are not yet entirely clear. Several studies indicate that autophagy, which plays an important role in degradation pathways of proteins, organelles and protein aggregates, may be impaired in AD. In the present study, we investigated the effects of ApoE4 versus the ApoE3 isoform on the process of autophagy in mouse-derived astrocytes. The results obtained reveal that under several autophagy-inducing conditions, astrocytes expressing ApoE4 exhibit lower autophagic flux compared to astrocytes expressing ApoE3. Using an in situ model, we examined the role of autophagy and the effects thereon of ApoE4 in the elimination of Aβ plaques from isolated brain sections of transgenic 5xFAD mice. This revealed that ApoE4 astrocytes eliminate Aβ plaques less effectively than the corresponding ApoE3 astrocytes. Additional experiments showed that the autophagy inducer, rapamycin, enhances Aβ plaque degradation by ApoE4 astrocytes whereas the autophagy inhibitor, chloroquine, blocks Aβ plaque degradation by ApoE3 astrocytes. Taken together, these findings show that ApoE4 impairs autophagy in astrocyte cultures and that this effect is associated with reduced capacity to clear Aβ plaques. This suggests that impaired autophagy may play a role in mediating the pathological effects of ApoE4 in AD.

  10. Astrocytic vesicle mobility in health and disease.

    PubMed

    Potokar, Maja; Vardjan, Nina; Stenovec, Matjaž; Gabrijel, Mateja; Trkov, Saša; Jorgačevski, Jernej; Kreft, Marko; Zorec, Robert

    2013-01-01

    Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes through the trafficking of intracellular vesicles. Recent studies of single vesicle mobility in astrocytes have prompted new views of how astrocytes contribute to information processing in nervous tissue. Here, we review the trafficking of several types of membrane-bound vesicles that are specifically involved in the processes of (i) intercellular communication by gliotransmitters (glutamate, adenosine 5'-triphosphate, atrial natriuretic peptide), (ii) plasma membrane exchange of transporters and receptors (EAAT2, MHC-II), and (iii) the involvement of vesicle mobility carrying aquaporins (AQP4) in water homeostasis. The properties of vesicle traffic in astrocytes are discussed in respect to networking with neighboring cells in physiologic and pathologic conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, and states in which astrocytes contribute to neuroinflammatory conditions.

  11. Astrocytic Vesicle Mobility in Health and Disease

    PubMed Central

    Potokar, Maja; Vardjan, Nina; Stenovec, Matjaž; Gabrijel, Mateja; Trkov, Saša; Jorgačevski, Jernej; Kreft, Marko; Zorec, Robert

    2013-01-01

    Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes through the trafficking of intracellular vesicles. Recent studies of single vesicle mobility in astrocytes have prompted new views of how astrocytes contribute to information processing in nervous tissue. Here, we review the trafficking of several types of membrane-bound vesicles that are specifically involved in the processes of (i) intercellular communication by gliotransmitters (glutamate, adenosine 5′-triphosphate, atrial natriuretic peptide), (ii) plasma membrane exchange of transporters and receptors (EAAT2, MHC-II), and (iii) the involvement of vesicle mobility carrying aquaporins (AQP4) in water homeostasis. The properties of vesicle traffic in astrocytes are discussed in respect to networking with neighboring cells in physiologic and pathologic conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, and states in which astrocytes contribute to neuroinflammatory conditions. PMID:23712361

  12. Astrocyte Aquaporin Dynamics in Health and Disease.

    PubMed

    Potokar, Maja; Jorgačevski, Jernej; Zorec, Robert

    2016-01-01

    The family of aquaporins (AQPs), membrane water channels, consists of diverse types of proteins that are mainly permeable to water; some are also permeable to small solutes, such as glycerol and urea. They have been identified in a wide range of organisms, from microbes to vertebrates and plants, and are expressed in various tissues. Here, we focus on AQP types and their isoforms in astrocytes, a major glial cell type in the central nervous system (CNS). Astrocytes have anatomical contact with the microvasculature, pia, and neurons. Of the many roles that astrocytes have in the CNS, they are key in maintaining water homeostasis. The processes involved in this regulation have been investigated intensively, in particular regulation of the permeability and expression patterns of different AQP types in astrocytes. Three aquaporin types have been described in astrocytes: aquaporins AQP1 and AQP4 and aquaglyceroporin AQP9. The aim here is to review their isoforms, subcellular localization, permeability regulation, and expression patterns in the CNS. In the human CNS, AQP4 is expressed in normal physiological and pathological conditions, but astrocytic expression of AQP1 and AQP9 is mainly associated with a pathological state. PMID:27420057

  13. Stargazing: Monitoring subcellular dynamics of brain astrocytes.

    PubMed

    Benjamin Kacerovsky, J; Murai, K K

    2016-05-26

    Astrocytes are major non-neuronal cell types in the central nervous system that regulate a variety of processes in the brain including synaptic transmission, neurometabolism, and cerebrovasculature tone. Recent discoveries have revealed that astrocytes perform very specialized and heterogeneous roles in brain homeostasis and function. Exactly how astrocytes fulfill such diverse roles in the brain remains to be fully understood and is an active area of research. In this review, we focus on the complex subcellular anatomical features of protoplasmic gray matter astrocytes in the mature, healthy brain that likely empower these cells with the ability to detect and respond to changes in neuronal and synaptic activity. In particular, we discuss how intricate processes on astrocytes allow these cells to communicate with neurons and their synapses and strategically deliver specific cellular organelles such as mitochondria and ribosomes to active compartments within the neuropil. Understanding the properties of these structural elements will lead to a better understanding of how astrocytes function in the healthy and diseased brain. PMID:26162237

  14. Astrocyte Aquaporin Dynamics in Health and Disease

    PubMed Central

    Potokar, Maja; Jorgačevski, Jernej; Zorec, Robert

    2016-01-01

    The family of aquaporins (AQPs), membrane water channels, consists of diverse types of proteins that are mainly permeable to water; some are also permeable to small solutes, such as glycerol and urea. They have been identified in a wide range of organisms, from microbes to vertebrates and plants, and are expressed in various tissues. Here, we focus on AQP types and their isoforms in astrocytes, a major glial cell type in the central nervous system (CNS). Astrocytes have anatomical contact with the microvasculature, pia, and neurons. Of the many roles that astrocytes have in the CNS, they are key in maintaining water homeostasis. The processes involved in this regulation have been investigated intensively, in particular regulation of the permeability and expression patterns of different AQP types in astrocytes. Three aquaporin types have been described in astrocytes: aquaporins AQP1 and AQP4 and aquaglyceroporin AQP9. The aim here is to review their isoforms, subcellular localization, permeability regulation, and expression patterns in the CNS. In the human CNS, AQP4 is expressed in normal physiological and pathological conditions, but astrocytic expression of AQP1 and AQP9 is mainly associated with a pathological state. PMID:27420057

  15. Human astrocytes inhibit Cryptococcus neoformans growth by a nitric oxide-mediated mechanism

    PubMed Central

    1994-01-01

    Cryptococcus neoformans is an opportunistic fungus that causes life- threatening meningoencephalitis in 5-10% of patients with acquired immune deficiency syndrome. Cryptococcal meningoencephalitis is characterized by a lymphohistiocytic infiltrate, accumulation of encapsulated forms of C. neoformans, and varying degrees of glial reaction. Little is known about the contribution of endogenous central nervous system cells to the pathogenesis of cryptococcal infections. In this study, we investigated the role of astrocytes as potential effector cells against C. neoformans. Primary cultures of human fetal astrocytes, activated with interleukin 1 beta plus interferon gamma inhibited the growth of C. neoformans. The inhibition of C. neoformans growth was paralleled by production of nitrite, and reversed by the inhibitors of nitric oxide (NO.) synthase, NG-methyl-mono-arginine and NG-nitro-arginine methyl ester. The results suggest a novel function for human astrocytes in host defence and provide a precedent for the use of NO. as an antimicrobial effector molecule by human cells. PMID:8006595

  16. Opioid-dependent growth of glial cultures: Suppression of astrocyte DNA synthesis by met-enkephalin

    SciTech Connect

    Stiene-Martin, A.; Hauser, K.F. )

    1990-01-01

    The action of met-enkephalin on the growth of astrocytes in mixed-glial cultures was examined. Primary, mixed-glial cultures were isolated from 1 day-old mouse cerebral hemispheres and continuously treated with either basal growth media, 1 {mu}M met-enkephalin, 1 {mu}M met-enkephalin plus the opioid antagonist naloxone, or naloxone alone. Absolute numbers of neural cells were counted in unstained preparations, while combined ({sup 3}H)-thymidine autoradiography and glial fibrillary acid protein (GFAP) immunocytochemistry was performed to identify specific changes in astrocytes. When compared to control and naloxone treated cultures, met-enkephalin caused a significant decrease in both total cell numbers, and in ({sup 3}H)-thymidine incorporation by GFAP-positive cells with flat morphology. These results indicate that met-enkephalin suppresses astrocyte growth in culture.

  17. Glycolysis and oxidative phosphorylation in neurons and astrocytes during network activity in hippocampal slices.

    PubMed

    Ivanov, Anton I; Malkov, Anton E; Waseem, Tatsiana; Mukhtarov, Marat; Buldakova, Svetlana; Gubkina, Olena; Zilberter, Misha; Zilberter, Yuri

    2014-03-01

    Network activation triggers a significant energy metabolism increase in both neurons and astrocytes. Questions of the primary neuronal energy substrate (e.g., glucose vs. lactate) as well as the relative contributions of glycolysis and oxidative phosphorylation and their cellular origin (neurons vs. astrocytes) are still a matter of debates. Using simultaneous measurements of electrophysiological and metabolic parameters during synaptic stimulation in hippocampal slices from mature mice, we show that neurons and astrocytes use both glycolysis and oxidative phosphorylation to meet their energy demands. Supplementation or replacement of glucose in artificial cerebrospinal fluid (ACSF) with pyruvate or lactate strongly modifies parameters related to network activity-triggered energy metabolism. These effects are not induced by changes in ATP content, pH(i), [Ca(2+)](i) or accumulation of reactive oxygen species. Our results suggest that during network activation, a significant fraction of NAD(P)H response (its overshoot phase) corresponds to glycolysis and the changes in cytosolic NAD(P)H and mitochondrial FAD are coupled. Our data do not support the hypothesis of a preferential utilization of astrocyte-released lactate by neurons during network activation in slices--instead, we show that during such activity glucose is an effective energy substrate for both neurons and astrocytes.

  18. l-Theanine protects against excess dopamine-induced neurotoxicity in the presence of astrocytes

    PubMed Central

    Takeshima, Mika; Miyazaki, Ikuko; Murakami, Shinki; Kita, Taizo; Asanuma, Masato

    2016-01-01

    l-Theanine (γ-glutamylethylamide), a component of green tea, is considered to have regulatory and neuroprotective roles in the brain. The present study was designed to determine the effect of l-theanine on excess dopamine-induced neurotoxicity in both cell culture and animal experiments. The primary cultured mesencephalic neurons or co-cultures of mesencephalic neurons and striatal astrocytes were pretreated with l-theanine for 72 h, and then treated with excess dopamine for further 24 h. The cell viability of dopamine neurons and levels of glutathione were evaluated. Excess dopamine-induced neurotoxicity was significantly attenuated by 72 h preincubation with l-theanine in neuron-astrocyte co-cultures but not in neuron-rich cultures. Exposure to l-theanine increased the levels of glutathione in both astrocytes and glial conditioned medium. The glial conditioned medium from l-theanine-pretreated striatal astrocytes attenuated dopamine-induced neurotoxicity and quinoprotein formation in mesencephalic neurons. In addition, replacement of l-glutamate with l-theanine in an in vitro cell-free glutathione-synthesis system produced glutathione-like thiol compounds. Furthermore, l-theanine administration (4 mg/kg, p.o.) for 14 days significantly increased glutathione levels in the striatum of mice. The results suggest that l-theanine provides neuroprotection against oxidative stress-induced neuronal damage by humoral molecules released from astrocytes, probably including glutathione. PMID:27698535

  19. Protective and Antioxidant Effects of a Chalconoid from Pulicaria incisa on Brain Astrocytes

    PubMed Central

    Telerman, Alona; Erlank, Hilla; Mordechay, Sharon; Rindner, Miriam; Ofir, Rivka; Kashman, Yoel

    2013-01-01

    Oxidative stress is involved in the pathogenesis of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. Astrocytes, the most abundant glial cells in the brain, protect neurons from reactive oxygen species (ROS) and provide them with trophic support, such as glial-derived neurotrophic factor (GDNF). Thus, any damage to astrocytes will affect neuronal survival. In the present study, by activity-guided fractionation, we have purified from the desert plant Pulicaria incisa two protective compounds and determined their structures by spectroscopic methods. The compounds were found to be new chalcones—pulichalconoid B and pulichalconoid C. This is the first study to characterize the antioxidant and protective effects of these compounds in any biological system. Using primary cultures of astrocytes, we have found that pulichalconoid B attenuated the accumulation of ROS following treatment of these cells with hydrogen peroxide by 89% and prevented 89% of the H2O2-induced death of astrocytes. Pulichalconoid B exhibited an antioxidant effect both in vitro and in the cellular antioxidant assay in astrocytes and microglial cells. Pulichalconoid B also caused a fourfold increase in GDNF transcription in these cells. Thus, this chalcone deserves further studies in order to evaluate if beneficial therapeutic effect exists. PMID:24069503

  20. Protective and antioxidant effects of a chalconoid from Pulicaria incisa on brain astrocytes.

    PubMed

    Elmann, Anat; Telerman, Alona; Erlank, Hilla; Mordechay, Sharon; Rindner, Miriam; Ofir, Rivka; Kashman, Yoel

    2013-01-01

    Oxidative stress is involved in the pathogenesis of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. Astrocytes, the most abundant glial cells in the brain, protect neurons from reactive oxygen species (ROS) and provide them with trophic support, such as glial-derived neurotrophic factor (GDNF). Thus, any damage to astrocytes will affect neuronal survival. In the present study, by activity-guided fractionation, we have purified from the desert plant Pulicaria incisa two protective compounds and determined their structures by spectroscopic methods. The compounds were found to be new chalcones-pulichalconoid B and pulichalconoid C. This is the first study to characterize the antioxidant and protective effects of these compounds in any biological system. Using primary cultures of astrocytes, we have found that pulichalconoid B attenuated the accumulation of ROS following treatment of these cells with hydrogen peroxide by 89% and prevented 89% of the H2O2-induced death of astrocytes. Pulichalconoid B exhibited an antioxidant effect both in vitro and in the cellular antioxidant assay in astrocytes and microglial cells. Pulichalconoid B also caused a fourfold increase in GDNF transcription in these cells. Thus, this chalcone deserves further studies in order to evaluate if beneficial therapeutic effect exists.

  1. Glycolysis and oxidative phosphorylation in neurons and astrocytes during network activity in hippocampal slices

    PubMed Central

    Ivanov, Anton I; Malkov, Anton E; Waseem, Tatsiana; Mukhtarov, Marat; Buldakova, Svetlana; Gubkina, Olena; Zilberter, Misha; Zilberter, Yuri

    2014-01-01

    Network activation triggers a significant energy metabolism increase in both neurons and astrocytes. Questions of the primary neuronal energy substrate (e.g., glucose vs. lactate) as well as the relative contributions of glycolysis and oxidative phosphorylation and their cellular origin (neurons vs. astrocytes) are still a matter of debates. Using simultaneous measurements of electrophysiological and metabolic parameters during synaptic stimulation in hippocampal slices from mature mice, we show that neurons and astrocytes use both glycolysis and oxidative phosphorylation to meet their energy demands. Supplementation or replacement of glucose in artificial cerebrospinal fluid (ACSF) with pyruvate or lactate strongly modifies parameters related to network activity-triggered energy metabolism. These effects are not induced by changes in ATP content, pHi, [Ca2+]i or accumulation of reactive oxygen species. Our results suggest that during network activation, a significant fraction of NAD(P)H response (its overshoot phase) corresponds to glycolysis and the changes in cytosolic NAD(P)H and mitochondrial FAD are coupled. Our data do not support the hypothesis of a preferential utilization of astrocyte-released lactate by neurons during network activation in slices—instead, we show that during such activity glucose is an effective energy substrate for both neurons and astrocytes. PMID:24326389

  2. l-Theanine protects against excess dopamine-induced neurotoxicity in the presence of astrocytes

    PubMed Central

    Takeshima, Mika; Miyazaki, Ikuko; Murakami, Shinki; Kita, Taizo; Asanuma, Masato

    2016-01-01

    l-Theanine (γ-glutamylethylamide), a component of green tea, is considered to have regulatory and neuroprotective roles in the brain. The present study was designed to determine the effect of l-theanine on excess dopamine-induced neurotoxicity in both cell culture and animal experiments. The primary cultured mesencephalic neurons or co-cultures of mesencephalic neurons and striatal astrocytes were pretreated with l-theanine for 72 h, and then treated with excess dopamine for further 24 h. The cell viability of dopamine neurons and levels of glutathione were evaluated. Excess dopamine-induced neurotoxicity was significantly attenuated by 72 h preincubation with l-theanine in neuron-astrocyte co-cultures but not in neuron-rich cultures. Exposure to l-theanine increased the levels of glutathione in both astrocytes and glial conditioned medium. The glial conditioned medium from l-theanine-pretreated striatal astrocytes attenuated dopamine-induced neurotoxicity and quinoprotein formation in mesencephalic neurons. In addition, replacement of l-glutamate with l-theanine in an in vitro cell-free glutathione-synthesis system produced glutathione-like thiol compounds. Furthermore, l-theanine administration (4 mg/kg, p.o.) for 14 days significantly increased glutathione levels in the striatum of mice. The results suggest that l-theanine provides neuroprotection against oxidative stress-induced neuronal damage by humoral molecules released from astrocytes, probably including glutathione.

  3. Label-free optical activation of astrocyte in vivo

    NASA Astrophysics Data System (ADS)

    Choi, Myunghwan; Yoon, Jonghee; Ku, Taeyun; Choi, Kyungsun; Choi, Chulhee

    2011-07-01

    As the most abundant cell type in the central nervous system, astrocyte has been one of main research topics in neuroscience. Although various tools have been developed, at present, there is no tool that allows noninvasive activation of astrocyte in vivo without genetic or pharmacological perturbation. Here we report a noninvasive label-free optical method for physiological astrocyte activation in vivo using a femtosecond pulsed laser. We showed the laser stimulation robustly induced astrocytic calcium activation in vivo and further verified physiological relevance of the calcium increase by demonstrating astrocyte mediated vasodilation in the brain. This novel optical method will facilitate noninvasive physiological study on astrocyte function.

  4. PFKFB3-mediated glycolysis is involved in reactive astrocyte proliferation after oxygen-glucose deprivation/reperfusion and is regulated by Cdh1.

    PubMed

    Lv, Youyou; Zhang, Bo; Zhai, Chunchun; Qiu, Jin; Zhang, Yue; Yao, Wenlong; Zhang, Chuanhan

    2015-12-01

    Reactive astrocyte proliferation is involved in many central degenerative diseases. The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3), an allosteric activator of 6-phosphofructo-1-kinase (PFK1), controls glycolytic flux. Furthermore, APC/C-Cdh1 plays a crucial role in brain metabolism by regulating PFKFB3 expression. Previous studies have defined the roles of PFKFB3-mediated glycolysis in pathological angiogenesis, cell autophagy, and amyloid plaque deposition in proliferating cells. However, the role of PFKFB3 in reactive astrocyte proliferation after cerebral ischemia is unknown. In this study, we cultured rat primary cortical astrocytes and established an oxygen-glucose deprivation/reperfusion (OGD/R) model to mimic cerebral ischemia in vivo. Astrocyte proliferation was measured by western blotting for proliferating cell nuclear antigen (PCNA) and by EdU incorporation. We found that OGD/R up-regulated PFKFB3 and PFK1 expression, which was accompanied by reactive astrocyte proliferation. Knockdown of PFKFB3 by siRNA transfection significantly inhibited reactive astrocyte proliferation and lactate release, an indicator of glycolysis. We found that PFKFB3 and PFK1 expression were down-regulated and lactate release was decreased when OGD/R-induced astrocyte proliferation was inhibited by a Cdh1-expressing lentivirus. Thus, reactive astrocyte proliferation can be effectively suppressed by down-regulation of PFKFB3 through control of glycolytic flux, which is downstream of APC/C-Cdh1.

  5. In vivo expression of polyglutamine-expanded huntingtin by mouse striatal astrocytes impairs glutamate transport: a correlation with Huntington's disease subjects.

    PubMed

    Faideau, Mathilde; Kim, Jinho; Cormier, Kerry; Gilmore, Richard; Welch, Mackenzie; Auregan, Gwennaelle; Dufour, Noelle; Guillermier, Martine; Brouillet, Emmanuel; Hantraye, Philippe; Déglon, Nicole; Ferrante, Robert J; Bonvento, Gilles

    2010-08-01

    Huntington's disease (HD) is a neurodegenerative disorder previously thought to be of primary neuronal origin, despite ubiquitous expression of mutant huntingtin (mHtt). We tested the hypothesis that mHtt expressed in astrocytes may contribute to the pathogenesis of HD. To better understand the contribution of astrocytes in HD in vivo, we developed a novel mouse model using lentiviral vectors that results in selective expression of mHtt into striatal astrocytes. Astrocytes expressing mHtt developed a progressive phenotype of reactive astrocytes that was characterized by a marked decreased expression of both glutamate transporters, GLAST and GLT-1, and of glutamate uptake. These effects were associated with neuronal dysfunction, as observed by a reduction in DARPP-32 and NR2B expression. Parallel studies in brain samples from HD subjects revealed early glial fibrillary acidic protein expression in striatal astrocytes from Grade 0 HD cases. Astrogliosis was associated with morphological changes that increased with severity of disease, from Grades 0 through 4 and was more prominent in the putamen. Combined immunofluorescence showed co-localization of mHtt in astrocytes in all striatal HD specimens, inclusive of Grade 0 HD. Consistent with the findings from experimental mice, there was a significant grade-dependent decrease in striatal GLT-1 expression from HD subjects. These findings suggest that the presence of mHtt in astrocytes alters glial glutamate transport capacity early in the disease process and may contribute to HD pathogenesis. PMID:20494921

  6. Decreased astrocytic thrombospondin-1 secretion after chronic ammonia treatment reduces the level of synaptic proteins: in vitro and in vivo studies.

    PubMed

    Jayakumar, Arumugam R; Tong, Xiao Y; Curtis, Kevin M; Ruiz-Cordero, Roberto; Shamaladevi, Nagarajarao; Abuzamel, Missa; Johnstone, Joshua; Gaidosh, Gabriel; Rama Rao, Kakulavarapu V; Norenberg, Michael D

    2014-11-01

    Chronic hepatic encephalopathy (CHE) is a major complication in patients with severe liver disease. Elevated blood and brain ammonia levels have been implicated in its pathogenesis, and astrocytes are the principal neural cells involved in this disorder. Since defective synthesis and release of astrocytic factors have been shown to impair synaptic integrity in other neurological conditions, we examined whether thrombospondin-1 (TSP-1), an astrocytic factor involved in the maintenance of synaptic integrity, is also altered in CHE. Cultured astrocytes were exposed to ammonia (NH₄Cl, 0.5-2.5 mM) for 1-10 days, and TSP-1 content was measured in cell extracts and culture media. Astrocytes exposed to ammonia exhibited a reduction in intra- and extracellular TSP-1 levels. Exposure of cultured neurons to conditioned media from ammonia-treated astrocytes showed a decrease in synaptophysin, PSD95, and synaptotagmin levels. Conditioned media from TSP-1 over-expressing astrocytes that were treated with ammonia, when added to cultured neurons, reversed the decline in synaptic proteins. Recombinant TSP-1 similarly reversed the decrease in synaptic proteins. Metformin, an agent known to increase TSP-1 synthesis in other cell types, also reversed the ammonia-induced TSP-1 reduction. Likewise, we found a significant decline in TSP-1 level in cortical astrocytes, as well as a reduction in synaptophysin content in vivo in a rat model of CHE. These findings suggest that TSP-1 may represent an important therapeutic target for CHE. Defective release of astrocytic factors may impair synaptic integrity in chronic hepatic encephalopathy. We found a reduction in the release of the astrocytic matricellular proteins thrombospondin-1 (TSP-1) in ammonia-treated astrocytes; such reduction was associated with a decrease in synaptic proteins caused by conditioned media from ammonia-treated astrocytes. Exposure of neurons to CM from ammonia-treated astrocytes, in which TSP-1 is over

  7. Specialized contacts of astrocytes with astrocytes and with other cell types in the hypothalamus of the hamster.

    PubMed Central

    Suarez Najera, I; Fernandez Ruiz, B; Garcia Segura, L M

    1980-01-01

    Adult hamsters were used for this electron microscopic study of the hypothalamic region. Specialized contacts between astrocytes and astrocytes, and between astrocytes and other cellular elements, are described and illustrated. The specialized inter-astrocytic junctions occur primarily in perivascular and subpial regions, but also in areas of high synaptic density. The junctions between astrocytic processes are of hemidesmosomal type. Astrocytes are connected to oligodendroglial cells by means of desmosomes, and to neuronal processes by means of zonulae occludens. The functional significance of these arrangements is discussed. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7364663

  8. Sodium signaling and astrocyte energy metabolism.

    PubMed

    Chatton, Jean-Yves; Magistretti, Pierre J; Barros, L Felipe

    2016-10-01

    The Na(+) gradient across the plasma membrane is constantly exploited by astrocytes as a secondary energy source to regulate the intracellular and extracellular milieu, and discard waste products. One of the most prominent roles of astrocytes in the brain is the Na(+) -dependent clearance of glutamate released by neurons during synaptic transmission. The intracellular Na(+) load collectively generated by these processes converges at the Na,K-ATPase pump, responsible for Na(+) extrusion from the cell, which is achieved at the expense of cellular ATP. These processes represent pivotal mechanisms enabling astrocytes to increase the local availability of metabolic substrates in response to neuronal activity. This review presents basic principles linking the intracellular handling of Na(+) following activity-related transmembrane fluxes in astrocytes and the energy metabolic pathways involved. We propose a role of Na(+) as an energy currency and as a mediator of metabolic signals in the context of neuron-glia interactions. We further discuss the possible impact of the astrocytic syncytium for the distribution and coordination of the metabolic response, and the compartmentation of these processes in cellular microdomains and subcellular organelles. Finally, we illustrate future avenues of investigation into signaling mechanisms aimed at bridging the gap between Na(+) and the metabolic machinery. GLIA 2016;64:1667-1676. PMID:27027636

  9. Unraveling the complex metabolic nature of astrocytes.

    PubMed

    Bouzier-Sore, Anne-Karine; Pellerin, Luc

    2013-01-01

    Since the initial description of astrocytes by neuroanatomists of the nineteenth century, a critical metabolic role for these cells has been suggested in the central nervous system. Nonetheless, it took several technological and conceptual advances over many years before we could start to understand how they fulfill such a role. One of the important and early recognized metabolic function of astrocytes concerns the reuptake and recycling of the neurotransmitter glutamate. But the description of this initial property will be followed by several others including an implication in the supply of energetic substrates to neurons. Indeed, despite the fact that like most eukaryotic non-proliferative cells, astrocytes rely on oxidative metabolism for energy production, they exhibit a prominent aerobic glycolysis capacity. Moreover, this unusual metabolic feature was found to be modulated by glutamatergic activity constituting the initial step of the neurometabolic coupling mechanism. Several approaches, including biochemical measurements in cultured cells, genetic screening, dynamic cell imaging, nuclear magnetic resonance spectroscopy and mathematical modeling, have provided further insights into the intrinsic characteristics giving rise to these key features of astrocytes. This review will provide an account of the different results obtained over several decades that contributed to unravel the complex metabolic nature of astrocytes that make this cell type unique.

  10. Unraveling the complex metabolic nature of astrocytes

    PubMed Central

    Bouzier-Sore, Anne-Karine; Pellerin, Luc

    2013-01-01

    Since the initial description of astrocytes by neuroanatomists of the nineteenth century, a critical metabolic role for these cells has been suggested in the central nervous system. Nonetheless, it took several technological and conceptual advances over many years before we could start to understand how they fulfill such a role. One of the important and early recognized metabolic function of astrocytes concerns the reuptake and recycling of the neurotransmitter glutamate. But the description of this initial property will be followed by several others including an implication in the supply of energetic substrates to neurons. Indeed, despite the fact that like most eukaryotic non-proliferative cells, astrocytes rely on oxidative metabolism for energy production, they exhibit a prominent aerobic glycolysis capacity. Moreover, this unusual metabolic feature was found to be modulated by glutamatergic activity constituting the initial step of the neurometabolic coupling mechanism. Several approaches, including biochemical measurements in cultured cells, genetic screening, dynamic cell imaging, nuclear magnetic resonance spectroscopy and mathematical modeling, have provided further insights into the intrinsic characteristics giving rise to these key features of astrocytes. This review will provide an account of the different results obtained over several decades that contributed to unravel the complex metabolic nature of astrocytes that make this cell type unique. PMID:24130515

  11. Disentangling the Role of Astrocytes in Alcohol Use Disorder.

    PubMed

    Adermark, Louise; Bowers, M Scott

    2016-09-01

    Several laboratories recently identified that astrocytes are critical regulators of addiction machinery. It is now known that astrocyte pathology is a common feature of ethanol (EtOH) exposure in both humans and animal models, as even brief EtOH exposure is sufficient to elicit long-lasting perturbations in astrocyte gene expression, activity, and proliferation. Astrocytes were also recently shown to modulate the motivational properties of EtOH and other strongly reinforcing stimuli. Given the role of astrocytes in regulating glutamate homeostasis, a crucial component of alcohol use disorder (AUD), astrocytes might be an important target for the development of next-generation alcoholism treatments. This review will outline some of the more prominent features displayed by astrocytes, how these properties are influenced by acute and long-term EtOH exposure, and future directions that may help to disentangle astrocytic from neuronal functions in the etiology of AUD. PMID:27476876

  12. Molecular approaches for manipulating astrocytic signaling in vivo

    PubMed Central

    Xie, Alison X.; Petravicz, Jeremy; McCarthy, Ken D.

    2015-01-01

    Astrocytes are the predominant glial type in the central nervous system and play important roles in assisting neuronal function and network activity. Astrocytes exhibit complex signaling systems that are essential for their normal function and the homeostasis of the neural network. Altered signaling in astrocytes is closely associated with neurological and psychiatric diseases, suggesting tremendous therapeutic potential of these cells. To further understand astrocyte function in health and disease, it is important to study astrocytic signaling in vivo. In this review, we discuss molecular tools that enable the selective manipulation of astrocytic signaling, including the tools to selectively activate and inactivate astrocyte signaling in vivo. Lastly, we highlight a few tools in development that present strong potential for advancing our understanding of the role of astrocytes in physiology, behavior, and pathology. PMID:25941472

  13. Disentangling the Role of Astrocytes in Alcohol Use Disorder.

    PubMed

    Adermark, Louise; Bowers, M Scott

    2016-09-01

    Several laboratories recently identified that astrocytes are critical regulators of addiction machinery. It is now known that astrocyte pathology is a common feature of ethanol (EtOH) exposure in both humans and animal models, as even brief EtOH exposure is sufficient to elicit long-lasting perturbations in astrocyte gene expression, activity, and proliferation. Astrocytes were also recently shown to modulate the motivational properties of EtOH and other strongly reinforcing stimuli. Given the role of astrocytes in regulating glutamate homeostasis, a crucial component of alcohol use disorder (AUD), astrocytes might be an important target for the development of next-generation alcoholism treatments. This review will outline some of the more prominent features displayed by astrocytes, how these properties are influenced by acute and long-term EtOH exposure, and future directions that may help to disentangle astrocytic from neuronal functions in the etiology of AUD.

  14. Differential IL-1 synthesis by astrocytes from Theiler's murine encephalomyelitis virus-susceptible and -resistant strains of mice.

    PubMed

    Rubio, N; Capa, L

    1993-07-01

    Increasing evidence shows that interleukin-1 (IL-1) contributes to inflammatory processes, such as experimental allergic encephalomyelitis (EAE) and virus-induced demyelination, inside the central nervous system (CNS). Using primary cultures of mouse astrocytes, we show that these glial cells can be induced to produce IL-1 alpha when infected with Theiler's murine encephalomyelitis virus (TMEV). This was true for astrocytes from SJL/J mice, a strain susceptible to TMEV-induced demyelination. Conversely, BALB/c astrocytes, derived from animals genetically resistant to demyelination, did not produce IL-1 alpha in detectable amounts. Therefore, a differential IL-1 gene expression, which is strain specific, is demonstrated after TMEV infection in astrocytes. The release of IL-1 alpha by SJL astrocytes was studied from kinetic, infectivity, and immunochemical points of view. Since IL-1 plays a critical role in the immune response, its production by astrocytes in some strains of mice may contribute to virus-induced susceptibility and to inflammation associated with this experimental model of multiple sclerosis.

  15. Antioxidants and NOS inhibitors selectively targets manganese-induced cell volume via Na-K-Cl cotransporter-1 in astrocytes.

    PubMed

    Alahmari, Khalid A; Prabhakaran, Harini; Prabhakaran, Krishnan; Chandramoorthy, Harish C; Ramugounder, Ramakrishnan

    2015-06-12

    Manganese has shown to be involved in astrocyte swelling. Several factors such as transporters, exchangers and ion channels are attributed to astrocyte swelling as a result in the deregulation of cell volume. Products of oxidation and nitration have been implied to be involved in the pathophysiology of swelling; however, the direct link and mechanism of manganese induced astrocyte swelling has not been fully elucidated. In the current study, we used rat primary astrocyte cultures to investigate the activation of Na-K-Cl cotransporter-1 (NKCC1) a downstream mechanism for free radical induced astrocyte swelling as a result of manganese toxicity. Our results showed manganese, oxidants and NO donors as potent inducer of oxidation and nitration of NKCC1. Our results further confirmed that manganese (50 μM) increased the total protein, phosphorylation and activity of NKCC1 as well as cell volume (p < 0.05 vs. control). NKCC1 inhibitor (bumetanide), NKCC1-siRNA, antioxidants; DMTU, MnTBAP, tempol, catalase and Vit-E, NOS inhibitor; L-NAME, peroxinitrite scavenger; uric acid all significantly reversed the effects of NKCC1 activation (p < 0.05). From the current investigation we infer that manganese or oxidants and NO induced activation, oxidation/nitration of NKCC1 play an important role in the astrocyte swelling.

  16. Elucidating the Role of Injury-Induced Electric Fields (EFs) in Regulating the Astrocytic Response to Injury in the Mammalian Central Nervous System.

    PubMed

    Baer, Matthew L; Henderson, Scott C; Colello, Raymond J

    2015-01-01

    Injury to the vertebrate central nervous system (CNS) induces astrocytes to change their morphology, to increase their rate of proliferation, and to display directional migration to the injury site, all to facilitate repair. These astrocytic responses to injury occur in a clear temporal sequence and, by their intensity and duration, can have both beneficial and detrimental effects on the repair of damaged CNS tissue. Studies on highly regenerative tissues in non-mammalian vertebrates have demonstrated that the intensity of direct-current extracellular electric fields (EFs) at the injury site, which are 50-100 fold greater than in uninjured tissue, represent a potent signal to drive tissue repair. In contrast, a 10-fold EF increase has been measured in many injured mammalian tissues where limited regeneration occurs. As the astrocytic response to CNS injury is crucial to the reparative outcome, we exposed purified rat cortical astrocytes to EF intensities associated with intact and injured mammalian tissues, as well as to those EF intensities measured in regenerating non-mammalian vertebrate tissues, to determine whether EFs may contribute to the astrocytic injury response. Astrocytes exposed to EF intensities associated with uninjured tissue showed little change in their cellular behavior. However, astrocytes exposed to EF intensities associated with injured tissue showed a dramatic increase in migration and proliferation. At EF intensities associated with regenerating non-mammalian vertebrate tissues, these cellular responses were even more robust and included morphological changes consistent with a regenerative phenotype. These findings suggest that endogenous EFs may be a crucial signal for regulating the astrocytic response to injury and that their manipulation may be a novel target for facilitating CNS repair. PMID:26562295

  17. Elucidating the Role of Injury-Induced Electric Fields (EFs) in Regulating the Astrocytic Response to Injury in the Mammalian Central Nervous System

    PubMed Central

    Baer, Matthew L.; Henderson, Scott C.; Colello, Raymond J.

    2015-01-01

    Injury to the vertebrate central nervous system (CNS) induces astrocytes to change their morphology, to increase their rate of proliferation, and to display directional migration to the injury site, all to facilitate repair. These astrocytic responses to injury occur in a clear temporal sequence and, by their intensity and duration, can have both beneficial and detrimental effects on the repair of damaged CNS tissue. Studies on highly regenerative tissues in non-mammalian vertebrates have demonstrated that the intensity of direct-current extracellular electric fields (EFs) at the injury site, which are 50–100 fold greater than in uninjured tissue, represent a potent signal to drive tissue repair. In contrast, a 10-fold EF increase has been measured in many injured mammalian tissues where limited regeneration occurs. As the astrocytic response to CNS injury is crucial to the reparative outcome, we exposed purified rat cortical astrocytes to EF intensities associated with intact and injured mammalian tissues, as well as to those EF intensities measured in regenerating non-mammalian vertebrate tissues, to determine whether EFs may contribute to the astrocytic injury response. Astrocytes exposed to EF intensities associated with uninjured tissue showed little change in their cellular behavior. However, astrocytes exposed to EF intensities associated with injured tissue showed a dramatic increase in migration and proliferation. At EF intensities associated with regenerating non-mammalian vertebrate tissues, these cellular responses were even more robust and included morphological changes consistent with a regenerative phenotype. These findings suggest that endogenous EFs may be a crucial signal for regulating the astrocytic response to injury and that their manipulation may be a novel target for facilitating CNS repair. PMID:26562295

  18. Fast Subplasma Membrane Ca2+ Transients Control Exo-Endocytosis of Synaptic-Like Microvesicles in Astrocytes

    PubMed Central

    Marchaland, Julie; Calì, Corrado; Voglmaier, Susan M.; Li, Haiyan; Regazzi, Romano; Edwards, Robert H.; Bezzi, Paola

    2009-01-01

    Astrocytes are the most abundant glial cell type in the brain. Although not apposite for long-range rapid electrical communication, astrocytes share with neurons the capacity of chemical signaling via Ca2+-dependent transmitter exocytosis. Despite this recent finding, little is known about the specific properties of regulated secretion and vesicle recycling in astrocytes. Important differences may exist with the neuronal exocytosis, starting from the fact that stimulus-secretion coupling in astrocytes is voltage independent, mediated by G-protein-coupled receptors and the release of Ca2+ from internal stores. Elucidating the spatiotemporal properties of astrocytic exo-endocytosis is, therefore, of primary importance for understanding the mode of communication of these cells and their role in brain signaling. We here take advantage of fluorescent tools recently developed for studying recycling of glutamatergic vesicles at synapses (Voglmaier et al., 2006; Balaji and Ryan, 2007); we combine epifluorescence and total internal reflection fluorescence imaging to investigate with unprecedented temporal and spatial resolution, the stimulus-secretion coupling underlying exo-endocytosis of glutamatergic synaptic-like microvesicles (SLMVs) in astrocytes. Our main findings indicate that (1) exo-endocytosis in astrocytes proceeds with a time course on the millisecond time scale (τexocytosis = 0.24 ± 0.017 s; τendocytosis = 0.26 ± 0.03 s) and (2) exocytosis is controlled by local Ca2+ microdomains. We identified submicrometer cytosolic compartments delimited by endoplasmic reticulum tubuli reaching beneath the plasma membrane and containing SLMVs at which fast (time-to-peak, ∼50 ms) Ca2+ events occurred in precise spatial-temporal correlation with exocytic fusion events. Overall, the above characteristics of transmitter exocytosis from astrocytes support a role of this process in fast synaptic modulation. PMID:18784293

  19. Astrocyte glycogenolysis is triggered by store-operated calcium entry and provides metabolic energy for cellular calcium homeostasis.

    PubMed

    Müller, Margit S; Fox, Rebecca; Schousboe, Arne; Waagepetersen, Helle S; Bak, Lasse K

    2014-04-01

    Astrocytic glycogen, the only storage form of glucose in the brain, has been shown to play a fundamental role in supporting learning and memory, an effect achieved by providing metabolic support for neurons. We have examined the interplay between glycogenolysis and the bioenergetics of astrocytic Ca(2+) homeostasis, by analyzing interdependency of glycogen and store-operated Ca(2+) entry (SOCE), a mechanism in cellular signaling that maintains high endoplasmatic reticulum (ER) Ca(2+) concentration and thus provides the basis for store-dependent Ca(2+) signaling. We stimulated SOCE in primary cultures of murine cerebellar and cortical astrocytes, and determined glycogen content to investigate the effects of SOCE on glycogen metabolism. By blocking glycogenolysis, we tested energetic dependency of SOCE-related Ca(2+) dynamics on glycogenolytic ATP. Our results show that SOCE triggers astrocytic glycogenolysis. Upon inhibition of adenylate cyclase with 2',5'-dideoxyadenosine, glycogen content was no longer significantly different from that in unstimulated control cells, indicating that SOCE triggers astrocytic glycogenolysis in a cAMP-dependent manner. When glycogenolysis was inhibited in cortical astrocytes by 1,4-dideoxy-1,4-imino-D-arabinitol, the amount of Ca(2+) loaded into ER via sarco/endoplasmic reticulum Ca(2)-ATPase (SERCA) was reduced, which suggests that SERCA pumps preferentially metabolize glycogenolytic ATP. Our study demonstrates SOCE as a novel pathway in stimulating astrocytic glycogenolysis. We also provide first evidence for a new functional role of brain glycogen, in providing local ATP to SERCA, thus establishing the bioenergetic basis for astrocytic Ca(2+) signaling. This mechanism could offer a novel explanation for the impact of glycogen on learning and memory.

  20. IL-17 enhancement of the IL-6 signaling cascade in astrocytes.

    PubMed

    Ma, Xiangyu; Reynolds, Stephanie L; Baker, Brandi J; Li, Xingang; Benveniste, Etty N; Qin, Hongwei

    2010-05-01

    Astrocytes have important physiological roles in CNS homeostasis and serve as a bridge between the CNS and immune system. IL-17 and IL-6 are important in many CNS disorders characterized by neuroinflammation. We examined the role of IL-17 on the IL-6 signaling cascade in primary astrocytes. IL-17 functioned in a synergistic manner with IL-6 to induce IL-6 expression in astrocytes. The synergistic effect involved numerous signaling pathways including NF-kappaB, JNK MAPK, and p38 MAPK. The NF-kappaB pathway inhibitor BAY-11, JNK inhibitor JNKi II, and p38 inhibitor SB203580 suppressed the synergistic effect of IL-6 and IL-17 on IL-6 expression. IL-17 synergized with IL-6 to enhance the recruitment of activated NF-kappaB p65, c-Fos, c-Jun, and the histone acetyltransferases CREB-binding protein and p300 to the IL-6 promoter in vivo to induce IL-6 transcription. This was accompanied by enhanced acetylation of histones H3 and H4 on the IL-6 promoter. Moreover, we elucidated an important role for suppressor of cytokine signaling (SOCS) 3 in IL-17 enhancement of IL-6 signaling in astrocytes. SOCS3 small interfering RNA knockdown and SOCS3 deletion in astrocytes augmented the synergistic effect of IL-6 and IL-17 due to an enhancement of activation of the NF-kappaB and MAPK pathways. These results indicate that astrocytes can serve as a target of Th17 cells and IL-17 in the CNS, and SOCS3 participates in IL-17 functions in the CNS as a negative feedback regulator.

  1. Astrocytic transactivation by alpha2A-adrenergic and 5-HT2B serotonergic signaling.

    PubMed

    Peng, Liang; Li, Baoman; Du, Ting; Kong, Ebenezer K C; Hu, Xiaoling; Zhang, Shiquen; Shan, Xiaolei; Zhang, Meixia

    2010-11-01

    EGF receptor transactivation has been known for more than ten years. It is a signal pathway in which a G-protein-coupled receptor (GPCR) signal leads to release of a growth factor, which in turn activates the EGF receptor-tyrosine kinase in the same or adjacent cells. Astrocytes express a number of GPCRs and play key roles in brain function. Astrocytic transactivation is of special interest, since its autocrine effect may regulate gene expression and alter cell functions in the cells themselves and its paracrine effect may provide additional opportunities for cross-talk between astrocytes and their neighbors, such as neurons. The signal pathways of EGF transactivation are complicated. This does not only apply to the pathways leading to shedding of growth factor(s), but also to the downstream signal pathways of the EGF receptor, i.e., MAPK and PI3K. The latter may vary according to the type of growth factor released, the sites of tyrosine phosphorylation on the EGF receptor, and the duration of the phosphorylation. Using primary cell cultures we have found that dexmedetomidine, a specific alpha(2)-adrenergic receptor, induced shedding of HB-EGF from astrocytes, which in turn transactivated EGF receptors and stimulated astrocytic c-Fos and FosB expression. At the same time released HB-EGF protected neurons from injury caused by H(2)O(2). We have also confirmed dexmedetomidine transactivation in the brain in vivo. EGF transactivation by 5-HT(2B) receptor stimulation was responsible for up-regulation of cPLA(2) in astrocytes by fluoxetine, an antidepressant and inhibitor of the serotonin transporter, which also is a specific 5-HT(2B) agonist. PMID:20450946

  2. Effects of lipopolysaccharide on oligodendrocyte progenitor cells are mediated by astrocytes and microglia.

    PubMed

    Pang, Y; Cai, Z; Rhodes, P G

    2000-11-15

    Oligodendrocytes are the primary cells injured in periventricular leukomalacia (PVL), a predominant form of brain white matter lesion in preterm infants. To explore the possible linkage between white matter injury and maternal infection, purified rat O-2A progenitor (Oligodendrocyte-type 2 astrocyte progenitor) cell cultures were used as a model in studying the effects of lipopolysaccharide (LPS), an endotoxin, on survival and differentiation of oligodendrocytes and the involvement of other glial cells in the effects of LPS. O-2A progenitor cells were cultured from optic nerves of 7-day-old rat pups in a chemically defined medium (CDM). Astrocyte and microglia cell cultures were prepared from the cortex of 1-day-old rat brains in the CDM. Direct treatment of LPS (1 microg/ml) to O-2A cells had no effect on viability or differentiation of these cells. When O-2A progenitor cells were cultured in the conditioned medium obtained from either astrocyte or microglial cell cultures for 48 hr, survival rate and differentiation of O-2A cells into mature oligodendrocytes were greatly enhanced as measured by the MTT assay and immunocytochemistry. The conditioned medium obtained from astrocytes or microglia treated with LPS for 48 hr, however, failed to show such a promotional effect on viability and differentiation of O-2A cells. When 5 microg/ml LPS was used to stimulate astrocytes or microglia, the conditioned medium from these glial cell cultures caused O-2A cell injury. The overall results indicate that astrocytes and microglia may promote viability and differentiation of O-2A progenitor cells under physiological conditions, but they may also mediate cytotoxic effects of LPS on oligodendrocytes under an infectious disease biochemical environment.

  3. Neuroinflammatory TNFα Impairs Memory via Astrocyte Signaling.

    PubMed

    Habbas, Samia; Santello, Mirko; Becker, Denise; Stubbe, Hiltrud; Zappia, Giovanna; Liaudet, Nicolas; Klaus, Federica R; Kollias, George; Fontana, Adriano; Pryce, Christopher R; Suter, Tobias; Volterra, Andrea

    2015-12-17

    The occurrence of cognitive disturbances upon CNS inflammation or infection has been correlated with increased levels of the cytokine tumor necrosis factor-α (TNFα). To date, however, no specific mechanism via which this cytokine could alter cognitive circuits has been demonstrated. Here, we show that local increase of TNFα in the hippocampal dentate gyrus activates astrocyte TNF receptor type 1 (TNFR1), which in turn triggers an astrocyte-neuron signaling cascade that results in persistent functional modification of hippocampal excitatory synapses. Astrocytic TNFR1 signaling is necessary for the hippocampal synaptic alteration and contextual learning-memory impairment observed in experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis (MS). This process may contribute to the pathogenesis of cognitive disturbances in MS, as well as in other CNS conditions accompanied by inflammatory states or infections. PMID:26686654

  4. Activity-dependent regulation of astrocyte GAT levels during synaptogenesis.

    PubMed

    Muthukumar, Allie K; Stork, Tobias; Freeman, Marc R

    2014-10-01

    Astrocytic uptake of GABA through GABA transporters (GATs) is an important mechanism regulating excitatory/inhibitory balance in the nervous system; however, mechanisms by which astrocytes regulate GAT levels are undefined. We found that at mid-pupal stages the Drosophila melanogaster CNS neuropil was devoid of astrocyte membranes and synapses. Astrocyte membranes subsequently infiltrated the neuropil coordinately with synaptogenesis, and astrocyte ablation reduced synapse numbers by half, indicating that Drosophila astrocytes are pro-synaptogenic. Shortly after synapses formed in earnest, GAT was upregulated in astrocytes. Ablation or silencing of GABAergic neurons or disruption of metabotropic GABA receptor 1 and 2 (GABA(B)R1/2) signaling in astrocytes led to a decrease in astrocytic GAT. Notably, developmental depletion of astrocytic GABA(B)R1/2 signaling suppressed mechanosensory-induced seizure activity in mutants with hyperexcitable neurons. These data reveal that astrocytes actively modulate GAT expression via metabotropic GABA receptor signaling and highlight the importance of precise regulation of astrocytic GAT in modulation of seizure activity.

  5. Uptake of dimercaptosuccinate-coated magnetic iron oxide nanoparticles by cultured brain astrocytes

    NASA Astrophysics Data System (ADS)

    Geppert, Mark; Hohnholt, Michaela C.; Thiel, Karsten; Nürnberger, Sylvia; Grunwald, Ingo; Rezwan, Kurosch; Dringen, Ralf

    2011-04-01

    Magnetic iron oxide nanoparticles (Fe-NP) are currently considered for various diagnostic and therapeutic applications in the brain. However, little is known on the accumulation and biocompatibility of such particles in brain cells. We have synthesized and characterized dimercaptosuccinic acid (DMSA) coated Fe-NP and have investigated their uptake by cultured brain astrocytes. DMSA-coated Fe-NP that were dispersed in physiological medium had an average hydrodynamic diameter of about 60 nm. Incubation of cultured astrocytes with these Fe-NP caused a time- and concentration-dependent accumulation of cellular iron, but did not lead within 6 h to any cell toxicity. After 4 h of incubation with 100-4000 µM iron supplied as Fe-NP, the cellular iron content reached levels between 200 and 2000 nmol mg - 1 protein. The cellular iron content after exposure of astrocytes to Fe-NP at 4 °C was drastically lowered compared to cells that had been incubated at 37 °C. Electron microscopy revealed the presence of Fe-NP-containing vesicles in cells that were incubated with Fe-NP at 37 °C, but not in cells exposed to the nanoparticles at 4 °C. These data demonstrate that cultured astrocytes efficiently take up DMSA-coated Fe-NP in a process that appears to be saturable and strongly depends on the incubation temperature.

  6. Differential microglial and astrocytic response to bacterial and viral infection in the developing hippocampus of neonatal rats.

    PubMed

    Patro, Nisha; Singh, Kavita; Patro, Ishan

    2013-08-01

    Polyinosinic:polycytidylic acid (Poly I:C; 5 mg/kg body weight, ip) and lipopolysaccharide (LPS; 0.3 mg/kg body weight, ip) induced microglial and astrocytic activation in Sprague Dawley rats. Higher microglial and astrocytic activities were noticed in Poly I:C infused rats throughout the hippocampus till postnatal day 21 with a comparatively weaker response in LPS group. However, LPS induced inflammation persisted even after postnatal day 21, indicating thereby, that the Poly I:C (viral mimic) produces an acute inflammation, while LPS (bacterial endotoxin) produces chronic inflammation when exposed during early neonatal life.

  7. Label-free based quantitative proteomics analysis of primary neonatal porcine Leydig cells exposed to the persistent contaminant 3-methylsulfonyl-DDE.

    PubMed

    Kalayou, Shewit; Granum, Cesilie; Berntsen, Hanne Friis; Groseth, Per Kristian; Verhaegen, Steven; Connolly, Lisa; Brandt, Ingvar; de Souza, Gustavo Antonio; Ropstad, Erik

    2016-03-30

    Evidence that persistent environmental pollutants may target the male reproductive system is increasing. The male reproductive system is regulated by secretion of testosterone by testicular Leydig cells, and perturbation of Leydig cell function may have ultimate consequences. 3-Methylsulfonyl-DDE (3-MeSO2-DDE) is a potent adrenal toxicants formed from the persistent insecticide DDT. Although studies have revealed the endocrine disruptive effect of 3-MeSO2-DDE, the underlying mechanisms at cellular level in steroidogenic Leydig cells remains to be established. The current study addresses the effect of 3-MeSO2-DDE on viability, hormone production and proteome response of primary neonatal porcine Leydig cells. The AlamarBlue™ assay was used to evaluate cell viability. Solid phase radioimmunoassay was used to measure concentration of hormones produced by both unstimulated and Luteinizing hormone (LH)-stimulated Leydig cells following 48h exposure. Protein samples from Leydig cells exposed to a non-cytotoxic concentration of 3-MeSO2-DDE (10 μM) were subjected to nano-LC-MS/MS and analyzed on a Q Exactive mass spectrometer and quantified using label-free quantitative algorithm. Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) were carried out for functional annotation and identification of protein interaction networks. 3-MeSO2-DDE regulated Leydig cell steroidogenesis differentially depending on cell culture condition. Whereas its effect on testosterone secretion at basal condition was stimulatory, the effect on LH-stimulated cells was inhibitory. From triplicate experiments, a total of 6804 proteins were identified in which the abundance of 86 proteins in unstimulated Leydig cells and 145 proteins in LH-stimulated Leydig cells was found to be significantly regulated in response to 3-MeSO2-DDE exposure. These proteins not only are the first reported in relation to 3-MeSO2-DDE exposure, but also display small number of proteins shared between culture conditions

  8. Target cell-specific modulation of neuronal activity by astrocytes

    NASA Astrophysics Data System (ADS)

    Kozlov, A. S.; Angulo, M. C.; Audinat, E.; Charpak, S.

    2006-06-01

    Interaction between astrocytes and neurons enriches the behavior of brain circuits. By releasing glutamate and ATP, astrocytes can directly excite neurons and modulate synaptic transmission. In the rat olfactory bulb, we demonstrate that the release of GABA by astrocytes causes long-lasting and synchronous inhibition of mitral and granule cells. In addition, astrocytes release glutamate, leading to a selective activation of granule-cell NMDA receptors. Thus, by releasing excitatory and inhibitory neurotransmitters, astrocytes exert a complex modulatory control on the olfactory network. glutamate | GABA | inhibition | olfactory bulb | synchronization

  9. Role of astrocytes in brain function and disease.

    PubMed

    Sidoryk-Wegrzynowicz, Marta; Wegrzynowicz, Michal; Lee, Eunsook; Bowman, Aaron B; Aschner, Michael

    2011-01-01

    Astrocytes assume multiple roles in maintaining an optimally suited milieu for neuronal function. Select astrocytic functions include the maintenance of redox potential, the production of trophic factors, the regulation of neurotransmitter and ion concentrations, and the removal of toxins and debris from the cerebrospinal fluid (CSF). Impairments in these and other functions, as well as physiological reactions of astrocytes to injury, can trigger or exacerbate neuronal dysfunction. This review addresses select metabolic interactions between neurons and astrocytes and emphasizes the role of astrocytes in mediating and amplifying the progression of several neurodegenerative disorders, such as Parkinson's disease (PD), hepatic encephalopathy (HE), hyperammonemia (HA), Alzheimer's disease (AD), and ischemia.

  10. Regional astrocyte allocation regulates CNS synaptogenesis and repair.

    PubMed

    Tsai, Hui-Hsin; Li, Huiliang; Fuentealba, Luis C; Molofsky, Anna V; Taveira-Marques, Raquel; Zhuang, Helin; Tenney, April; Murnen, Alice T; Fancy, Stephen P J; Merkle, Florian; Kessaris, Nicoletta; Alvarez-Buylla, Arturo; Richardson, William D; Rowitch, David H

    2012-07-20

    Astrocytes, the most abundant cell population in the central nervous system (CNS), are essential for normal neurological function. We show that astrocytes are allocated to spatial domains in mouse spinal cord and brain in accordance with their embryonic sites of origin in the ventricular zone. These domains remain stable throughout life without evidence of secondary tangential migration, even after acute CNS injury. Domain-specific depletion of astrocytes in ventral spinal cord resulted in abnormal motor neuron synaptogenesis, which was not rescued by immigration of astrocytes from adjoining regions. Our findings demonstrate that region-restricted astrocyte allocation is a general CNS phenomenon and reveal intrinsic limitations of the astroglial response to injury.

  11. New tools for investigating astrocyte-to-neuron communication

    PubMed Central

    Li, Dongdong; Agulhon, Cendra; Schmidt, Elke; Oheim, Martin; Ropert, Nicole

    2013-01-01

    Gray matter protoplasmic astrocytes extend very thin processes and establish close contacts with synapses. It has been suggested that the release of neuroactive gliotransmitters at the tripartite synapse contributes to information processing. However, the concept of calcium (Ca2+)-dependent gliotransmitter release from astrocytes, and the release mechanisms are being debated. Studying astrocytes in their natural environment is challenging because: (i) astrocytes are electrically silent; (ii) astrocytes and neurons express an overlapping repertoire of transmembrane receptors; (iii) the size of astrocyte processes in contact with synapses are below the resolution of confocal and two-photon microscopes (iv) bulk-loading techniques using fluorescent Ca2+ indicators lack cellular specificity. In this review, we will discuss some limitations of conventional methodologies and highlight the interest of novel tools and approaches for studying gliotransmission. Genetically encoded Ca2+ indicators (GECIs), light-gated channels, and exogenous receptors are being developed to selectively read out and stimulate astrocyte activity. Our review discusses emerging perspectives on: (i) the complexity of astrocyte Ca2+ signaling revealed by GECIs; (ii) new pharmacogenetic and optogenetic approaches to activate specific Ca2+ signaling pathways in astrocytes; (iii) classical and new techniques to monitor vesicle fusion in cultured astrocytes; (iv) possible strategies to express specifically reporter genes in astrocytes. PMID:24194698

  12. Activity-dependent regulation of astrocyte GAT levels during synaptogenesis

    PubMed Central

    Muthukumar, Allie K.; Stork, Tobias; Freeman, Marc R.

    2014-01-01

    Astrocytic uptake of GABA through GABA transporters (GATs) is an important mechanism regulating excitatory/inhibitory balance in the nervous system, however mechanisms by which astrocytes regulate GAT levels are undefined. Here we show at mid-pupal stages the Drosophila CNS neuropil is devoid of astrocyte membranes and synapses. Astrocyte membranes subsequently infiltrate the neuropil coordinate with synaptogenesis and a strocyte ablation reduces synapse numbers by half, indicating that Drosophila astrocytes are pro-synaptogenic. Shortly after synapses form in earnest, the GABA transporter, GAT, is up-regulated in astrocytes. Ablation or silencing of GABAergic neurons or disruption of metabotropic GABA receptor (GABABR1/2) signaling in astrocytes leads to decreased astrocytic GAT levels. Interestingly, developmental depletion of astrocytic GABABR1/2 signaling suppresses mechanosensory-induced seizure activity in mutants with hyperexcitable neurons. These data reveal astrocytes actively modulate GAT expression via metabotropic GABA receptor signaling, and highlight the importance of precise regulation of astrocytic GAT in modulation of seizure activity. PMID:25151265

  13. Recent molecular approaches to understanding astrocyte function in vivo

    PubMed Central

    Davila, David; Thibault, Karine; Fiacco, Todd A.; Agulhon, Cendra

    2013-01-01

    Astrocytes are a predominant glial cell type in the nervous systems, and are becoming recognized as important mediators of normal brain function as well as neurodevelopmental, neurological, and neurodegenerative brain diseases. Although numerous potential mechanisms have been proposed to explain the role of astrocytes in the normal and diseased brain, research into the physiological relevance of these mechanisms in vivo is just beginning. In this review, we will summarize recent developments in innovative and powerful molecular approaches, including knockout mouse models, transgenic mouse models, and astrocyte-targeted gene transfer/expression, which have led to advances in understanding astrocyte biology in vivo that were heretofore inaccessible to experimentation. We will examine the recently improved understanding of the roles of astrocytes – with an emphasis on astrocyte signaling – in the context of both the healthy and diseased brain, discuss areas where the role of astrocytes remains debated, and suggest new research directions. PMID:24399932

  14. Metabolic pathways for glucose in astrocytes.

    PubMed

    Wiesinger, H; Hamprecht, B; Dringen, R

    1997-09-01

    Cultured astroglial cells are able to utilize the monosaccharides glucose, mannose, or fructose as well as the sugar alcohol sorbitol as energy fuel. Astroglial uptake of the aldoses is carrier-mediated, whereas a non-saturable transport mechanism is operating for fructose and sorbitol. The first metabolic step for all sugars, including fructose being generated by enzymatic oxidation of sorbitol, is phosphorylation by hexokinase. Besides glucose only mannose may serve as substrate for build-up of astroglial glycogen. Whereas glycogen synthase appears to be present in astrocytes as well as neurons, the exclusive localization of glycogen phosphorylase in astrocytes and ependymal cells of central nervous tissue correlates well with the occurrence of glycogen in these cells. The identification of lactic acid rather than glucose as degradation product of astroglial glycogen appears to render the presence of glucose-6-phosphatase in cultured astrocytes an enigma. The colocalization of pyruvate carboxylase, phosphenolpyruvate carboxykinase and fructose-1,6-bisphosphatase points to astrocytes as being the gluconeogenic cell type of the CNS. PMID:9298844

  15. Astrocytic adaptation during cerebral angiogenesis follows the new vessel formation induced through chronic hypoxia in adult mouse cortex

    NASA Astrophysics Data System (ADS)

    Masamoto, Kazuto; Kanno, Iwao

    2014-03-01

    We examined longitudinal changes of the neuro-glia-vascular unit during cerebral angiogenesis induced through chronic hypoxia in the adult mouse cortex. Tie2-GFP mice in which the vascular endothelial cells expressed green fluorescent proteins (GFP) were exposed to chronic hypoxia, while the spatiotemporal developments of the cortical capillary sprouts and the neighboring astrocytic remodeling were characterized with repeated two-photon microscopy. The capillary sprouts appeared at early phases of the hypoxia adaptation (1-2 weeks), while the morphological changes of the astrocytic soma and processes were not detected in this phase. In the later phases of the hypoxia adaptation (> 2 weeks), the capillary sprouts created a new connection with existing capillaries, and its neighboring astrocytes extended their processes to the newly-formed vessels. The findings show that morphological adaptation of the astrocytes follow the capillary development during the hypoxia adaptation, which indicate that the newly-formed vessels provoke cellular interactions with the neighboring astrocytes to strengthen the functional blood-brain barrier.

  16. Activated microglia impairs neuroglial interaction by opening Cx43 hemichannels in hippocampal astrocytes.

    PubMed

    Abudara, Verónica; Roux, Lisa; Dallérac, Glenn; Matias, Isabelle; Dulong, Jérôme; Mothet, Jean Pierre; Rouach, Nathalie; Giaume, Christian

    2015-05-01

    Glia plays an active role in neuronal functions and dysfunctions, some of which depend on the expression of astrocyte connexins, the gap junction channel and hemichannel proteins. Under neuroinflammation triggered by the endotoxin lipopolysacharide (LPS), microglia is primary stimulated and releases proinflammatory agents affecting astrocytes and neurons. Here, we investigate the effects of such microglial activation on astrocyte connexin-based channel functions and their consequences on synaptic activity in an ex vivo model. We found that LPS induces astroglial hemichannel opening in acute hippocampal slices while no change is observed in gap junctional communication. Based on pharmacological and genetic approaches we found that the LPS-induced hemichannel opening is mainly due to Cx43 hemichannel activity. This process primarily requires a microglial stimulation resulting in the release of at least two proinflammatory cytokines, IL-1β and TNF-α. Consequences of the hemichannel-mediated increase in membrane permeability are a calcium rise in astrocytes and an enhanced glutamate release associated to a reduction in excitatory synaptic activity of pyramidal neurons in response to Schaffer's collateral stimulation. As a whole our findings point out astroglial hemichannels as key determinants of the impairment of synaptic transmission during neuroinflammation.

  17. Methamphetamine Alters the Normal Progression by Inducing Cell Cycle Arrest in Astrocytes

    PubMed Central

    Jackson, Austin R.; Shah, Ankit; Kumar, Anil

    2014-01-01

    Methamphetamine (MA) is a potent psychostimulant with a high addictive capacity, which induces many deleterious effects on the brain. Chronic MA abuse leads to cognitive dysfunction and motor impairment. MA affects many cells in the brain, but the effects on astrocytes of repeated MA exposure is not well understood. In this report, we used Gene chip array to analyze the changes in the gene expression profile of primary human astrocytes treated with MA for 3 days. Range of genes were found to be differentially regulated, with a large number of genes significantly downregulated, including NEK2, TTK, TOP2A, and CCNE2. Gene ontology and pathway analysis showed a highly significant clustering of genes involved in cell cycle progression and DNA replication. Further pathway analysis showed that the genes downregulated by multiple MA treatment were critical for G2/M phase progression and G1/S transition. Cell cycle analysis of SVG astrocytes showed a significant reduction in the percentage of cell in the G2/M phase with a concomitant increase in G1 percentage. This was consistent with the gene array and validation data, which showed that repeated MA treatment downregulated the genes associated with cell cycle regulation. This is a novel finding, which explains the effect of MA treatment on astrocytes and has clear implication in neuroinflammation among the drug abusers. PMID:25290377

  18. Ammonium Activates Ouabain-Activated Signalling Pathway in Astrocytes: Therapeutic Potential of Ouabain Antagonist

    PubMed Central

    Song, Dan; Du, Ting

    2014-01-01

    The causal role of ammonium in hepatic encephalopathy was identified in 1930s. Astroglial cells are primary cellular elements of hepatic encephalopathy which conceptually, can be considered a toxic astrogliopathology. Previously we have reported that acute exposure to ammonium activated ouabain/Na,K-ATPase signalling pathway, which includes Src, EGF receptor, Raf, Ras, MEK and ERK1/2. Chronic incubation of astrocytes with ammonium increased production of endogenous ouabain-like compound. Ouabain antagonist canrenone abolished effects of ammonium on astrocytic swelling, ROS production, and upregulation of gene expression and function of TRPC1 and Cav1.2. However, ammonium induces multiple pathological modifications in astrocytes, and some of them may be not related to this signalling pathway. In this review, we focus on the effect of ammonium on ouabain/Na,K-ATPase signalling pathway and its involvement in ammonium-induced ROS production, cell swelling and aberration of Ca2+ signals in astrocytes. We also briefly discuss Na,K-ATPase, EGF receptor, endogenous ouabain and ouabain antagonist. PMID:25342941

  19. Ammonium activates ouabain-activated signalling pathway in astrocytes: therapeutic potential of ouabain antagonist.

    PubMed

    Song, Dan; Du, Ting

    2014-07-01

    The causal role of ammonium in hepatic encephalopathy was identified in 1930s. Astroglial cells are primary cellular elements of hepatic encephalopathy which conceptually, can be considered a toxic astrogliopathology. Previously we have reported that acute exposure to ammonium activated ouabain/Na,K-ATPase signalling pathway, which includes Src, EGF receptor, Raf, Ras, MEK and ERK1/2. Chronic incubation of astrocytes with ammonium increased production of endogenous ouabain-like compound. Ouabain antagonist canrenone abolished effects of ammonium on astrocytic swelling, ROS production, and upregulation of gene expression and function of TRPC1 and Cav1.2. However, ammonium induces multiple pathological modifications in astrocytes, and some of them may be not related to this signalling pathway. In this review, we focus on the effect of ammonium on ouabain/Na,K-ATPase signalling pathway and its involvement in ammonium-induced ROS production, cell swelling and aberration of Ca(2+) signals in astrocytes. We also briefly discuss Na,K-ATPase, EGF receptor, endogenous ouabain and ouabain antagonist. PMID:25342941

  20. The connexin43 mimetic peptide Gap19 inhibits hemichannels without altering gap junctional communication in astrocytes

    PubMed Central

    Abudara, Verónica; Bechberger, John; Freitas-Andrade, Moises; De Bock, Marijke; Wang, Nan; Bultynck, Geert; Naus, Christian C.; Leybaert, Luc; Giaume, Christian

    2014-01-01

    In the brain, astrocytes represent the cellular population that expresses the highest amount of connexins (Cxs). This family of membrane proteins is the molecular constituent of gap junction channels and hemichannels that provide pathways for direct cytoplasm-to-cytoplasm and inside-out exchange, respectively. Both types of Cx channels are permeable to ions and small signaling molecules allowing astrocytes to establish dynamic interactions with neurons. So far, most pharmacological approaches currently available do not distinguish between these two channel functions, stressing the need to develop new specific molecular tools. In astrocytes two major Cxs are expressed, Cx43 and Cx30, and there is now evidence indicating that at least Cx43 operates as a gap junction channel as well as a hemichannel in these cells. Based on studies in primary cultures as well as in acute hippocampal slices, we report here that Gap19, a nonapeptide derived from the cytoplasmic loop of Cx43, inhibits astroglial Cx43 hemichannels in a dose-dependent manner, without affecting gap junction channels. This peptide, which not only selectively inhibits hemichannels but is also specific for Cx43, can be delivered in vivo in mice as TAT-Gap19, and displays penetration into the brain parenchyma. As a result, Gap19 combined with other tools opens up new avenues to decipher the role of Cx43 hemichannels in interactions between astrocytes and neurons in physiological as well as pathological situations. PMID:25374505

  1. Proton Fall or Bicarbonate Rise: GLYCOLYTIC RATE IN MOUSE ASTROCYTES IS PAVED BY INTRACELLULAR ALKALINIZATION.

    PubMed

    Theparambil, Shefeeq M; Weber, Tobias; Schmälzle, Jana; Ruminot, Ivàn; Deitmer, Joachim W

    2016-09-01

    Glycolysis is the primary step for major energy production in the cell. There is strong evidence suggesting that glucose consumption and rate of glycolysis are highly modulated by cytosolic pH/[H(+)], but those can also be stimulated by an increase in the intracellular [HCO3 (-)]. Because proton and bicarbonate shift concomitantly, it remained unclear whether enhanced glucose consumption and glycolytic rate were mediated by the changes in intracellular [H(+)] or [HCO3 (-)]. We have asked whether glucose metabolism is enhanced by either a fall in intracellular [H(+)] or a rise in intracellular [HCO3 (-)], or by both, in mammalian astrocytes. We have recorded intracellular glucose in mouse astrocytes using a FRET-based nanosensor, while imposing different intracellular [H(+)] and [CO2]/[HCO3 (-)]. Glucose consumption and glycolytic rate were augmented by a fall in intracellular [H(+)], irrespective of a concomitant rise or fall in intracellular [HCO3 (-)]. Transport of HCO3 (-) into and out of astrocytes by the electrogenic sodium bicarbonate cotransporter (NBCe1) played a crucial role in causing changes in intracellular pH and [HCO3 (-)], but was not obligatory for the pH-dependent changes in glucose metabolism. Our results clearly show that it is the cytosolic pH that modulates glucose metabolism in cortical astrocytes, and possibly also in other cell types. PMID:27422823

  2. Proton Fall or Bicarbonate Rise: GLYCOLYTIC RATE IN MOUSE ASTROCYTES IS PAVED BY INTRACELLULAR ALKALINIZATION.

    PubMed

    Theparambil, Shefeeq M; Weber, Tobias; Schmälzle, Jana; Ruminot, Ivàn; Deitmer, Joachim W

    2016-09-01

    Glycolysis is the primary step for major energy production in the cell. There is strong evidence suggesting that glucose consumption and rate of glycolysis are highly modulated by cytosolic pH/[H(+)], but those can also be stimulated by an increase in the intracellular [HCO3 (-)]. Because proton and bicarbonate shift concomitantly, it remained unclear whether enhanced glucose consumption and glycolytic rate were mediated by the changes in intracellular [H(+)] or [HCO3 (-)]. We have asked whether glucose metabolism is enhanced by either a fall in intracellular [H(+)] or a rise in intracellular [HCO3 (-)], or by both, in mammalian astrocytes. We have recorded intracellular glucose in mouse astrocytes using a FRET-based nanosensor, while imposing different intracellular [H(+)] and [CO2]/[HCO3 (-)]. Glucose consumption and glycolytic rate were augmented by a fall in intracellular [H(+)], irrespective of a concomitant rise or fall in intracellular [HCO3 (-)]. Transport of HCO3 (-) into and out of astrocytes by the electrogenic sodium bicarbonate cotransporter (NBCe1) played a crucial role in causing changes in intracellular pH and [HCO3 (-)], but was not obligatory for the pH-dependent changes in glucose metabolism. Our results clearly show that it is the cytosolic pH that modulates glucose metabolism in cortical astrocytes, and possibly also in other cell types.

  3. Blast shockwaves propagate Ca(2+) activity via purinergic astrocyte networks in human central nervous system cells.

    PubMed

    Ravin, Rea; Blank, Paul S; Busse, Brad; Ravin, Nitay; Vira, Shaleen; Bezrukov, Ludmila; Waters, Hang; Guerrero-Cazares, Hugo; Quinones-Hinojosa, Alfredo; Lee, Philip R; Fields, R Douglas; Bezrukov, Sergey M; Zimmerberg, Joshua

    2016-01-01

    In a recent study of the pathophysiology of mild, blast-induced traumatic brain injury (bTBI) the exposure of dissociated, central nervous system (CNS) cells to simulated blast resulted in propagating waves of elevated intracellular Ca(2+). Here we show, in dissociated human CNS cultures, that these calcium waves primarily propagate through astrocyte-dependent, purinergic signaling pathways that are blocked by P2 antagonists. Human, compared to rat, astrocytes had an increased calcium response and prolonged calcium wave propagation kinetics, suggesting that in our model system rat CNS cells are less responsive to simulated blast. Furthermore, in response to simulated blast, human CNS cells have increased expressions of a reactive astrocyte marker, glial fibrillary acidic protein (GFAP) and a protease, matrix metallopeptidase 9 (MMP-9). The conjoint increased expression of GFAP and MMP-9 and a purinergic ATP (P2) receptor antagonist reduction in calcium response identifies both potential mechanisms for sustained changes in brain function following primary bTBI and therapeutic strategies targeting abnormal astrocyte activity. PMID:27162174

  4. Blast shockwaves propagate Ca2+ activity via purinergic astrocyte networks in human central nervous system cells

    PubMed Central

    Ravin, Rea; Blank, Paul S.; Busse, Brad; Ravin, Nitay; Vira, Shaleen; Bezrukov, Ludmila; Waters, Hang; Guerrero-Cazares, Hugo; Quinones-Hinojosa, Alfredo; Lee, Philip R.; Fields, R. Douglas; Bezrukov, Sergey M.; Zimmerberg, Joshua

    2016-01-01

    In a recent study of the pathophysiology of mild, blast-induced traumatic brain injury (bTBI) the exposure of dissociated, central nervous system (CNS) cells to simulated blast resulted in propagating waves of elevated intracellular Ca2+. Here we show, in dissociated human CNS cultures, that these calcium waves primarily propagate through astrocyte-dependent, purinergic signaling pathways that are blocked by P2 antagonists. Human, compared to rat, astrocytes had an increased calcium response and prolonged calcium wave propagation kinetics, suggesting that in our model system rat CNS cells are less responsive to simulated blast. Furthermore, in response to simulated blast, human CNS cells have increased expressions of a reactive astrocyte marker, glial fibrillary acidic protein (GFAP) and a protease, matrix metallopeptidase 9 (MMP-9). The conjoint increased expression of GFAP and MMP-9 and a purinergic ATP (P2) receptor antagonist reduction in calcium response identifies both potential mechanisms for sustained changes in brain function following primary bTBI and therapeutic strategies targeting abnormal astrocyte activity. PMID:27162174

  5. Chronic stress-induced disruption of the astrocyte network is driven by structural atrophy and not loss of astrocytes.

    PubMed

    Tynan, Ross J; Beynon, Sarah B; Hinwood, Madeleine; Johnson, Sarah J; Nilsson, Michael; Woods, Jason J; Walker, Frederick R

    2013-07-01

    Chronic stress is well recognized to decrease the number of GFAP⁺ astrocytes within the prefrontal cortex (PFC). Recent research, however, has suggested that our understanding of how stress alters astrocytes may be incomplete. Specifically, chronic stress has been shown to induce a unique form of microglial remodelling, but it is not yet clear whether astrocytes also undergo similar structural modifications. Such alterations may be significant given the role of astrocytes in modulating synaptic function. Accordingly, in the current study we have examined changes in astrocyte morphology following exposure to chronic stress in adult rats, using three-dimensional digital reconstructions of astrocytes. Our analysis indicated that chronic stress produced profound atrophy of astrocyte process length, branching and volume. We additionally examined changes in astrocyte-specific S100β, which are both a putative astrocyte marker and a protein whose expression is associated with astrocyte distress. While we found that S100β levels were increased by stress, this increase was not correlated with atrophy. We further established that while chronic stress was associated with a decrease in astrocyte numbers when GFAP labelling was used as a marker, we could find no evidence of a decrease in the total number of cells, based on Nissl staining, or in the number of S100β⁺ cells. This finding suggests that chronic stress may not actually reduce astrocyte numbers and may instead selectively decrease GFAP expression. The results of the current study are significant as they indicate stress-induced astrocyte-mediated disturbances may not be due to a loss of cells but rather due to significant remodeling of the astrocyte network.

  6. Astrocyte Reactivity Following Blast Exposure Involves Aberrant Histone Acetylation

    PubMed Central

    Bailey, Zachary S.; Grinter, Michael B.; VandeVord, Pamela J.

    2016-01-01

    Blast induced neurotrauma (BINT) is a prevalent injury within military and civilian populations. The injury is characterized by persistent inflammation at the cellular level which manifests as a multitude of cognitive and functional impairments. Epigenetic regulation of transcription offers an important control mechanism for gene expression and cellular function which may underlie chronic inflammation and result in neurodegeneration. We hypothesize that altered histone acetylation patterns may be involved in blast induced inflammation and the chronic activation of glial cells. This study aimed to elucidate changes to histone acetylation occurring following injury and the roles these changes may have within the pathology. Sprague Dawley rats were subjected to either a 10 or 17 psi blast overpressure within an Advanced Blast Simulator (ABS). Sham animals underwent the same procedures without blast exposure. Memory impairments were measured using the Novel Object Recognition (NOR) test at 2 and 7 days post-injury. Tissues were collected at 7 days for Western blot and immunohistochemistry (IHC) analysis. Sham animals showed intact memory at each time point. The novel object discrimination decreased significantly between two and 7 days for each injury group (p < 0.05). This is indicative of the onset of memory impairment. Western blot analysis showed glial fibrillary acidic protein (GFAP), a known marker of activated astrocytes, was elevated in the prefrontal cortex (PFC) following blast exposure for both injury groups. Analysis of histone protein extract showed no changes in the level of any total histone proteins within the PFC. However, acetylation levels of histone H2b, H3, and H4 were decreased in both groups (p < 0.05). Co-localization immunofluorescence was used to further investigate any potential correlation between decreased histone acetylation and astrocyte activation. These experiments showed a similar decrease in H3 acetylation in astrocytes exposed to a 17

  7. Astrocyte Reactivity Following Blast Exposure Involves Aberrant Histone Acetylation.

    PubMed

    Bailey, Zachary S; Grinter, Michael B; VandeVord, Pamela J

    2016-01-01

    Blast induced neurotrauma (BINT) is a prevalent injury within military and civilian populations. The injury is characterized by persistent inflammation at the cellular level which manifests as a multitude of cognitive and functional impairments. Epigenetic regulation of transcription offers an important control mechanism for gene expression and cellular function which may underlie chronic inflammation and result in neurodegeneration. We hypothesize that altered histone acetylation patterns may be involved in blast induced inflammation and the chronic activation of glial cells. This study aimed to elucidate changes to histone acetylation occurring following injury and the roles these changes may have within the pathology. Sprague Dawley rats were subjected to either a 10 or 17 psi blast overpressure within an Advanced Blast Simulator (ABS). Sham animals underwent the same procedures without blast exposure. Memory impairments were measured using the Novel Object Recognition (NOR) test at 2 and 7 days post-injury. Tissues were collected at 7 days for Western blot and immunohistochemistry (IHC) analysis. Sham animals showed intact memory at each time point. The novel object discrimination decreased significantly between two and 7 days for each injury group (p < 0.05). This is indicative of the onset of memory impairment. Western blot analysis showed glial fibrillary acidic protein (GFAP), a known marker of activated astrocytes, was elevated in the prefrontal cortex (PFC) following blast exposure for both injury groups. Analysis of histone protein extract showed no changes in the level of any total histone proteins within the PFC. However, acetylation levels of histone H2b, H3, and H4 were decreased in both groups (p < 0.05). Co-localization immunofluorescence was used to further investigate any potential correlation between decreased histone acetylation and astrocyte activation. These experiments showed a similar decrease in H3 acetylation in astrocytes exposed to a 17

  8. Manganese suppresses ATP-dependent intercellular calcium waves in astrocyte networks through alteration of mitochondrial and endoplasmic reticulum calcium dynamics.

    PubMed

    Tjalkens, Ronald B; Zoran, Mark J; Mohl, Brianne; Barhoumi, Roula

    2006-10-01

    The neurotoxicity of manganese [Mn] is due in part to glutamate excitotoxicity. Release of ATP by astrocytes is a critical modulator of glutamatergic neurotransmission, which is regulated by calcium (Ca(2+)) waves that propagate through astrocytic networks in response to synaptic activity. It was postulated that Mn alters ATP-dependent intracellular Ca(2+) dynamics in astrocytes, thereby suppressing Ca(2+) wave activity. Confluent primary cultures of cortical astrocytes were loaded with the Ca(2+)-sensitive dye fluo-4 and examined by fluorescence microscopy for Ca(2+) wave activity following micropipet mechanical stimulation of a single cell. Mitochondrial Ca(2+) was evaluated by fluorescence microscopy following addition of ATP using the mitochondrial-specific Ca(2+) dye rhod-2-AM. Imaging studies revealed that pretreatment of astrocytes with 1-10 microM Mn significantly reduced the rate, area, and amplitude of mechanically induced Ca(2+) waves. This attenuation was not a result of inhibited mitochondrial calcium uptake because robust calcium waves were still observed following pretreatment of astrocytes with Ru360, an inhibitor of mitochondrial Ca(2+) uptake, either in coupling or uncoupling conditions. However, determination of endoplasmic reticulum (ER) Ca(2+) levels in cells using the sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin indicated that Mn reduced the available pool of releasable ER Ca(2+) at concentrations as low as 1 muM. Examination of ATP-stimulated changes in mitochondrial Ca(2+) indicated that, in cells pretreated with Mn, mitochondria retained high levels of Ca(2+). It is concluded that exposure of astrocytes to low concentrations of Mn(2+) results in sequestration of Ca(2+) within the mitochondria that reduces the available pool of releasable Ca(2+) within the ER, thereby inhibiting calcium wave activity. PMID:16934782

  9. P2Y1 receptor inhibits GABA transport through a calcium signalling-dependent mechanism in rat cortical astrocytes.

    PubMed

    Jacob, Pedro F; Vaz, Sandra H; Ribeiro, Joaquim A; Sebastião, Ana M

    2014-08-01

    Astrocytes express a variety of purinergic (P2) receptors, involved in astrocytic communication through fast increases in [Ca(2+) ]i . Of these, the metabotropic ATP receptors (P2Y) regulate cytoplasmic Ca(2+) levels through the PLC-PKC pathway. GABA transporters are a substrate for a number of Ca(2+) -related kinases, raising the possibility that calcium signalling in astrocytes impact the control of extracellular levels of the major inhibitory transmitter in the brain. To access this possibility we tested the influence of P2Y receptors upon GABA transport into astrocytes. Mature primary cortical astroglial-enriched cultures expressed functional P2Y receptors, as evaluated through Ca(2+) imaging, being P2Y1 the predominant P2Y receptor subtype. ATP (100 μM, for 1 min) caused an inhibition of GABA transport through either GAT-1 or GAT-3 transporters, decreasing the Vmax kinetic constant. ATP-induced inhibition of GATs activity was still evident in the presence of adenosine deaminase, precluding an adenosine-mediated effect. This, was mimicked by a specific agonist for the P2Y1,12,13 receptor (2-MeSADP). The effect of 2-MeSADP on GABA transport was blocked by the P2 (PPADS) and P2Y1 selective (MRS2179) receptor antagonists, as well as by the PLC inhibitor (U73122). 2-MeSADP failed to inhibit GABA transport in astrocytes where intracellular calcium had been chelated (BAPTA-AM) or where calcium stores were depleted (α-cyclopiazonic acid, CPA). In conclusion, P2Y1 receptors in astrocytes inhibit GABA transport through a mechanism dependent of P2Y1 -mediated calcium signalling, suggesting that astrocytic calcium signalling, which occurs as a consequence of neuronal firing, may operate a negative feedback loop to enhance extracellular levels of GABA. PMID:24733747

  10. Manganese inhibits the ability of astrocytes to promote neuronal differentiation

    SciTech Connect

    Giordano, Gennaro; Pizzurro, Daniella; VanDeMark, Kathryn; Guizzetti, Marina; Costa, Lucio G.

    2009-10-15

    Manganese (Mn) is a known neurotoxicant and developmental neurotoxicant. As Mn has been shown to accumulate in astrocytes, we sought to investigate whether Mn would alter astrocyte-neuronal interactions, specifically the ability of astrocytes to promote differentiation of neurons. We found that exposure of rat cortical astrocytes to Mn (50-500 {mu}M) impaired their ability to promote axonal and neurite outgrowth in hippocampal neurons. This effect of Mn appeared to be mediated by oxidative stress, as it was reversed by antioxidants (melatonin and PBN) and by increasing glutathione levels, while it was potentiated by glutathione depletion in astrocytes. As the extracellular matrix protein fibronectin plays an important role in astrocyte-mediated neuronal neurite outgrowth, we also investigated the effect of Mn on fibronectin. Mn caused a concentration-dependent decrease of fibronectin protein and mRNA in astrocytes lysate and of fibronectin protein in astrocyte medium; these effects were also antagonized by antioxidants. Exposure of astrocytes to two oxidants, H{sub 2}O{sub 2} and DMNQ, similarly impaired their neuritogenic action, and led to a decreased expression of fibronectin. Mn had no inhibitory effect on neurite outgrowth when applied directly onto hippocampal neurons, where it actually caused a small increase in neuritogenesis. These results indicate that Mn, by targeting astrocytes, affects their ability to promote neuronal differentiation by a mechanism which is likely to involve oxidative stress.

  11. Astrocytes increase the activity of synaptic GluN2B NMDA receptors

    PubMed Central

    Hahn, Junghyun; Wang, Xianhong; Margeta, Marta

    2015-01-01

    Astrocytes regulate excitatory synapse formation and surface expression of glutamate AMPA receptors (AMPARs) during development. Less is known about glial modulation of glutamate NMDA receptors (NMDARs), which mediate synaptic plasticity and regulate neuronal survival in a subunit- and subcellular localization-dependent manner. Using primary hippocampal cultures with mature synapses, we found that the density of NMDA-evoked whole-cell currents was approximately twice as large in neurons cultured in the presence of glia compared to neurons cultured alone. The glial effect was mediated by (an) astrocyte-secreted soluble factor(s), was Mg2+ and voltage independent, and could not be explained by a significant change in the synaptic density. Instead, we found that the peak amplitudes of total and NMDAR miniature excitatory postsynaptic currents (mEPSCs), but not AMPAR mEPSCs, were significantly larger in mixed than neuronal cultures, resulting in a decreased synaptic AMPAR/NMDAR ratio. Astrocytic modulation was restricted to synaptic NMDARs that contain the GluN2B subunit, did not involve an increase in the cell surface expression of NMDAR subunits, and was mediated by protein kinase C (PKC). Taken together, our findings indicate that astrocyte-secreted soluble factor(s) can fine-tune synaptic NMDAR activity through the PKC-mediated regulation of GluN2B NMDAR channels already localized at postsynaptic sites, presumably on a rapid time scale. Given that physiologic activation of synaptic NMDARs is neuroprotective and that an increase in the synaptic GluN2B current is associated with improved learning and memory, the astrocyte-induced potentiation of synaptic GluN2B receptor activity is likely to enhance cognitive function while simultaneously strengthening neuroprotective signaling pathways. PMID:25941471

  12. Astrocyte Transcriptome from the Mecp2(308)-Truncated Mouse Model of Rett Syndrome.

    PubMed

    Delépine, Chloé; Nectoux, Juliette; Letourneur, Franck; Baud, Véronique; Chelly, Jamel; Billuart, Pierre; Bienvenu, Thierry

    2015-12-01

    Mutations in the gene encoding the transcriptional modulator methyl-CpG binding protein 2 (MeCP2) are responsible for the neurodevelopmental disorder Rett syndrome which is one of the most frequent sources of intellectual disability in women. Recent studies showed that loss of Mecp2 in astrocytes contributes to Rett-like symptoms and restoration of Mecp2 can rescue some of these defects. The goal of this work is to compare gene expression profiles of wild-type and mutant astrocytes from Mecp2(308/y) mice (B6.129S-MeCP2/J) by using Affymetrix mouse 2.0 microarrays. Results were confirmed by quantitative real-time RT-PCR and by Western blot analysis. Gene set enrichment analysis utilizing Ingenuity Pathways was employed to identify pathways disrupted by Mecp2 deficiency. A total of 2152 genes were statistically differentially expressed between wild-type and mutated samples, including 1784 coding transcripts. However, only 257 showed fold changes >1.2. We confirmed our data by replicative studies in independent primary cultures of cortical astrocytes from Mecp2-deficient mice. Interestingly, two genes known to encode secreted proteins, chromogranin B and lipocalin-2, showed significant dysregulation. These proteins secreted from Mecp2-deficient glia may exert negative non-cell autonomous effects on neuronal properties, including dendritic morphology. Moreover, transcriptional profiling revealed altered Nr2f2 expression which may explain down- and upregulation of several target genes in astrocytes such as Ccl2, Lcn2 and Chgb. Unraveling Nr2f2 involvement in Mecp2-deficient astrocytes could pave the way for a better understanding of Rett syndrome pathophysiology and offers new therapeutic perspectives.

  13. Andrographolide attenuates interleukin-1β-stimulated upregulation of chemokine CCL5 and glial fibrillary acidic protein in astrocytes.

    PubMed

    Wong, Siew-Ying; Chan, Su-Jing; Wong, W S Fred; Wong, Peter T-H; Lai, Mitchell K P

    2014-08-20

    Andrographolide is a bioactive molecule isolated from Andrographis paniculata with anticancer and anti-inflammatory activities. In this study, we tested the effects of andrographolide on astrocyte-mediated neuroinflammatory responses. Cultured rat primary astrocytes were treated with proinflammatory cytokine interleukin 1β with or without pretreatment with andrographolide, and then processed for measurements of chemokine C-C motif ligand 5 (CCL5) and glial fibrillary acidic protein. The activation status of nuclear factor-κB activation that may underlie CCL5 upregulation was also measured. Andrographolide pretreatment was found to attenuate the upregulation of CCL5 and glial fibrillary basic protein as well as reduce the phosphorylation of nuclear factor-κB p65 and IκBα after interleukin 1β stimulation. These data suggest that andrographolide should be evaluated further as a therapeutic for central nervous system diseases characterized by astrocyte-mediated neuroinflammatory processes.

  14. Evidence for involvement of the astrocytic benzodiazepine receptor in the mechanism of action of convulsant and anticonvulsant drugs

    SciTech Connect

    Bender, A.S.; Hertz, L.

    1988-01-01

    The anticonvulsant drugs carbamazepine, phenobarbital, trimethadione, valproic acid and ethosuximide at pharmacologically relevant concentrations inhibit (/sup 3/H)diazepam binding to astrocytes in primary cultures but have much less effect on a corresponding preparation of neurons. Phenytoin as well as pentobarbital (which is not used chronically as an anticonvulsant) are equipotent in the two cell types. The convulsants picrotoxinin and pentylenetetrazol, the convulsant benzodiazepine RO 5-3663 and the two convulsant barbiturates DMBB and CHEB similarly inhibit diazepam binding to astrocytes but have little effect on neurons. On the basis of these findings it is suggested that these convulsants and anticonvulsants owe at least part of their effect to an interaction with the astrocytic benzodiazepine receptor, perhaps by interference with a calcium channel.

  15. Motor neuron-astrocyte interactions and levels of Cu,Zn superoxide dismutase in sporadic amyotrophic lateral sclerosis.

    PubMed

    O'Reilly, S A; Roedica, J; Nagy, D; Hallewell, R A; Alderson, K; Marklund, S L; Kuby, J; Kushner, P D

    1995-02-01

    Copper, zinc superoxide dismutase (SOD1) is involved in neutralizing free radicals within cells, and mutant forms of the enzyme have recently been shown to occur in about 20% of familial cases of amyotrophic lateral sclerosis (ALS). To explore the mechanism of SOD1 involvement in ALS, we have analyzed SOD1 in sporadic ALS using activity assays and immunocyto-chemistry. Analyses of SOD1 activity in washed erythrocytes revealed no difference between 13 ALS cases and 4 controls. Spinal cord sections from 6 ALS cases, 1 primary lateral sclerosis (PLS) case, and 1 control case were stained using three different antibodies to SOD1. Since astrocytes are closely associated with motor neurons, antibodies to glial fibrillary acidic protein (GFAP) and vimentin were used as independent monitors of astrocytes. The principal findings from localizations are: (1) normal motor neurons do not have higher levels of SOD1 than other neurons, (2) there was no detectable difference in SOD1 levels in motor neurons of ALS cases and controls, (3) ALS spinal cord displayed a reduction or absence of SOD1-reactive astrocytes compared to the control and PLS cases, and (4) examination of GFAP-stained sections and morphometry showed that the normal close association between astrocytic processes and motor neuron somata was decreased in the ALS and PLS cases. These results indicate the disease mechanism in sporadic ALS may involve alterations in spinal cord astrocytes.

  16. Glutamine synthetase stability and subcellular distribution in astrocytes are regulated by γ-aminobutyric type B receptors.

    PubMed

    Huyghe, Deborah; Nakamura, Yasuko; Terunuma, Miho; Faideau, Mathilde; Haydon, Philip; Pangalos, Menelas N; Moss, Stephen J

    2014-10-17

    Emerging evidence suggests that functional γ-aminobutyric acid B receptors (GABABRs) are expressed by astrocytes within the mammalian brain. GABABRs are heterodimeric G-protein-coupled receptors that are composed of R1/R2 subunits. To date, they have been characterized in neurons as the principal mediators of sustained inhibitory signaling; however their roles in astrocytic physiology have been ill defined. Here we reveal that the cytoplasmic tail of the GABABR2 subunit binds directly to the astrocytic protein glutamine synthetase (GS) and that this interaction determines the subcellular localization of GS. We further demonstrate that the binding of GS to GABABR2 increases the steady state expression levels of GS in heterologous cells and in mouse primary astrocyte culture. Mechanistically this increased stability of GS in the presence of GABABR2 occurs via reduced proteasomal degradation. Collectively, our results suggest a novel role for GABABRs as regulators of GS stability. Given the critical role that GS plays in the glutamine-glutamate cycle, astrocytic GABABRs may play a critical role in supporting both inhibitory and excitatory neurotransmission.

  17. Mutant Copper-Zinc Superoxide Dismutase (SOD1) Induces Protein Secretion Pathway Alterations and Exosome Release in Astrocytes

    PubMed Central

    Basso, Manuela; Pozzi, Silvia; Tortarolo, Massimo; Fiordaliso, Fabio; Bisighini, Cinzia; Pasetto, Laura; Spaltro, Gabriella; Lidonnici, Dario; Gensano, Francesco; Battaglia, Elisa; Bendotti, Caterina; Bonetto, Valentina

    2013-01-01

    Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known. The interplay between motor neurons and astrocytes is crucial in the outcome of the disease. We show that mutant copper-zinc superoxide dismutase (SOD1) overexpression in primary astrocyte cultures is associated with decreased levels of proteins involved in secretory pathways. This is linked to a general reduction of total secreted proteins, except for specific enrichment in a number of proteins in the media, such as mutant SOD1 and valosin-containing protein (VCP)/p97. Because there was also an increase in exosome release, we can deduce that astrocytes expressing mutant SOD1 activate unconventional secretory pathways, possibly as a protective mechanism. This may help limit the formation of intracellular aggregates and overcome mutant SOD1 toxicity. We also found that astrocyte-derived exosomes efficiently transfer mutant SOD1 to spinal neurons and induce selective motor neuron death. We conclude that the expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread. PMID:23592792

  18. Physiopathologic dynamics of vesicle traffic in astrocytes.

    PubMed

    Potokar, Maja; Stenovec, Matjaž; Kreft, Marko; Gabrijel, Mateja; Zorec, Robert

    2011-02-01

    The view of how astrocytes, a type of glial cells, contribute to the functioning of the central nervous system (CNS) has changed greatly in the last decade. Although glial cells outnumber neurons in the mammalian brain, it was considered for over a century that they played a subservient role to neurons. This view changed. Functions thought to be exclusively present in neurons, i.e. excitability mediated release of chemical messengers, has also been demonstrated in astrocytes. In this process, following an increase in cytosolic calcium activity, membrane bound vesicles, storing chemical messengers (gliotransmitters), fuse with the plasma membrane, a process known as exocytosis, permitting the exit of vesicle cargo into the extracellular space. Vesicles are delivered to and are removed from the site of exocytosis by an amazingly complex set of processes that we have only started to learn about recently. In this paper we review vesicle traffic, which is subject to physiological regulation and may be changed under pathological conditions.

  19. Functions of astrocytes and their potential as therapeutic targets

    PubMed Central

    Kimelberg, Harold K.; Nedergaard, Maiken

    2010-01-01

    Astrocytes are often referred to, and historically have been regarded as, support cells of the mammalian CNS. Work over the last decade suggests otherwise, that astrocytes may in fact play a more active role in higher neural processing than previously recognized. Because astrocytes can potentially serve as novel therapeutic targets, it is critical to understand how astrocytes execute their diverse supportive tasks while maintaining neuronal health. To that end, this review will focus on the supportive roles of astrocytes, a line of study relevant to essentially all acute and chronic neurological diseases. Furthermore, this review will critically re-evaluate our concepts of the functional properties of astrocytes and relate these tasks to their intricate morphology. PMID:20880499

  20. Astrocyte-neuronal interactions in epileptogenesis.

    PubMed

    Hadera, Mussie Ghezu; Eloqayli, Haytham; Jaradat, Saied; Nehlig, Astrid; Sonnewald, Ursula

    2015-07-01

    Pentylenetetrazol, kainic acid, or pilocarpine can be used to induce seizures in animal models of epilepsy. The present Review describes disturbances in astrocyte-neuron interactions in the acute, latent, and chronic phases analyzed by magnetic resonance spectroscopy of brain tissue extracts from rats injected with [1-(13)C]glucose and [1,2-(13)C]acetate. The most consistent change after onset of seizures was the decrease in (13)C labeling of glutamate (GLU) from [1-(13) C]glucose regardless of brain area, severity, or duration of the period with seizures and toxin used. In most cases this decrease was accompanied by a reduction in glutamine (GLN) labeling from [1-(13)C]glucose, presumably as a direct consequence of the reduction in labeling of GLU and the GLU-GLN cycle. Amounts of GLN were never changed. Reduction in the content of N-acetyl aspartate (NAA) was first detectable some time after status epilepticus but before the occurrence of spontaneous seizures. This decrease can be an indication of neuronal death and/or mitochondrial impairment and might indicate beginning gliosis. It is known that gliosis occurs in the chronic phase of temporal lobe epilepsy in hippocampus, but astrocyte metabolism appears normal in this phase, indicating that the gliotic astrocytes have a somewhat reduced metabolism per volume. A decrease in (13)C labeling of GLU from [1-(13)C]glucose is a very sensitive measure for the onset of epileptogenesis, whereas reduction of NAA is first detectable later. In the chronic phases of the hippocampal formation, astrocyte metabolism is upregulated given that the number of neurons is reduced. PMID:25782696

  1. Sodium para-aminosalicylate protected cultured basal ganglia astrocytes from manganese-induced DNA damages and alteration of amino acid neurotransmitter levels.

    PubMed

    Li, Shao-Jun; Luo, Yi-Ni; Li, Yong; Chen, Jing-Wen; Mo, Yu-Huan; Yuan, Zong-Xiang; Ou, Shi-Yan; Ou, Chao-Yan; Jiang, Yue-Ming; Deng, Xiang-Fa

    2016-01-01

    Sodium para-aminosalicylate (PAS-Na) was first applied successfully in clinical treatment of two manganism patients with good prognosis. However, the mechanism of how PAS-Na protects against Mn-induced neurotoxicity is still elusive. The current study was conducted to explore the effects of PAS-Na on Mn-induced basal ganglia astrocyte injury, and the involvement of amino acid neurotransmitter in vitro. Basal ganglia astrocytes were exposed to 500 μM manganese chloride (MnCl2) for 24 hr, following by 50, 150, or 450 μM PAS-Na treatment for another 24 hr. MnCl2 significantly decreased viability of astrocytes and induced DNA damages via increasing the percentage of tail DNA and Olive tail moment of DNA. Moreover, Mn interrupted amino acid neurotransmitters by decreasing Gln levels and increasing Glu, Gly levels. In contrast, PAS-Na treatment reversed the aforementioned Mn-induced toxic effects on basal ganglia astrocytes. Taken together, our results demonstrated that excessive Mn exposure may induce toxic effects on basal ganglia astrocytes, while PAS-Na could protect basal ganglia astrocytes from Mn-induced neurotoxicity. PMID:27665767

  2. Imaging neurotransmitter uptake and depletion in astrocytes

    SciTech Connect

    Tan, W. |; Haydon, P.G.; Yeung, E.S.

    1997-08-01

    An ultraviolet (UV) laser-based optical microscope and charge-coupled device (CCD) detection system was used to obtain chemical images of biological cells. Subcellular structures can be easily seen in both optical and fluorescence images. Laser-induced native fluorescence detection provides high sensitivity and low limits of detection, and it does not require coupling to fluorescent dyes. We were able to quantitatively monitor serotonin that has been taken up into and released from individual astrocytes on the basis of its native fluorescence. Different regions of the cells took up different amounts of serotonin with a variety of uptake kinetics. Similarly, we observed different serotonin depletion dynamics in different astrocyte regions. There were also some astrocyte areas where no serotonin uptake or depletion was observed. Potential applications include the mapping of other biogenic species in cells as well as the ability to image their release from specific regions of cells in response to external stimuli. {copyright} {ital 1997} {ital Society for Applied Spectroscopy}

  3. Mechanisms of Astrocyte-Mediated Cerebral Edema

    PubMed Central

    Stokum, Jesse A.; Kurland, David B.; Gerzanich, Volodymyr; Simard, J. Marc

    2014-01-01

    Cerebral edema formation stems from disruption of blood brain barrier (BBB) integrity and occurs after injury to the CNS. Due to the restrictive skull, relatively small increases in brain volume can translate into impaired tissue perfusion and brain herniation. In excess, cerebral edema can be gravely harmful. Astrocytes are key participants in cerebral edema by virtue of their relationship with the cerebral vasculature, their unique compliment of solute and water transport proteins, and their general role in brain volume homeostasis. Following the discovery of aquaporins, passive conduits of water flow, aquaporin 4 (AQP4) was identified as the predominant astrocyte water channel. Normally, AQP4 is highly enriched at perivascular endfeet, the outermost layer of the BBB, whereas after injury, AQP4 expression disseminates to the entire astrocytic plasmalemma, a phenomenon termed dysregulation. Arguably, the most important role of AQP4 is to rapidly neutralize osmotic gradients generated by ionic transporters. In pathological conditions, AQP4 is believed to be intimately involved in the formation and clearance of cerebral edema. In this review, we discuss aquaporin function and localization in the BBB during health and injury, and we examine post-injury ionic events that modulate AQP4- dependent edema formation. PMID:24996934

  4. A Triple Culture Model of the Blood-Brain Barrier Using Porcine Brain Endothelial cells, Astrocytes and Pericytes.

    PubMed

    Thomsen, Louiza Bohn; Burkhart, Annette; Moos, Torben

    2015-01-01

    In vitro blood-brain barrier (BBB) models based on primary brain endothelial cells (BECs) cultured as monoculture or in co-culture with primary astrocytes and pericytes are useful for studying many properties of the BBB. The BECs retain their expression of tight junction proteins and efflux transporters leading to high trans-endothelial electric resistance (TEER) and low passive paracellular permeability. The BECs, astrocytes and pericytes are often isolated from small rodents. Larger species as cows and pigs however, reveal a higher yield, are readily available and have a closer resemblance to humans, which make them favorable high-throughput sources for cellular isolation. The aim of the present study has been to determine if the preferable combination of purely porcine cells isolated from the 6 months old domestic pigs, i.e. porcine brain endothelial cells (PBECs) in co-culture with porcine astrocytes and pericytes, would compare with PBECs co-cultured with astrocytes and pericytes isolated from newborn rats with respect to TEER value and low passive permeability. The astrocytes and pericytes were grown both as contact and non-contact co-cultures as well as in triple culture to examine their effects on the PBECs for barrier formation as revealed by TEER, passive permeability, and expression patterns of tight junction proteins, efflux transporters and the transferrin receptor. This syngenic porcine in vitro BBB model is comparable to triple cultures using PBECs, rat astrocytes and rat pericytes with respect to TEER formation, low passive permeability, and expression of hallmark proteins signifying the brain endothelium (tight junction proteins claudin 5 and occludin, the efflux transporters P-glycoprotein (PgP) and breast cancer related protein (BCRP), and the transferrin receptor).

  5. Astrocyte Contributions to Flow/Pressure-Evoked Parenchymal Arteriole Vasoconstriction

    PubMed Central

    Kim, Ki Jung; Iddings, Jennifer A.; Stern, Javier E.; Blanco, Víctor M.; Croom, Deborah; Kirov, Sergei A.

    2015-01-01

    Basal and activity-dependent cerebral blood flow changes are coordinated by the action of critical processes, including cerebral autoregulation, endothelial-mediated signaling, and neurovascular coupling. The goal of our study was to determine whether astrocytes contribute to the regulation of parenchymal arteriole (PA) tone in response to hemodynamic stimuli (pressure/flow). Cortical PA vascular responses and astrocytic Ca2+ dynamics were measured using an in vitro rat/mouse brain slice model of perfused/pressurized PAs; studies were supplemented with in vivo astrocytic Ca2+ imaging. In vitro, astrocytes responded to PA flow/pressure increases with an increase in intracellular Ca2+. Astrocytic Ca2+ responses were corroborated in vivo, where acute systemic phenylephrine-induced increases in blood pressure evoked a significant increase in astrocytic Ca2+. In vitro, flow/pressure-evoked vasoconstriction was blunted when the astrocytic syncytium was loaded with BAPTA (chelating intracellular Ca2+) and enhanced when high Ca2+ or ATP were introduced to the astrocytic syncytium. Bath application of either the TRPV4 channel blocker HC067047 or purinergic receptor antagonist suramin blunted flow/pressure-evoked vasoconstriction, whereas K+ and 20-HETE signaling blockade showed no effect. Importantly, we found TRPV4 channel expression to be restricted to astrocytes and not the endothelium of PA. We present evidence for a novel role of astrocytes in PA flow/pressure-evoked vasoconstriction. Our data suggest that astrocytic TRPV4 channels are key molecular sensors of hemodynamic stimuli and that a purinergic, glial-derived signal contributes to flow/pressure-induced adjustments in PA tone. Together our results support bidirectional signaling within the neurovascular unit and astrocytes as key modulators of PA tone. PMID:26019339

  6. Astrocyte contributions to flow/pressure-evoked parenchymal arteriole vasoconstriction.

    PubMed

    Kim, Ki Jung; Iddings, Jennifer A; Stern, Javier E; Blanco, Víctor M; Croom, Deborah; Kirov, Sergei A; Filosa, Jessica A

    2015-05-27

    Basal and activity-dependent cerebral blood flow changes are coordinated by the action of critical processes, including cerebral autoregulation, endothelial-mediated signaling, and neurovascular coupling. The goal of our study was to determine whether astrocytes contribute to the regulation of parenchymal arteriole (PA) tone in response to hemodynamic stimuli (pressure/flow). Cortical PA vascular responses and astrocytic Ca(2+) dynamics were measured using an in vitro rat/mouse brain slice model of perfused/pressurized PAs; studies were supplemented with in vivo astrocytic Ca(2+) imaging. In vitro, astrocytes responded to PA flow/pressure increases with an increase in intracellular Ca(2+). Astrocytic Ca(2+) responses were corroborated in vivo, where acute systemic phenylephrine-induced increases in blood pressure evoked a significant increase in astrocytic Ca(2+). In vitro, flow/pressure-evoked vasoconstriction was blunted when the astrocytic syncytium was loaded with BAPTA (chelating intracellular Ca(2+)) and enhanced when high Ca(2+) or ATP were introduced to the astrocytic syncytium. Bath application of either the TRPV4 channel blocker HC067047 or purinergic receptor antagonist suramin blunted flow/pressure-evoked vasoconstriction, whereas K(+) and 20-HETE signaling blockade showed no effect. Importantly, we found TRPV4 channel expression to be restricted to astrocytes and not the endothelium of PA. We present evidence for a novel role of astrocytes in PA flow/pressure-evoked vasoconstriction. Our data suggest that astrocytic TRPV4 channels are key molecular sensors of hemodynamic stimuli and that a purinergic, glial-derived signal contributes to flow/pressure-induced adjustments in PA tone. Together our results support bidirectional signaling within the neurovascular unit and astrocytes as key modulators of PA tone. PMID:26019339

  7. Responses of astrocytes in culture after low dose laser irradiation

    SciTech Connect

    Yew, D.T.; Zheng, D.R.; Au, C.; Li, W.W. )

    1990-03-01

    The effect of Helium-Neon low dose laser on astrocytes was investigated in cultures of isolated astrocytes from albino neonatal rats. The laser appeared to inhibit the growth of astrocytes as exemplified by the smaller sizes of the cells and the decreased leucine uptake in each cell after treatment. Temporary decrease in the number of mitoses was also observed, but this trend was reversed soon after. Electron microscopic studies revealed an increase in buddings from cell bodies and processes (branches) after irradiation.

  8. Development of a method for the purification and culture of rodent astrocytes.

    PubMed

    Foo, Lynette C; Allen, Nicola J; Bushong, Eric A; Ventura, P Britten; Chung, Won-Suk; Zhou, Lu; Cahoy, John D; Daneman, Richard; Zong, Hui; Ellisman, Mark H; Barres, Ben A

    2011-09-01

    The inability to purify and culture astrocytes has long hindered studies of their function. Whereas astrocyte progenitor cells can be cultured from neonatal brain, culture of mature astrocytes from postnatal brain has not been possible. Here, we report a new method to prospectively purify astrocytes by immunopanning. These astrocytes undergo apoptosis in culture, but vascular cells and HBEGF promote their survival in serum-free culture. We found that some developing astrocytes normally undergo apoptosis in vivo and that the vast majority of astrocytes contact blood vessels, suggesting that astrocytes are matched to blood vessels by competing for vascular-derived trophic factors such as HBEGF. Compared to traditional astrocyte cultures, the gene profiles of the cultured purified postnatal astrocytes much more closely resemble those of in vivo astrocytes. Although these astrocytes strongly promote synapse formation and function, they do not secrete glutamate in response to stimulation.

  9. Handling of iron oxide and silver nanoparticles by astrocytes.

    PubMed

    Hohnholt, Michaela C; Geppert, Mark; Luther, Eva M; Petters, Charlotte; Bulcke, Felix; Dringen, Ralf

    2013-02-01

    Metal-containing nanoparticles (NPs) are currently used for various biomedical applications. Since such NPs are able to enter the brain, the cells of this organ have to deal with NPs and with NP-derived metal ions. In brain, astrocytes are considered to play a key function in regulating metal homeostasis and in protecting other brain cells against metal toxicity. Thus, among the different types of brain cells, especially astrocytes are of interest regarding the uptake and the handling of metal-containing NPs. This article summarizes the current knowledge on the consequences of an exposure of astrocytes to NPs. Special focus will be given to magnetic iron oxide nanoparticles (IONPs) and silver nanoparticles (AgNPs), since the biocompatibility of these NPs has been studied for astrocytes in detail. Cultured astrocytes efficiently accumulate IONPs and AgNPs in a time-, concentration- and temperature-dependent manner by endocytotic processes. Astrocytes are neither acutely damaged by the exposure to high concentrations of NPs nor by the prolonged intracellular presence of large amounts of accumulated NPs. Although metal ions are liberated from accumulated NPs, NP-derived iron and silver ions are not exported from astrocytes but are rather stored in proteins such as ferritin and metallothioneins which are synthesized in NP-treated astrocytes. The efficient accumulation of large amounts of metal-containing NPs and the upregulation of proteins that safely store NP-derived metal ions suggest that astrocytes protect the brain against the potential toxicity of metal-containing NPs.

  10. Elusive roles for reactive astrocytes in neurodegenerative diseases

    PubMed Central

    Ben Haim, Lucile; Carrillo-de Sauvage, Maria-Angeles; Ceyzériat, Kelly; Escartin, Carole

    2015-01-01

    Astrocytes play crucial roles in the brain and are involved in the neuroinflammatory response. They become reactive in response to virtually all pathological situations in the brain such as axotomy, ischemia, infection, and neurodegenerative diseases (ND). Astrocyte reactivity was originally characterized by morphological changes (hypertrophy, remodeling of processes) and the overexpression of the intermediate filament glial fibrillary acidic protein (GFAP). However, it is unclear how the normal supportive functions of astrocytes are altered by their reactive state. In ND, in which neuronal dysfunction and astrocyte reactivity take place over several years or decades, the issue is even more complex and highly debated, with several conflicting reports published recently. In this review, we discuss studies addressing the contribution of reactive astrocytes to ND. We describe the molecular triggers leading to astrocyte reactivity during ND, examine how some key astrocyte functions may be enhanced or altered during the disease process, and discuss how astrocyte reactivity may globally affect ND progression. Finally we will consider the anticipated developments in this important field. With this review, we aim to show that the detailed study of reactive astrocytes may open new perspectives for ND. PMID:26283915

  11. Astrocyte scar formation aids central nervous system axon regeneration.

    PubMed

    Anderson, Mark A; Burda, Joshua E; Ren, Yilong; Ao, Yan; O'Shea, Timothy M; Kawaguchi, Riki; Coppola, Giovanni; Khakh, Baljit S; Deming, Timothy J; Sofroniew, Michael V

    2016-04-14

    Transected axons fail to regrow in the mature central nervous system. Astrocytic scars are widely regarded as causal in this failure. Here, using three genetically targeted loss-of-function manipulations in adult mice, we show that preventing astrocyte scar formation, attenuating scar-forming astrocytes, or ablating chronic astrocytic scars all failed to result in spontaneous regrowth of transected corticospinal, sensory or serotonergic axons through severe spinal cord injury (SCI) lesions. By contrast, sustained local delivery via hydrogel depots of required axon-specific growth factors not present in SCI lesions, plus growth-activating priming injuries, stimulated robust, laminin-dependent sensory axon regrowth past scar-forming astrocytes and inhibitory molecules in SCI lesions. Preventing astrocytic scar formation significantly reduced this stimulated axon regrowth. RNA sequencing revealed that astrocytes and non-astrocyte cells in SCI lesions express multiple axon-growth-supporting molecules. Our findings show that contrary to the prevailing dogma, astrocyte scar formation aids rather than prevents central nervous system axon regeneration. PMID:27027288

  12. The multifaceted role of astrocytes in regulating myelination.

    PubMed

    Kıray, Hülya; Lindsay, Susan L; Hosseinzadeh, Sara; Barnett, Susan C

    2016-09-01

    Astrocytes are the major glial cell of the central nervous system (CNS), providing both metabolic and physical support to other neural cells. After injury, astrocytes become reactive and express a continuum of phenotypes which may be supportive or inhibitory to CNS repair. This review will focus on the ability of astrocytes to influence myelination in the context of specific secreted factors, cytokines and other neural cell targets within the CNS. In particular, we focus on how astrocytes provide energy and cholesterol to neurons, influence synaptogenesis, affect oligodendrocyte biology and instigate cross-talk between the many cellular components of the CNS.

  13. A Digital Realization of Astrocyte and Neural Glial Interactions.

    PubMed

    Hayati, Mohsen; Nouri, Moslem; Haghiri, Saeed; Abbott, Derek

    2016-04-01

    The implementation of biological neural networks is a key objective of the neuromorphic research field. Astrocytes are the largest cell population in the brain. With the discovery of calcium wave propagation through astrocyte networks, now it is more evident that neuronal networks alone may not explain functionality of the strongest natural computer, the brain. Models of cortical function must now account for astrocyte activities as well as their relationships with neurons in encoding and manipulation of sensory information. From an engineering viewpoint, astrocytes provide feedback to both presynaptic and postsynaptic neurons to regulate their signaling behaviors. This paper presents a modified neural glial interaction model that allows a convenient digital implementation. This model can reproduce relevant biological astrocyte behaviors, which provide appropriate feedback control in regulating neuronal activities in the central nervous system (CNS). Accordingly, we investigate the feasibility of a digital implementation for a single astrocyte constructed by connecting a two coupled FitzHugh Nagumo (FHN) neuron model to an implementation of the proposed astrocyte model using neuron-astrocyte interactions. Hardware synthesis, physical implementation on FPGA, and theoretical analysis confirm that the proposed neuron astrocyte model, with significantly low hardware cost, can mimic biological behavior such as the regulation of postsynaptic neuron activity and the synaptic transmission mechanisms. PMID:26390499

  14. SLAM- and Nectin-4-Independent Noncytolytic Spread of Canine Distemper Virus in Astrocytes

    PubMed Central

    Alves, Lisa; Khosravi, Mojtaba; Avila, Mislay; Ader-Ebert, Nadine; Bringolf, Fanny; Zurbriggen, Andreas; Vandevelde, Marc

    2015-01-01

    ABSTRACT Measles and canine distemper viruses (MeV and CDV, respectively) first replicate in lymphatic and epithelial tissues by using SLAM and nectin-4 as entry receptors, respectively. The viruses may also invade the brain to establish persistent infections, triggering fatal complications, such as subacute sclerosis pan-encephalitis (SSPE) in MeV infection or chronic, multiple sclerosis-like, multifocal demyelinating lesions in the case of CDV infection. In both diseases, persistence is mediated by viral nucleocapsids that do not require packaging into particles for infectivity but are directly transmitted from cell to cell (neurons in SSPE or astrocytes in distemper encephalitis), presumably by relying on restricted microfusion events. Indeed, although morphological evidence of fusion remained undetectable, viral fusion machineries and, thus, a putative cellular receptor, were shown to contribute to persistent infections. Here, we first showed that nectin-4-dependent cell-cell fusion in Vero cells, triggered by a demyelinating CDV strain, remained extremely limited, thereby supporting a potential role of nectin-4 in mediating persistent infections in astrocytes. However, nectin-4 could not be detected in either primary cultured astrocytes or the white matter of tissue sections. In addition, a bioengineered “nectin-4-blind” recombinant CDV retained full cell-to-cell transmission efficacy in primary astrocytes. Combined with our previous report demonstrating the absence of SLAM expression in astrocytes, these findings are suggestive for the existence of a hitherto unrecognized third CDV receptor expressed by glial cells that contributes to the induction of noncytolytic cell-to-cell viral transmission in astrocytes. IMPORTANCE While persistent measles virus (MeV) infection induces SSPE in humans, persistent canine distemper virus (CDV) infection causes chronic progressive or relapsing demyelination in carnivores. Common to both central nervous system (CNS

  15. Oxidative damage of copper chloride overload to the cultured rat astrocytes

    PubMed Central

    Hu, Hao-Lu; Ni, Xiu-Shi; Duff-Canning, Sarah; Wang, Xiao-Ping

    2016-01-01

    Disorders of copper metabolism are associated with neurological dysfunction including Wilson’s disease (WD). WD is a autosomal recessive disorder caused by mutations in the ATP7B gene resulting in the inability of the hepatocytes to remove excess copper. Gradual copper accumulation causes damage to liver, brain and other organs manifesting in liver disease, neurological and psychiatric symptoms. Also scond copper-neurometaboic disorder: Menkes disease charaterized with mutated ATP7A gene, is ralated with abnormally neuroal transmission and synaptogenesis. Parkinson’s disease and Alzheimer’s disease both are refered to some degree of copper/iron metabolism changes. The precise mechanisms by which excess copper causes neurological damage remain to be elucidated. In this study, we aimed to investigate the influence of excessive amounts of Cu2+ on the oxidative damage response and survival of primary astrocytes from newborn rats. Primary cultured rat astrocytes were divided into three groups: 30 μmol/L CuCl2, 100 μmol/L CuCl2 and control. At 12, 24, 48, 96 and 120 hours of CuCl2 intervention, cell viability, intracellular reduced glutathione level and glutathion reductase activity, and nitric oxide secretion were determined. It was found that 30 μmol/L CuCl2 might stimulate the exaltation and the compensatory proliferation of astrocytes. The survival rate of astrocytes in the 100 μmol/L CuCl2 group was significantly decreased relative to the 30 μmol/L CuCl2 group. At 24 hours of CuCl2 intervention, intracellular reduced glutathione level and glutathion reductase activity were significantly decreased in the 100 μmol/L CuCl2 group compared to the control group. At 120 hours of CuCl2 intervention, nitric oxide secretion in the 100 μmol/L CuCl2 group was significantly greater than in the control group. Under pathological conditions, excessive amounts of Cu2+ greatly damaged the growth and proliferation of astrocytes, reduced the anti-oxidative capacity of

  16. Oxidative damage of copper chloride overload to the cultured rat astrocytes.

    PubMed

    Hu, Hao-Lu; Ni, Xiu-Shi; Duff-Canning, Sarah; Wang, Xiao-Ping

    2016-01-01

    Disorders of copper metabolism are associated with neurological dysfunction including Wilson's disease (WD). WD is a autosomal recessive disorder caused by mutations in the ATP7B gene resulting in the inability of the hepatocytes to remove excess copper. Gradual copper accumulation causes damage to liver, brain and other organs manifesting in liver disease, neurological and psychiatric symptoms. Also scond copper-neurometaboic disorder: Menkes disease charaterized with mutated ATP7A gene, is ralated with abnormally neuroal transmission and synaptogenesis. Parkinson's disease and Alzheimer's disease both are refered to some degree of copper/iron metabolism changes. The precise mechanisms by which excess copper causes neurological damage remain to be elucidated. In this study, we aimed to investigate the influence of excessive amounts of Cu(2+) on the oxidative damage response and survival of primary astrocytes from newborn rats. Primary cultured rat astrocytes were divided into three groups: 30 μmol/L CuCl2, 100 μmol/L CuCl2 and control. At 12, 24, 48, 96 and 120 hours of CuCl2 intervention, cell viability, intracellular reduced glutathione level and glutathion reductase activity, and nitric oxide secretion were determined. It was found that 30 μmol/L CuCl2 might stimulate the exaltation and the compensatory proliferation of astrocytes. The survival rate of astrocytes in the 100 μmol/L CuCl2 group was significantly decreased relative to the 30 μmol/L CuCl2 group. At 24 hours of CuCl2 intervention, intracellular reduced glutathione level and glutathion reductase activity were significantly decreased in the 100 μmol/L CuCl2 group compared to the control group. At 120 hours of CuCl2 intervention, nitric oxide secretion in the 100 μmol/L CuCl2 group was significantly greater than in the control group. Under pathological conditions, excessive amounts of Cu(2+) greatly damaged the growth and proliferation of astrocytes, reduced the anti-oxidative capacity of

  17. The expression of CAP1 after traumatic brain injury and its role in astrocyte proliferation.

    PubMed

    Zhang, Haiyan; Liu, Yonghua; Li, Yao; Zhou, Ying; Chen, Dongjian; Shen, Jianhong; Yan, Yaohua; Yan, Song; Wu, Xinmin; Li, Aihong; Guo, Aisong; Cheng, Chun

    2014-12-01

    Adenylate cyclase-associated protein 1 (CAP1), a member of cyclase-associated proteins involved in the regulation of actin filaments, was recently reported to play a role in the pathology of sciatic nerves injury. However, the distribution and function of CAP1 in the central nervous system (CNS) remain unclear. To investigate whether CAP1 is involved in CNS injury and repair, we used an acute traumatic brain injury (TBI) model in adult rats. Western blot analysis and immunohistochemistry showed a significant upregulation of CAP1 in ipsilateral peritrauma cortex compared with the contralateral and sham-operated ones. Double immunofluorescence staining showed that CAP1 was co-expressed with glial fibrillary acidic protein (GFAP). In addition, we detected that Ki-67 had colocalization with GFAP and CAP1 after TBI. In vitro, during the process of lipopolysaccharide (LPS)-induced primary astrocyte proliferation, we observed enhanced expression of CAP1. Specially, CAP1-specific siRNA-transfected primary astrocytes show significantly decreased ability for proliferation. Together, all these data indicated that the change of CAP1 protein expression was associated with astrocyte proliferation after the trauma of the central nervous system (CNS).

  18. Adenosine A2A receptors modulate glutamate uptake in cultured astrocytes and gliosomes.

    PubMed

    Matos, Marco; Augusto, Elisabete; Santos-Rodrigues, Alexandre Dos; Schwarzschild, Michael A; Chen, Jiang-Fan; Cunha, Rodrigo A; Agostinho, Paula

    2012-05-01

    Glutamate is the primary excitatory neurotransmitter in the central nervous system, where its toxic build-up leads to synaptic dysfunction and excitotoxic cell death that underlies many neurodegenerative diseases. Therefore, efforts have been made to understand the regulation of glutamate transporters, which are responsible for the clearance of extracellular glutamate. We now report that adenosine A(2A) receptors (A(2A) R) control the uptake of D-aspartate in primary cultured astrocytes as well as in an ex vivo preparation enriched in glial plasmalemmal vesicles (gliosomes) from adult rats, whereas A(1) R and A(3) R were devoid of effects. Thus, the acute exposure to the A(2A) R agonist, CGS 21680, inhibited glutamate uptake, an effect prevented by the A(2A) R antagonist, SCH 58261, and abbrogated in cultured astrocytes from A(2A) R knockout mice. Furthermore, the prolonged activation of A(2A) R lead to a cAMP/protein kinase A-dependent reduction of GLT-I and GLAST mRNA and protein levels, which leads to a sustained decrease of glutamate uptake. This dual mechanism of inhibition of glutamate transporters by astrocytic A(2A) R provides a novel candidate mechanism to understand the ability of A(2) (A) R to control synaptic plasticity and neurodegeneration, two conditions tightly associated with the control of extracellular glutamate levels by glutamate transporters.

  19. Astrocyte morphology is confined by cortical functional boundaries in mammals ranging from mice to human

    PubMed Central

    Eilam, Raya; Aharoni, Rina; Arnon, Ruth; Malach, Rafael

    2016-01-01

    Cortical blood flow can be modulated by local activity across a range of species; from barrel-specific blood flow in the rodent somatosensory cortex to the human cortex, where BOLD-fMRI reveals numerous functional borders. However, it appears that the distribution of blood capillaries largely ignores these functional boundaries. Here we report that, by contrast, astrocytes, a major player in blood-flow control, show a striking morphological sensitivity to functional borders. Specifically, we show that astrocyte processes are structurally confined by barrel boundaries in the mouse, by the border of primary auditory cortex in the rat and by layers IIIa/b and Cytochrome Oxidase (CO)-blobs boundaries in the human primary visual cortex. Thus, astrocytes which are critical elements in neuro-hemodynamic coupling show a significant anatomical segregation along functional boundaries across different mammalian species. These results may open a new anatomical marker for delineating functional borders across species, including post-mortem human brains. DOI: http://dx.doi.org/10.7554/eLife.15915.001 PMID:27282388

  20. Identifying and Intervening with Substance-Using Women Exposed to Intimate Partner Violence: Phenomenology, Comorbidities, and Integrated Approaches Within Primary Care and Other Agency Settings

    PubMed Central

    Gilbert, Louisa; El-Bassel, Nabila; Resnick, Heidi S.; Noursi, Samia

    2015-01-01

    Abstract Substance use and/or disorders (SUDs) have been identified as a significant correlate of intimate partner violence (IPV) exposure and present complex issues that intersect with the topography of IPV, attendant mental health, and physical co-morbidities and may pose barriers to primary care- and other agency-based screening and intervention efforts. Despite substantial research indicating significantly higher rates of all types and severity of IPV victimization among women with SUDs and bidirectional associations between partner or self-use of drugs or alcohol and IPV victimization, effective screening, brief interventions, coordinated systems of care, and treatment approaches to address these co-occurring problems remain very limited. We integrated select research examining the intersection of IPV victimization and SUDs and several comorbidities that have significant public health impact and provided recommendations for scaling up targeted interventions to redress these co-occurring problems among women in primary, emergency, and other care settings. PMID:25554915

  1. Selenoprotein S expression in reactive astrocytes following brain injury.

    PubMed

    Fradejas, Noelia; Serrano-Pérez, Maria Del Carmen; Tranque, Pedro; Calvo, Soledad

    2011-06-01

    Selenoprotein S (SelS) is an endoplasmic reticulum (ER)-resident protein involved in the unfolded protein response. Besides reducing ER-stress, SelS attenuates inflammation by decreasing pro-inflammatory cytokines. We have recently shown that SelS is responsive to ischemia in cultured astrocytes. To check the possible association of SelS with astrocyte activation, here we investigate the expression of SelS in two models of brain injury: kainic acid (KA) induced excitotoxicity and cortical mechanical lesion. The regulation of SelS and its functional consequences for neuroinflammation, ER-stress, and cell survival were further analyzed using cultured astrocytes from mouse and human. According to our immunofluorescence analysis, SelS expression is prominent in neurons and hardly detectable in astrocytes from control mice. However, brain injury intensely upregulates SelS, specifically in reactive astrocytes. SelS induction by KA was evident at 12 h and faded out after reaching maximum levels at 3-4 days. Analysis of mRNA and protein expression in cultured astrocytes showed SelS upregulation by inflammatory stimuli as well as ER-stress inducers. In turn, siRNA-mediated SelS silencing combined with adenoviral overexpression assays demonstrated that SelS reduces ER-stress markers CHOP and spliced XBP-1, as well as inflammatory cytokines IL-1β and IL-6 in stimulated astrocytes. SelS overexpression increased astrocyte resistance to ER-stress and inflammatory stimuli. Conversely, SelS suppression compromised astrocyte viability. In summary, our results reveal the upregulation of SelS expression in reactive astrocytes, as well as a new protective role for SelS against inflammation and ER-stress that can be relevant to astrocyte function in the context of inflammatory neuropathologies. PMID:21456042

  2. Minocycline attenuates bone cancer pain in rats by inhibiting NF-κB in spinal astrocytes

    PubMed Central

    Song, Zhen-peng; Xiong, Bing-rui; Guan, Xue-hai; Cao, Fei; Manyande, Anne; Zhou, Ya-qun; Zheng, Hua; Tian, Yu-ke

    2016-01-01

    Aim: To investigate the mechanisms underlying the anti-nociceptive effect of minocycline on bone cancer pain (BCP) in rats. Methods: A rat model of BCP was established by inoculating Walker 256 mammary carcinoma cells into tibial medullary canal. Two weeks later, the rats were injected with minocycline (50, 100 μg, intrathecally; or 40, 80 mg/kg, ip) twice daily for 3 consecutive days. Mechanical paw withdrawal threshold (PWT) was used to assess pain behavior. After the rats were euthanized, spinal cords were harvested for immunoblotting analyses. The effects of minocycline on NF-κB activation were also examined in primary rat astrocytes stimulated with IL-1β in vitro. Results: BCP rats had marked bone destruction, and showed mechanical tactile allodynia on d 7 and d 14 after the operation. Intrathecal injection of minocycline (100 μg) or intraperitoneal injection of minocycline (80 mg/kg) reversed BCP-induced mechanical tactile allodynia. Furthermore, intraperitoneal injection of minocycline (80 mg/kg) reversed BCP-induced upregulation of GFAP (astrocyte marker) and PSD95 in spinal cord. Moreover, intraperitoneal injection of minocycline (80 mg/kg) reversed BCP-induced upregulation of NF-κB, p-IKKα and IκBα in spinal cord. In IL-1β-stimulated primary rat astrocytes, pretreatment with minocycline (75, 100 μmol/L) significantly inhibited the translocation of NF-κB to nucleus. Conclusion: Minocycline effectively alleviates BCP by inhibiting the NF-κB signaling pathway in spinal astrocytes. PMID:27157092

  3. Regulation of NF-{kappa}B activity in astrocytes: effects of flavonoids at dietary-relevant concentrations

    SciTech Connect

    Spilsbury, Alison; Vauzour, David; Spencer, Jeremy P.E.; Rattray, Marcus

    2012-02-17

    Highlights: Black-Right-Pointing-Pointer We tested the hypothesis that low concentrations of flavonoids inhibit NF-{kappa}B in astrocytes. Black-Right-Pointing-Pointer Primary cultured astrocytes possess a functional {kappa}B-system, measured using luciferase assays. Black-Right-Pointing-Pointer Seven flavonoids (100 nM-1 {mu}M) failed to reduce NF-{kappa}B activity in astrocytes. Black-Right-Pointing-Pointer Four flavonoids (100 nM-1 {mu}M) failed to reduce TNFa-stimulated NF-{kappa}B activity in astrocytes. Black-Right-Pointing-Pointer (-)-Epicatechin did not regulate nuclear translocation of the NF-{kappa}B subunit, p65. -- Abstract: Neuroinflammation plays an important role in the progression of neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Sustained activation of nuclear transcription factor {kappa}B (NF-{kappa}B) is thought to play an important role in the pathogenesis of neurodegenerative disorders. Flavonoids have been shown to possess antioxidant and anti-inflammatory properties and we investigated whether flavonoids, at submicromolar concentrations relevant to their bioavailability from the diet, were able to modulate NF-{kappa}B signalling in astrocytes. Using luciferase reporter assays, we found that tumour necrosis factor (TNF{alpha}, 150 ng/ml) increased NF-{kappa}B-mediated transcription in primary cultures of mouse cortical astrocytes, which was abolished on co-transfection of a dominant-negative I{kappa}B{alpha} construct. In addition, TNF{alpha} increased nuclear localisation of p65 as shown by immunocytochemistry. To investigate potential flavonoid modulation of NF-{kappa}B activity, astrocytes were treated with flavonoids from different classes; flavan-3-ols ((-)-epicatechin and (+)-catechin), flavones (luteolin and chrysin), a flavonol (kaempferol) or the flavanones (naringenin and hesperetin) at dietary-relevant concentrations (0.1-1 {mu}M) for 18 h. None of the flavonoids modulated constitutive or TNF

  4. Involvement of the Notch pathway in terminal astrocytic differentiation: role of PKA.

    PubMed

    Angulo-Rojo, Carla; Manning-Cela, Rebeca; Aguirre, Adán; Ortega, Arturo; López-Bayghen, Esther

    2013-12-23

    The Notch pathway is a highly conserved signaling system essential for modulating neurogenesis and promoting astrogenesis. Similarly, the cAMP signaling cascade can promote astrocytic commitment in several cell culture models, such as the C6 glioma cell line. These cells have the capacity to differentiate into oligodendrocytes or astrocytes, characteristics that allow their use as a glial progenitor model. In this context, we explore here the plausible involvement of cAMP in Notch-dependent signal transactions. The exposure of C6 cells to a non-hydrolysable cAMP analogue resulted in a sustained augmentation of Notch activity, as detected by nuclear translocation of its intracellular domain portion (NICD) and transcriptional activity. The cAMP effect is mediated through the activation of the γ-secretase complex, responsible for Notch cleavage and is sensitive to inhibitors of the cAMP-dependent protein kinase, PKA. As expected, Notch cleavage and nuclear translocation resulted in the up-regulation of the mRNA levels of one of its target genes, the transcription factor Hair and enhancer of split 5. Moreover, the glutamate uptake activity, as well as the expression of astrocytic markers such as glial fibrillary acidic protein, S100β protein and GLAST was also enhanced in cAMP-exposed cells. Our results clearly suggest that during the process of C6 astrocytic differentiation, cAMP activates the PKA/γ-secretase/NICD/RBPJ(κ) pathway and Notch1 expression, leading to transcriptional activation of the genes responsible for glial progenitor cell fate decision.

  5. Involvement of the Notch pathway in terminal astrocytic differentiation: role of PKA

    PubMed Central

    Angulo-Rojo, Carla; Manning-Cela, Rebeca; Aguirre, Adán; Ortega, Arturo; López-Bayghen, Esther

    2013-01-01

    The Notch pathway is a highly conserved signaling system essential for modulating neurogenesis and promoting astrogenesis. Similarly, the cAMP signaling cascade can promote astrocytic commitment in several cell culture models, such as the C6 glioma cell line. These cells have the capacity to differentiate into oligodendrocytes or astrocytes, characteristics that allow their use as a glial progenitor model. In this context, we explore here the plausible involvement of cAMP in Notch-dependent signal transactions. The exposure of C6 cells to a non-hydrolysable cAMP analogue resulted in a sustained augmentation of Notch activity, as detected by nuclear translocation of its intracellular domain portion (NICD) and transcriptional activity. The cAMP effect is mediated through the activation of the γ-secretase complex, responsible for Notch cleavage and is sensitive to inhibitors of the cAMP-dependent protein kinase, PKA. As expected, Notch cleavage and nuclear translocation resulted in the up-regulation of the mRNA levels of one of its target genes, the transcription factor Hair and enhancer of split 5. Moreover, the glutamate uptake activity, as well as the expression of astrocytic markers such as glial fibrillary acidic protein, S100β protein and GLAST was also enhanced in cAMP-exposed cells. Our results clearly suggest that during the process of C6 astrocytic differentiation, cAMP activates the PKA/γ-secretase/NICD/RBPJκ pathway and Notch1 expression, leading to transcriptional activation of the genes responsible for glial progenitor cell fate decision. PMID:24286475

  6. Curcumin Protects against 1-Methyl-4-phenylpyridinium Ion- and Lipopolysaccharide-Induced Cytotoxicities in the Mouse Mesencephalic Astrocyte via Inhibiting the Cytochrome P450 2E1

    PubMed Central

    Gui, Hai-Yan; Chen, Rui-Ni; Peng, Yan; Hu, Jin-Hua; Mao, Zhao; Ning, Rui; Shang, Wei; Liu, Wei; Xiong, Jing; Hu, Gang; Yang, Jian

    2013-01-01

    Curcumin is extracted from the rhizomes of the ginger family plant Curcuma longa L., which has a good protection for liver, kidney, and immune system. However, there is little information about its contribution in protection of astrocytes recently. The present study was undertaken to elucidate the protective effect of curcumin, an herbal antioxidant, on 1-methyl-4-phenylpyridinium ion- (MPP+-) and lipopolysaccharide- (LPS-) induced cytotoxicities, as well as the underlying mechanisms by using primary mouse mesencephalic astrocytes. The results showed that curcumin protected the mesencephalic astrocytes from MPP+- and LPS-induced toxicities along with reducing reactive oxygen species (P < 0.05) and maleic dialdehyde (P < 0.05) sufficiently. Moreover, curcumin significantly inhibited the cytochrome P450 2E1 (CYP2E1) expression (P < 0.01 at mRNA level, P < 0.05 at protein level) and its activity (P < 0.05) sufficiently induced by MPP+ and LPS in the mouse mesencephalic astrocytes. And curcumin as well as diallyl sulphide, a CYP2E1 positive inhibitor, ameliorated MPP+- and LPS-induced mouse mesencephalic astrocytes damage. Accordingly, curcumin protects against MPP+- and LPS-induced cytotoxicities in the mouse mesencephalic astrocyte via inhibiting the CYP2E1 expression and activity. PMID:23843878

  7. IL-1β and IL-6 activate inflammatory responses of astrocytes against Naegleria fowleri infection via the modulation of MAPKs and AP-1.

    PubMed

    Kim, J-H; Song, A-R; Sohn, H-J; Lee, J; Yoo, J-K; Kwon, D; Shin, H-J

    2013-01-01

    Naegleria fowleri, a free-living amoeba, has been found in diverse habitats throughout the world. It causes primary amoebic meningoencephalitis in children and young adults. The amoeba attaches to nasal mucosa, migrates along olfactory nerves and enters the brain. Astrocytes are involved in the defence against infection and produce inflammatory responses. In this study, we focus on the mechanism of immune responses in astrocytes. We showed, using RNase protection assay, RT-PCR and ELISA in an in vitro culture system, that N. fowleri lysates induce interleukin-1beta (IL-1β) and IL-6 expression of astrocytes. In addition, cytokine levels of astrocytes gradually decreased due to extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 inhibitors. To determine the transcription factor, we used transcription inhibitor (AP-1 inhibitor), which downregulated IL-1β and IL-6 expression. These results show that AP-1 is related to IL-1β and IL-6 production. N. fowleri-mediated IL-1β and IL-6 expression requires ERK, JNK and p38 mitogen-activated protein kinases (MAPKs) activation in astrocytes. These findings show that N. fowleri-stimulated astrocytes in an in vitro culture system lead to AP-1 activation and the subsequent expressions of IL-1β and IL-6, which are dependent on ERK, JNK and p38 MAPKs activation. These results may imply that proinflammatory cytokines have important roles in inflammatory responses to N. fowleri infection.

  8. Glucocorticoids facilitate astrocytic amyloid-β peptide deposition by increasing the expression of APP and BACE1 and decreasing the expression of amyloid-β-degrading proteases.

    PubMed

    Wang, Yanyan; Li, Maoquan; Tang, Jun; Song, Min; Xu, Xueqing; Xiong, Jiaxiang; Li, Junxia; Bai, Yun

    2011-07-01

    In most cases, the molecular mechanism underlying the pathogenesis of sporadic Alzheimer's disease (AD) is unknown. Elevated basal cortisol levels in AD patients suggest that glucocorticoids (GC) may contribute to the development and/or maintenance of AD. Amyloid plaques are the hallmark of AD, and they are considered to play an early role in the AD process. However, little is known about how their formation is regulated by stress and GC. Astrocyte accumulation is one of the earliest neuropathological changes in AD. Here, we report that GC elevated amyloid-β (Aβ) production in primary cultures of astrocytes by increasing amyloid precursor protein (APP) and β-site APP-cleaving enzyme 1 gene expression. Notably, GC administered to normal, middle-aged mice promoted the expression of APP and β-site APP-cleaving enzyme 1 in astrocytes, as determined by double immunofluorescence. Additionally, confocal microscopy and ELISA revealed that GC markedly reduced Aβ degradation and clearance by astrocytes in vitro, indicating a decreased neuroprotective capacity of the astrocytes. This may have been due to the decrease of several Aβ-degrading proteases, such as insulin-degrading enzyme and matrix metalloproteinase-9. These effects occurred through the activation of GC receptors. Taken together, our results demonstrate that GC can enhance the production of Aβ, reduce its degradation in astrocytes, and provide a molecular mechanism linking stress factors to AD. Our study suggests that GC can facilitate AD pathogenesis and that reducing GC in the elderly and early AD patients would be beneficial.

  9. Characterization of cellular immune response and innate immune signaling in human and nonhuman primate primary mononuclear cells exposed to Burkholderia mallei.

    PubMed

    Alam, Shahabuddin; Amemiya, Kei; Bernhards, Robert C; Ulrich, Robert G; Waag, David M; Saikh, Kamal U

    2015-01-01

    Burkholderia pseudomallei infection causes melioidosis and is often characterized by severe sepsis. Although rare in humans, Burkholderia mallei has caused infections in laboratory workers, and the early innate cellular response to B. mallei in human and nonhuman primates has not been characterized. In this study, we examined the primary cellular immune response to B. mallei in PBMC cultures of non-human primates (NHPs), Chlorocebus aethiops (African Green Monkeys), Macaca fascicularis (Cynomolgus macaque), and Macaca mulatta (Rhesus macaque) and humans. Our results demonstrated that B. mallei elicited strong primary pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1β, and IL-6) equivalent to the levels of B. pseudomallei in primary PBMC cultures of NHPs and humans. When we examined IL-1β and other cytokine responses by comparison to Escherichia coli LPS, African Green Monkeys appears to be most responsive to B. mallei than Cynomolgus or Rhesus. Characterization of the immune signaling mechanism for cellular response was conducted by using a ligand induced cell-based reporter assay, and our results demonstrated that MyD88 mediated signaling contributed to the B. mallei and B. pseudomallei induced pro-inflammatory responses. Notably, the induced reporter activity with B. mallei, B. pseudomallei, or purified LPS from these pathogens was inhibited and cytokine production was attenuated by a MyD88 inhibitor. Together, these results show that in the scenario of severe hyper-inflammatory responses to B. mallei infection, MyD88 targeted therapeutic intervention may be a successful strategy for therapy.

  10. Neuron-glia interactions through the Heartless FGF receptor signaling pathway mediate morphogenesis of Drosophila astrocytes.

    PubMed

    Stork, Tobias; Sheehan, Amy; Tasdemir-Yilmaz, Ozge E; Freeman, Marc R

    2014-07-16

    Astrocytes are critically important for neuronal circuit assembly and function. Mammalian protoplasmic astrocytes develop a dense ramified meshwork of cellular processes to form intimate contacts with neuronal cell bodies, neurites, and synapses. This close neuron-glia morphological relationship is essential for astrocyte function, but it remains unclear how astrocytes establish their intricate morphology, organize spatial domains, and associate with neurons and synapses in vivo. Here we characterize a Drosophila glial subtype that shows striking morphological and functional similarities to mammalian astrocytes. We demonstrate that the Fibroblast growth factor (FGF) receptor Heartless autonomously controls astrocyte membrane growth, and the FGFs Pyramus and Thisbe direct astrocyte processes to ramify specifically in CNS synaptic regions. We further show that the shape and size of individual astrocytes are dynamically sculpted through inhibitory or competitive astrocyte-astrocyte interactions and Heartless FGF signaling. Our data identify FGF signaling through Heartless as a key regulator of astrocyte morphological elaboration in vivo.

  11. Motor neuron death in ALS – programmed by astrocytes?

    PubMed Central

    Pirooznia, Sheila K.; Dawson, Valina L.; Dawson, Ted M.

    2014-01-01

    Motor neurons in ALS die via cell-autonomous and non-cell autonomous mechanisms. Using adult human astrocytes and motor neurons, Re et al (2014) discover that familial and sporadic ALS derived human adult astrocytes secrete neurotoxic factors that selectively kill motor neurons through necroptosis, suggesting a new therapeutic avenue. PMID:24607221

  12. Neuroimmunological Implications of AQP4 in Astrocytes.

    PubMed

    Ikeshima-Kataoka, Hiroko

    2016-01-01

    The brain has high-order functions and is composed of several kinds of cells, such as neurons and glial cells. It is becoming clear that many kinds of neurodegenerative diseases are more-or-less influenced by astrocytes, which are a type of glial cell. Aquaporin-4 (AQP4), a membrane-bound protein that regulates water permeability is a member of the aquaporin family of water channel proteins that is expressed in the endfeet of astrocytes in the central nervous system (CNS). Recently, AQP4 has been shown to function, not only as a water channel protein, but also as an adhesion molecule that is involved in cell migration and neuroexcitation, synaptic plasticity, and learning/memory through mechanisms involved in long-term potentiation or long-term depression. The most extensively examined role of AQP4 is its ability to act as a neuroimmunological inducer. Previously, we showed that AQP4 plays an important role in neuroimmunological functions in injured mouse brain in concert with the proinflammatory inducer osteopontin (OPN). The aim of this review is to summarize the functional implication of AQP4, focusing especially on its neuroimmunological roles. This review is a good opportunity to compile recent knowledge and could contribute to the therapeutic treatment of autoimmune diseases through strategies targeting AQP4. Finally, the author would like to hypothesize on AQP4's role in interaction between reactive astrocytes and reactive microglial cells, which might occur in neurodegenerative diseases. Furthermore, a therapeutic strategy for AQP4-related neurodegenerative diseases is proposed. PMID:27517922

  13. Neuroimmunological Implications of AQP4 in Astrocytes

    PubMed Central

    Ikeshima-Kataoka, Hiroko

    2016-01-01

    The brain has high-order functions and is composed of several kinds of cells, such as neurons and glial cells. It is becoming clear that many kinds of neurodegenerative diseases are more-or-less influenced by astrocytes, which are a type of glial cell. Aquaporin-4 (AQP4), a membrane-bound protein that regulates water permeability is a member of the aquaporin family of water channel proteins that is expressed in the endfeet of astrocytes in the central nervous system (CNS). Recently, AQP4 has been shown to function, not only as a water channel protein, but also as an adhesion molecule that is involved in cell migration and neuroexcitation, synaptic plasticity, and learning/memory through mechanisms involved in long-term potentiation or long-term depression. The most extensively examined role of AQP4 is its ability to act as a neuroimmunological inducer. Previously, we showed that AQP4 plays an important role in neuroimmunological functions in injured mouse brain in concert with the proinflammatory inducer osteopontin (OPN). The aim of this review is to summarize the functional implication of AQP4, focusing especially on its neuroimmunological roles. This review is a good opportunity to compile recent knowledge and could contribute to the therapeutic treatment of autoimmune diseases through strategies targeting AQP4. Finally, the author would like to hypothesize on AQP4’s role in interaction between reactive astrocytes and reactive microglial cells, which might occur in neurodegenerative diseases. Furthermore, a therapeutic strategy for AQP4-related neurodegenerative diseases is proposed. PMID:27517922

  14. Probing astrocytes with carbon nanotubes and assessing their effects on astrocytic structural and functional properties

    NASA Astrophysics Data System (ADS)

    Gottipati, Manoj K.

    Single-walled carbon nanotubes, chemically-functionalized with polyethylene glycol (SWCNT-PEG) have been shown to modulate the morphology and proliferation characteristics of astrocytes in culture, when applied to the cells as colloidal solutes or as films upon which the cells can attach and grow. These changes were associated with a change in the immunoreactivity of the astrocyte-specific protein, glial fibrillary acidic protein (GFAP); the solutes and films caused an increase and a decrease in GFAP levels, respectively. To assess if these morpho-functional changes induced by the SWCNT-PEG modalities are dependent on GFAP or if the changes in GFAP levels are independent events, I used astrocytes isolated from GFAP knockout mice and found that selected changes induced by the SWCNT-PEG modalities are mediated by GFAP, namely the changes in perimeter, shape and cell death for colloidal solutes and the rate of proliferation for films. Since the loss GFAP has been shown to hamper the trafficking of glutamate transporters to the surface of astrocytes, which plays a vital role in the uptake of extracellular glutamate and maintaining homeostasis in the brain and spinal cord, in a subsequent study, I assessed if the SWCNT-PEG solute causes any change in the glutamate uptake characteristics of astrocytes. Using a radioactive glutamate uptake assay and immunolabeling, I found that SWCNT-PEG solute causes an increase in the uptake of glutamate from the extracellular space along with an increase in the immunoreactivity of the glutamate transporter, L-glutamate L-aspartate transporter (GLAST), on their cell surface, a likely consequence of the increase in GFAP levels induced by the SWCNT-PEG solute. These results imply that SWCNT-PEG could potentially be used as a viable candidate in neural prosthesis applications to prevent glutamate excitotoxicity, a pathological process observed in brain and spinal cord injuries, and alleviate the death toll of neurons surrounding the injury

  15. The PI3K/Akt/mTOR pathway is implicated in the premature senescence of primary human endothelial cells exposed to chronic radiation.

    PubMed

    Yentrapalli, Ramesh; Azimzadeh, Omid; Sriharshan, Arundhathi; Malinowsky, Katharina; Merl, Juliane; Wojcik, Andrzej; Harms-Ringdahl, Mats; Atkinson, Michael J; Becker, Karl-Friedrich; Haghdoost, Siamak; Tapio, Soile

    2013-01-01

    The etiology of radiation-induced cardiovascular disease (CVD) after chronic exposure to low doses of ionizing radiation is only marginally understood. We have previously shown that a chronic low-dose rate exposure (4.1 mGy/h) causes human umbilical vein endothelial cells (HUVECs) to prematurely senesce. We now show that a dose rate of 2.4 mGy/h is also able to trigger premature senescence in HUVECs, primarily indicated by a loss of growth potential and the appearance of the senescence-associated markers ß-galactosidase (SA-ß-gal) and p21. In contrast, a lower dose rate of 1.4 mGy/h was not sufficient to inhibit cellular growth or increase SA-ß-gal-staining despite an increased expression of p21. We used reverse phase protein arrays and triplex Isotope Coded Protein Labeling with LC-ESI-MS/MS to study the proteomic changes associated with chronic radiation-induced senescence. Both technologies identified inactivation of the PI3K/Akt/mTOR pathway accompanying premature senescence. In addition, expression of proteins involved in cytoskeletal structure and EIF2 signaling was reduced. Age-related diseases such as CVD have been previously associated with increased endothelial cell senescence. We postulate that a similar endothelial aging may contribute to the increased rate of CVD seen in populations chronically exposed to low-dose-rate radiation.

  16. The PI3K/Akt/mTOR Pathway Is Implicated in the Premature Senescence of Primary Human Endothelial Cells Exposed to Chronic Radiation

    PubMed Central

    Yentrapalli, Ramesh; Azimzadeh, Omid; Sriharshan, Arundhathi; Malinowsky, Katharina; Merl, Juliane; Wojcik, Andrzej; Harms-Ringdahl, Mats; Atkinson, Michael J.; Becker, Karl-Friedrich; Haghdoost, Siamak; Tapio, Soile

    2013-01-01

    The etiology of radiation-induced cardiovascular disease (CVD) after chronic exposure to low doses of ionizing radiation is only marginally understood. We have previously shown that a chronic low-dose rate exposure (4.1 mGy/h) causes human umbilical vein endothelial cells (HUVECs) to prematurely senesce. We now show that a dose rate of 2.4 mGy/h is also able to trigger premature senescence in HUVECs, primarily indicated by a loss of growth potential and the appearance of the senescence-associated markers ß-galactosidase (SA-ß-gal) and p21. In contrast, a lower dose rate of 1.4 mGy/h was not sufficient to inhibit cellular growth or increase SA-ß-gal-staining despite an increased expression of p21. We used reverse phase protein arrays and triplex Isotope Coded Protein Labeling with LC-ESI-MS/MS to study the proteomic changes associated with chronic radiation-induced senescence. Both technologies identified inactivation of the PI3K/Akt/mTOR pathway accompanying premature senescence. In addition, expression of proteins involved in cytoskeletal structure and EIF2 signaling was reduced. Age-related diseases such as CVD have been previously associated with increased endothelial cell senescence. We postulate that a similar endothelial aging may contribute to the increased rate of CVD seen in populations chronically exposed to low-dose-rate radiation. PMID:23936371

  17. Calcium dynamics in astrocyte processes during neurovascular coupling

    PubMed Central

    Otsu, Yo; Couchman, Kiri; Lyons, Declan G; Collot, Mayeul; Agarwal, Amit; Mallet, Jean-Maurice; Pfrieger, Frank W; Bergles, Dwight E; Charpak, Serge

    2015-01-01

    Enhanced neuronal activity in the brain triggers a local increase in blood flow, termed functional hyperemia, via several mechanisms, including calcium (Ca2+) signaling in astrocytes. However, recent in vivo studies have questioned the role of astrocytes in functional hyperemia because of the slow and sparse dynamics of their somatic Ca2+ signals and the absence of glutamate metabotropic receptor 5 in adults. Here, we reexamined their role in neurovascular coupling by selectively expressing a genetically encoded Ca2+ sensor in astrocytes of the olfactory bulb. We show that in anesthetized mice, the physiological activation of olfactory sensory neuron (OSN) terminals reliably triggers Ca2+ increases in astrocyte processes but not in somata. These Ca2+ increases systematically precede the onset of functional hyperemia by 1–2 s, reestablishing astrocytes as potential regulators of neurovascular coupling. PMID:25531572

  18. Astrocyte arachidonate and palmitate uptake and metabolism is differentially modulated by dibutyryl-cAMP treatment.

    PubMed

    Seeger, D R; Murphy, C C; Murphy, E J

    2016-07-01

    Astrocytes play a vital role in brain lipid metabolism; however the impact of the phenotypic shift in astrocytes to a reactive state on arachidonic acid metabolism is unknown. Therefore, we determined the impact of dibutyryl-cAMP (dBcAMP) treatment on radiolabeled arachidonic acid ([1-(14)C]20:4n-6) and palmitic acid ([1-(14)C]16:0) uptake and metabolism in primary cultured murine cortical astrocytes. In dBcAMP treated astrocytes, total [1-(14)C]20:4n-6 uptake was increased 1.9-fold compared to control, while total [1-(14)C]16:0 uptake was unaffected. Gene expression of long-chain acyl-CoA synthetases (Acsl), acyl-CoA hydrolase (Acot7), fatty acid binding protein(s) (Fabp) and alpha-synuclein (Snca) were determined using qRT-PCR. dBcAMP treatment increased expression of Acsl3 (4.8-fold) and Acsl4 (1.3-fold), which preferentially use [1-(14)C]20:4n-6 and are highly expressed in astrocytes, consistent with the increase in [1-(14)C]20:4n-6 uptake. However, expression of Fabp5 and Fabp7 were significantly reduced by 25% and 45%, respectively. Acot7 (20%) was also reduced, suggesting dBcAMP treatment favors acyl-CoA formation. dBcAMP treatment enhanced [1-(14)C]20:4n-6 (2.2-fold) and [1-(14)C]16:0 (1.6-fold) esterification into total phospholipids, but the greater esterification of [1-(14)C]20:4n-6 is consistent with the observed uptake through increased Acsl, but not Fabp expression. Although total [1-(14)C]16:0 uptake was not affected, there was a dramatic decrease in [1-(14)C]16:0 in the free fatty acid pool as esterification into the phospholipid pool was increased, which is consistent with the increase in Acsl3 and Acsl4 expression. In summary, our data demonstrates that dBcAMP treatment increases [1-(14)C]20:4n-6 uptake in astrocytes and this increase appears to be due to increased expression of Acsl3 and Acsl4 coupled with a reduction in Acot7 expression. PMID:27255639

  19. Effects of Carbon Ions on Primary Cultures of Mouse Brain Cells

    NASA Astrophysics Data System (ADS)

    Nojima, K.; Ando, K.; Fujiwara, H.; Ando, S.

    Primary mixed cultures of astrocytes and microglia were obtained from neonatal mice, and were irradiated with high-LET carbon ions. Immunohistochemical staining showed astrocytes survived more prominently than microglia. Tagged with specific antibodies, astrocytes and microglia surviving after irradiation were counted by flow cytometry. Decreases in the number of microglia and astrocytes were detected at a dose as small as 2 Gy when Day 5 cultures were irradiated with 13 keV/μm carbon ions. When the cultures were irradiated on Day 10, the dose-dependent decrease of microglia was more prominent for 13 keV/μun carbon ions than 70 keV/μm carbon ions. Astrocytes showed a marginal decrease at Day 10 and Day 14. We concluded that microglia are more sensitive than astrocytes to carbon ions and X-rays, and that the radiosensitivity of microglia depends on both differentiation/proliferation status and radiation quality

  20. Study of red wine neuroprotection on astrocytes.

    PubMed

    Gómez-Serranillos, M Pilar; Martín, Sara; Ortega, Teresa; Palomino, Olga M; Prodanov, Marín; Vacas, Visitación; Hernández, Teresa; Estrella, Isabel; Carretero, M Emilia

    2009-12-01

    Phenolic composition of wine depends not only on the grape variety from which it is made, but on some external factors such as winemaking technology. Red wine possesses the most antioxidant effect because of its high polyphenolic content. The aim of this work is to study for the first time, the neuroprotective activity of four monovarietal Spanish red wines (Merlot (ME), Tempranillo (T), Garnacha (G) and Cabernet-Sauvignon (CS)) through its antioxidant ability, and to relate this neuroprotection to its polyphenolic composition, if possible. The wine effect on neuroprotection was studied through its effect as free radical scavenger against FeSO4, H2O2 and FeSO4 + H2O2. Effect on cell survival was determined by 3(4,5-dimethyltiazol-2-il)-2,5-diphenyltetrazolium reduction assay (MTT) and lactate dehydrogenase (LDH) release assay on astrocytes cultures. Results showed that most of the studied wine varieties induced neuroprotection through their antioxidant ability in astrocytes, Merlot being the most active; this variety is especially rich in phenolic compounds, mainly catechins and oligomeric proanthocyanidins. Our results show that red wine exerts a protection against oxidative stress generated by different toxic agents and that the observed neuroprotective activity is related to their polyphenolic content.

  1. Translational potential of astrocytes in brain disorders.

    PubMed

    Verkhratsky, Alexei; Steardo, Luca; Parpura, Vladimir; Montana, Vedrana

    2016-09-01

    Fundamentally, all brain disorders can be broadly defined as the homeostatic failure of this organ. As the brain is composed of many different cells types, including but not limited to neurons and glia, it is only logical that all the cell types/constituents could play a role in health and disease. Yet, for a long time the sole conceptualization of brain pathology was focused on the well-being of neurons. Here, we challenge this neuron-centric view and present neuroglia as a key element in neuropathology, a process that has a toll on astrocytes, which undergo complex morpho-functional changes that can in turn affect the course of the disorder. Such changes can be grossly identified as reactivity, atrophy with loss of function and pathological remodeling. We outline the pathogenic potential of astrocytes in variety of disorders, ranging from neurotrauma, infection, toxic damage, stroke, epilepsy, neurodevelopmental, neurodegenerative and psychiatric disorders, Alexander disease to neoplastic changes seen in gliomas. We hope that in near future we would witness glial-based translational medicine with generation of deliverables for the containment and cure of disorders. We point out that such as a task will require a holistic and multi-disciplinary approach that will take in consideration the concerted operation of all the cell types in the brain.

  2. Astrocytes refine cortical connectivity at dendritic spines

    PubMed Central

    Risher, W Christopher; Patel, Sagar; Kim, Il Hwan; Uezu, Akiyoshi; Bhagat, Srishti; Wilton, Daniel K; Pilaz, Louis-Jan; Singh Alvarado, Jonnathan; Calhan, Osman Y; Silver, Debra L; Stevens, Beth; Calakos, Nicole; Soderling, Scott H; Eroglu, Cagla

    2014-01-01

    During cortical synaptic development, thalamic axons must establish synaptic connections despite the presence of the more abundant intracortical projections. How thalamocortical synapses are formed and maintained in this competitive environment is unknown. Here, we show that astrocyte-secreted protein hevin is required for normal thalamocortical synaptic connectivity in the mouse cortex. Absence of hevin results in a profound, long-lasting reduction in thalamocortical synapses accompanied by a transient increase in intracortical excitatory connections. Three-dimensional reconstructions of cortical neurons from serial section electron microscopy (ssEM) revealed that, during early postnatal development, dendritic spines often receive multiple excitatory inputs. Immuno-EM and confocal analyses revealed that majority of the spines with multiple excitatory contacts (SMECs) receive simultaneous thalamic and cortical inputs. Proportion of SMECs diminishes as the brain develops, but SMECs remain abundant in Hevin-null mice. These findings reveal that, through secretion of hevin, astrocytes control an important developmental synaptic refinement process at dendritic spines. DOI: http://dx.doi.org/10.7554/eLife.04047.001 PMID:25517933

  3. Translational potential of astrocytes in brain disorders.

    PubMed

    Verkhratsky, Alexei; Steardo, Luca; Parpura, Vladimir; Montana, Vedrana

    2016-09-01

    Fundamentally, all brain disorders can be broadly defined as the homeostatic failure of this organ. As the brain is composed of many different cells types, including but not limited to neurons and glia, it is only logical that all the cell types/constituents could play a role in health and disease. Yet, for a long time the sole conceptualization of brain pathology was focused on the well-being of neurons. Here, we challenge this neuron-centric view and present neuroglia as a key element in neuropathology, a process that has a toll on astrocytes, which undergo complex morpho-functional changes that can in turn affect the course of the disorder. Such changes can be grossly identified as reactivity, atrophy with loss of function and pathological remodeling. We outline the pathogenic potential of astrocytes in variety of disorders, ranging from neurotrauma, infection, toxic damage, stroke, epilepsy, neurodevelopmental, neurodegenerative and psychiatric disorders, Alexander disease to neoplastic changes seen in gliomas. We hope that in near future we would witness glial-based translational medicine with generation of deliverables for the containment and cure of disorders. We point out that such as a task will require a holistic and multi-disciplinary approach that will take in consideration the concerted operation of all the cell types in the brain. PMID:26386136

  4. Experimental feature in the primary-proton flux at energies above 10 TeV according to the results of searches for primary particles in nuclear emulsions exposed in the stratosphere (RUNJOB Experiment)

    SciTech Connect

    Zayarnaya, I. S.

    2008-02-15

    In the RUNJOB experiment, a long-term exposure of x-ray emulsion chambers in the stratosphere from 1995 to 1999 with the aim of studying the composition and spectra of primary cosmic particles in the energy range 10-1000 TeV per nucleon revealed about 50% proton tracks. The remaining events of the proton group did not feature any candidate for a track of a singly charged particle within the search region determined from measurements of the coordinates of background nuclei going close to the sought track. Methodological factors that could explain this experimental observation are considered. A possible physical reason associated with the presence of a neutral component in the flux of primary protons in the energy region above 10 TeV is also analyzed.

  5. Comparative evaluation of antimicrobial activity of miswak, propolis, sodium hypochlorite and saline as root canal irrigants by microbial culturing and quantification in chronically exposed primary teeth.

    PubMed Central

    Shingare, Poonam; Chaugule, Vishwas

    2011-01-01

    Introduction: One of the essential factors for successful root canal therapy is elimination of bacterial contaminants using an effective root canal chemical irrigant such as sodium hypochlorite which inherently possesses certain disadvantages like irritation to periapical tissues, staining of the instruments, burning of surrounding tissues etc. The aim of the study was to explore newer irrigant agents which would probably be as effective or more and at the same time less irritating to the tissues than sodium hypochlorite. Our search included two such agents, propolis and miswak. Methods: The study was performed on 40 infected primary teeth (20 male and 20 female patients). The subjects were divided into 4 groups of 10 children. Group 1 received 3% sodium hypochlorite as irrigating solution, group 2 received 12.5% alcoholic extract of miswak, group 3 received 11% alcoholic extract of propolis and group 4, 0.9% saline. We used the antimicrobial activity of sodium hypochlorite as gold standard for comparing the activities of both propolis and miswak. We collected pre- and post-irrigation samples using sterile paper points. Samples were cultured on tryptose soya agar at a temperature of 37oC for 24-48 hours. The colonies were counted with a digital colony counter. For the statistical analysis, we used the unpaired t test at level of significance 0.05 and the ANOVA test for analysis of variance. Results: The differences in pre- and post- irrigation values were calculated for each group, the greatest difference being seen in group 1 (95.549%) followed by group 2 (89.794%), group 3 (34.735%) and group 4 (28.087%). When comparing the results between groups, there was no statistically significant difference between groups 3 and 4. Conclusions: The statistically analyzed results suggested that miswak could be a good natural substitute to sodium hypochlorite, while propolis showed results comparable to those of the negative control. PMID:24432254

  6. Curcumin exerts anti-inflammatory and antioxidative properties in 1-methyl-4-phenylpyridinium ion (MPP(+))-stimulated mesencephalic astrocytes by interference with TLR4 and downstream signaling pathway.

    PubMed

    Yu, Song; Wang, Xu; He, Xingliang; Wang, Yue; Gao, Sujie; Ren, Lu; Shi, Yan

    2016-07-01

    Neuroinflammation is closely associated with the pathophysiology of neurodegenerative diseases including Parkinson's disease (PD). Recent evidence indicates that astrocytes also play pro-inflammatory roles in the central nervous system (CNS) by activation with toll-like receptor (TLR) ligands. Therefore, targeting anti-inflammation may provide a promising therapeutic strategy for PD. Curcumin, a polyphenolic compound isolated from Curcuma longa root, has been commonly used for the treatment of neurodegenerative diseases. However, the details of how curcumin exerts neuroprotection remain uncertain. Here, we investigated the protective effect of curcumin on 1-methyl-4-phenylpyridinium ion-(MPP(+)-) stimulated primary astrocytes. Our results showed that MPP(+) stimulation resulted in significant production of tumor necrosis factor (TNF)-α, interleukin (IL-6), and reactive oxygen species (ROS) in primary mesencephalic astrocytes. Curcumin pretreatment decreased the levels of these pro-inflammatory cytokines while increased IL-10 expression in MPP(+)-stimulated astrocytes. In addition, curcumin increased the levels of antioxidant glutathione (GSH) and reduced ROS production. Our results further showed that curcumin decreased the levels of TLR4 and its downstream effectors including NF-κB, IRF3, MyD88, and TIRF that are induced by MPP(+) as well as inhibited the immunoreactivity of TLR4 and morphological activation in MPP(+)-stimulated astrocytes. Together, data suggest that curcumin might exert a beneficial effect on neuroinflammation in the pathophysiology of PD. PMID:27164829

  7. Extensive astrocyte infection is prominent in human immunodeficiency virus-associated dementia.

    PubMed

    Churchill, Melissa J; Wesselingh, Steven L; Cowley, Daniel; Pardo, Carlos A; McArthur, Justin C; Brew, Bruce J; Gorry, Paul R

    2009-08-01

    Astrocyte infection with human immunodeficiency virus (HIV) is considered rare, so astrocytes are thought to play a secondary role in HIV neuropathogenesis. By combining double immunohistochemistry, laser capture microdissection, and highly sensitive multiplexed polymerase chain reaction to detect HIV DNA in single astrocytes in vivo, we showed that astrocyte infection is extensive in subjects with HIV-associated dementia, occurring in up to 19% of GFAP+ cells. In addition, astrocyte infection frequency correlated with the severity of neuropathological changes and proximity to perivascular macrophages. Our data indicate that astrocytes can be extensively infected with HIV, and suggest an important role for HIV-infected astrocytes in HIV neuropathogenesis.

  8. Simultaneous neuron- and astrocyte-specific fluorescent marking

    SciTech Connect

    Schulze, Wiebke; Hayata-Takano, Atsuko; Kamo, Toshihiko; Nakazawa, Takanobu; Nagayasu, Kazuki; Kasai, Atsushi; Seiriki, Kaoru; Shintani, Norihito; Ago, Yukio; Farfan, Camille; and others

    2015-03-27

    Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignment of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains. - Highlights: • We develop a method for the specific fluorescent labeling of neurons and astrocytes. • Neuron-specific labeling is achieved using Scg10 and synapsin promoters. • Astrocyte-specific labeling is generated using the minimal GFAP promoter. • Nuclear localization of fluorescent proteins is achieved with histone 2B protein.

  9. Differentiation of purified astrocytes in a chemically defined medium

    SciTech Connect

    Morrison, R.S.; de Vellis, J.

    1981-01-01

    Homogeneous cultures of astrocytes and oligodendrocytes provide an excellent model system for studying the regulation of glial structure and function. Recently, a chemically defined (CD) medium was developed for purified cultures of astrocytes, thus eliminating the requirement for serum and providing a controlled system for the study of astroglial properties. Due to the widespread use of astrocyte cultures and the potential benefits to be gained from using a defined medium, astrocyte cultures raised in CD medium were analyzed for purity as well as morphological and biochemical properties. Purity was assessed using immunocytochemical staining for glial fibrillary acidic protein (GFAP) and fibronectin. Astrocytes raised in CD medium are 95% pure using the expression of GFAP as a criterion. Fewer than 1% of the cells in CD medium stained positive for fibronectin eliminating the possibility that CD medium is selective for meningeal or endothelial cells. Astrocytes raised in CD medium exhibit a striking degree of morphological differentiation as seen in scanning electron micrographs. They also exhibit a high degree of biochemical differentiation illustrated by increases in the specific activity of S-100 protein and the induction of glutamine synthetase by glucocorticoids. A defined medium that supports the proliferation of rat astrocytes and enhances numerous morphological and biochemical properties should greatly facilitate the study of factors controlling glial proliferation and differentiation.

  10. Curcumin alleviates oxidative stress and mitochondrial dysfunction in astrocytes.

    PubMed

    Daverey, Amita; Agrawal, Sandeep K

    2016-10-01

    Oxidative stress plays a critical role in various neurodegenerative diseases, thus alleviating oxidative stress is a potential strategy for therapeutic intervention and/or prevention of neurodegenerative diseases. In the present study, alleviation of oxidative stress through curcumin is investigated in A172 (human glioblastoma cell line) and HA-sp (human astrocytes cell line derived from the spinal cord) astrocytes. H2O2 was used to induce oxidative stress in astrocytes (A172 and HA-sp). Data show that H2O2 induces activation of astrocytes in dose- and time-dependent manner as evident by increased expression of GFAP in A172 and HA-sp cells after 24 and 12h respectively. An upregulation of Prdx6 was also observed in A172 and HA-sp cells after 24h of H2O2 treatment as compared to untreated control. Our data also showed that curcumin inhibits oxidative stress-induced cytoskeleton disarrangement, and impedes the activation of astrocytes by inhibiting upregulation of GFAP, vimentin and Prdx6. In addition, we observed an inhibition of oxidative stress-induced inflammation, apoptosis and mitochondria fragmentation after curcumin treatment. Therefore, our results suggest that curcumin not only protects astrocytes from H2O2-induced oxidative stress but also reverses the mitochondrial damage and dysfunction induced by oxidative stress. This study also provides evidence for protective role of curcumin on astrocytes by showing its effects on attenuating reactive astrogliosis and inhibiting apoptosis.

  11. Calcineurin proteolysis in astrocytes: Implications for impaired synaptic function.

    PubMed

    Pleiss, Melanie M; Sompol, Pradoldej; Kraner, Susan D; Abdul, Hafiz Mohmmad; Furman, Jennifer L; Guttmann, Rodney P; Wilcock, Donna M; Nelson, Peter T; Norris, Christopher M

    2016-09-01

    Mounting evidence suggests that astrocyte activation, found in most forms of neural injury and disease, is linked to the hyperactivation of the protein phosphatase calcineurin. In many tissues and cell types, calcineurin hyperactivity is the direct result of limited proteolysis. However, little is known about the proteolytic status of calcineurin in activated astrocytes. Here, we developed a polyclonal antibody to a high activity calcineurin proteolytic fragment in the 45-48kDa range (ΔCN) for use in immunohistochemical applications. When applied to postmortem human brain sections, the ΔCN antibody intensely labeled cell clusters in close juxtaposition to amyloid deposits and microinfarcts. Many of these cells exhibited clear activated astrocyte morphology. The expression of ΔCN in astrocytes near areas of pathology was further confirmed using confocal microscopy. Multiple NeuN-positive cells, particularly those within microinfarct core regions, also labeled positively for ΔCN. This observation suggests that calcineurin proteolysis can also occur within damaged or dying neurons, as reported in other studies. When a similar ΔCN fragment was selectively expressed in hippocampal astrocytes of intact rats (using adeno-associated virus), we observed a significant reduction in the strength of CA3-CA1 excitatory synapses, indicating that the hyperactivation of astrocytic calcineurin is sufficient for disrupting synaptic function. Together, these results suggest that proteolytic activation of calcineurin in activated astrocytes may be a central mechanism for driving and/or exacerbating neural dysfunction during neurodegenerative disease and injury. PMID:27212416

  12. Ethanol inhibits neuritogenesis induced by astrocyte muscarinic receptors.

    PubMed

    Guizzetti, Marina; Moore, Nadia H; Giordano, Gennaro; VanDeMark, Kathryn L; Costa, Lucio G

    2010-09-01

    In utero alcohol exposure can lead to fetal alcohol spectrum disorders, characterized by cognitive and behavioral deficits. In vivo and in vitro studies have shown that ethanol alters neuronal development. We have recently shown that stimulation of M(3) muscarinic receptors in astrocytes increases the synthesis and release of fibronectin, laminin, and plasminogen activator inhibitor-1, causing neurite outgrowth in hippocampal neurons. As M(3) muscarinic receptor signaling in astroglial cells is strongly inhibited by ethanol, we hypothesized that ethanol may also inhibit neuritogenesis in hippocampal neurons induced by carbachol-stimulated astrocytes. In the present study, we report that the effect of carbachol-stimulated astrocytes on hippocampal neuron neurite outgrowth was inhibited in a concentration-dependent manner (25-100 mM) by ethanol. This effect was because of the inhibition of the release of fibronectin, laminin, and plasminogen activator inhibitor-1. Similar effects on neuritogenesis and on the release of astrocyte extracellular proteins were observed after the incubation of astrocytes with carbachol in the presence of 1-butanol, another short-chain alcohol, which like ethanol is a competitive substrate for phospholipase D, but not by tert-butanol, its analog that is not a substrate for this enzyme. This study identifies a potential novel mechanism involved in the developmental effects of ethanol mediated by the interaction of ethanol with cell signaling in astrocytes, leading to an impairment in neuron-astrocyte communication.

  13. Protective role of astrocytic leptin signaling against excitotoxicity.

    PubMed

    Jayaram, Bhavaani; Khan, Reas S; Kastin, Abba J; Hsuchou, Hung; Wu, Xiaojun; Pan, Weihong

    2013-03-01

    Both proconvulsive and anticonvulsive roles of leptin have been reported, suggesting cell-specific actions of leptin in different models of seizure and epilepsy. The goal of our study was to determine the regulation and function of astrocytic leptin receptors in a mouse model of epilepsy and glutamate-induced cytotoxicity. We show that in pilocarpine-challenged mice developing epilepsy with recurrent seizures after a latent period of 2 weeks, hippocampal leptin receptor (ObR) immunofluorescence was increased at 6 weeks. This was more pronounced in astrocytes than in neurons. In cultured astrocytes, glutamate increased ObRa and ObRb expression, whereas leptin pretreatment attenuated glial cytotoxicity by excess glutamate, reflected by better preserved adenosine triphosphate production. The protective role of astrocytic leptin signaling is further supported by the higher lethality of the astrocyte-specific leptin receptor knockout mice in the initial phase of seizure production. Thus, leptin signaling in astrocytes plays a protective role against seizure, and the effects are at least partially mediated by attenuation of glutamate toxicity. Astrocytic leptin signaling, therefore, may be a novel therapeutic target.

  14. Calcium oscillations encoding neuron-to-astrocyte communication.

    PubMed

    Zonta, Micaela; Carmignoto, Giorgio

    2002-01-01

    The observation that the excitatory neurotransmitter glutamate released from presynaptic terminals can activate, beside the post-synaptic neuron, the glial cell astrocyte, stimulated glial cell research like no other event since the recognition in the 1980s that astrocytes can express on their membrane many receptors for classical neurotransmitters. The properties and the functional role(s) of such a neuron-to-astrocyte signaling have now become the focus of intense research in neurobiology. Indeed, a growing body of evidence has recently highlighted the ability of astrocytes to work as sophisticated detectors of synaptic activity: by changing the frequency of [Ca(2+)](i) oscillations evoked by the synaptic release of glutamate, these cells display the remarkable capacity to discriminate between different levels and patterns of synaptic activity. Furthermore, the observation that astrocytes increase the frequency of [Ca(2+)](i) oscillations in response to repetitive episodes of high neuronal activity challenges the common concept that memory function in the brain is an exclusive property of neuronal cells. Glutamate-mediated [Ca(2+)](i) elevations can also trigger in astrocytes the release of glutamate that can ultimately affect neuronal transmission. Given the wide role played by glutamate in brain physiology, our view on how the brain operates needs now to be revised taking into account the bi-directional, glutamatergic communication between neurons and astrocytes.

  15. Calcineurin proteolysis in astrocytes: Implications for impaired synaptic function.

    PubMed

    Pleiss, Melanie M; Sompol, Pradoldej; Kraner, Susan D; Abdul, Hafiz Mohmmad; Furman, Jennifer L; Guttmann, Rodney P; Wilcock, Donna M; Nelson, Peter T; Norris, Christopher M

    2016-09-01

    Mounting evidence suggests that astrocyte activation, found in most forms of neural injury and disease, is linked to the hyperactivation of the protein phosphatase calcineurin. In many tissues and cell types, calcineurin hyperactivity is the direct result of limited proteolysis. However, little is known about the proteolytic status of calcineurin in activated astrocytes. Here, we developed a polyclonal antibody to a high activity calcineurin proteolytic fragment in the 45-48kDa range (ΔCN) for use in immunohistochemical applications. When applied to postmortem human brain sections, the ΔCN antibody intensely labeled cell clusters in close juxtaposition to amyloid deposits and microinfarcts. Many of these cells exhibited clear activated astrocyte morphology. The expression of ΔCN in astrocytes near areas of pathology was further confirmed using confocal microscopy. Multiple NeuN-positive cells, particularly those within microinfarct core regions, also labeled positively for ΔCN. This observation suggests that calcineurin proteolysis can also occur within damaged or dying neurons, as reported in other studies. When a similar ΔCN fragment was selectively expressed in hippocampal astrocytes of intact rats (using adeno-associated virus), we observed a significant reduction in the strength of CA3-CA1 excitatory synapses, indicating that the hyperactivation of astrocytic calcineurin is sufficient for disrupting synaptic function. Together, these results suggest that proteolytic activation of calcineurin in activated astrocytes may be a central mechanism for driving and/or exacerbating neural dysfunction during neurodegenerative disease and injury.

  16. Contrasting effects of Krüppel-like factor 4 on X-ray-induced double-strand and single-strand DNA breaks in mouse astrocytes.

    PubMed

    Zhang, Ji; Cui, Fengmei; Li, Lei; Yang, Jiangtao; Zhang, Liyuan; Chen, Qiu; Tian, Ye

    2014-04-01

    Astrocytes, the most common cell type in the brain, play a principal role in the repair of damaged brain tissues during external radiotherapy of brain tumours. As a downstream gene of p53, the effects of Krüppel-like factor 4 (KLF4) in response to X-ray-induced DNA damage in astrocytes are unclear. In the present study, KLF4 expression was upregulated after the exposure of astrocytes isolated from the murine brain. Inhibition of KLF4 expression using lentiviral transduction produced less double-strand DNA breaks (DSB) determined by a neutral comet assay and flow cytometric analysis of phosphorylated histone family 2A variant and more single-strand DNA breaks (SSB) determined by a basic comet assay when the astrocytes were exposed to 4 Gy of X-ray radiation. These data suggest that radiation exposure of the tissues around brain tumour during radiation therapy causes KLF4 overexpression in astrocytes, which induces more DSB and reduces SSB. This causes the adverse effects of radiation therapy in the treatment of brain tumours.

  17. The phosphorylation status and cytoskeletal remodeling of striatal astrocytes treated with quinolinic acid

    SciTech Connect

    Pierozan, Paula; Ferreira, Fernanda; Ortiz de Lima, Bárbara; Gonçalves Fernandes, Carolina; Totarelli Monteforte, Priscila; Castro Medaglia, Natalia de; Bincoletto, Claudia; Soubhi Smaili, Soraya; Pessoa-Pureur, Regina

    2014-04-01

    Quinolinic acid (QUIN) is a glutamate agonist which markedly enhances the vulnerability of neural cells to excitotoxicity. QUIN is produced from the amino acid tryptophan through the kynurenine pathway (KP). Dysregulation of this pathway is associated with neurodegenerative conditions. In this study we treated striatal astrocytes in culture with QUIN and assayed the endogenous phosphorylating system associated with glial fibrillary acidic protein (GFAP) and vimentin as well as cytoskeletal remodeling. After 24 h incubation with 100 µM QUIN, cells were exposed to {sup 32}P-orthophosphate and/or protein kinase A (PKA), protein kinase dependent of Ca{sup 2+}/calmodulin II (PKCaMII) or protein kinase C (PKC) inhibitors, H89 (20 μM), KN93 (10 μM) and staurosporin (10 nM), respectively. Results showed that hyperphosphorylation was abrogated by PKA and PKC inhibitors but not by the PKCaMII inhibitor. The specific antagonists to ionotropic NMDA and non-NMDA (50 µM DL-AP5 and CNQX, respectively) glutamate receptors as well as to metabotropic glutamate receptor (mGLUR; 50 µM MCPG), mGLUR1 (100 µM MPEP) and mGLUR5 (10 µM 4C3HPG) prevented the hyperphosphorylation provoked by QUIN. Also, intra and extracellular Ca{sup 2+} quelators (1 mM EGTA; 10 µM BAPTA-AM, respectively) prevented QUIN-mediated effect, while Ca{sup 2+} influx through voltage-dependent Ca{sup 2+} channel type L (L-VDCC) (blocker: 10 µM verapamil) is not implicated in this effect. Morphological analysis showed dramatically altered actin cytoskeleton with concomitant change of morphology to fusiform and/or flattened cells with retracted cytoplasm and disruption of the GFAP meshwork, supporting misregulation of actin cytoskeleton. Both hyperphosphorylation and cytoskeletal remodeling were reversed 24 h after QUIN removal. Astrocytes are highly plastic cells and the vulnerability of astrocyte cytoskeleton may have important implications for understanding the neurotoxicity of QUIN in neurodegenerative

  18. Envelope glycoprotein gp120 of human immunodeficiency virus type 1 alters ion transport in astrocytes: implications for AIDS dementia complex.

    PubMed Central

    Benos, D J; Hahn, B H; Bubien, J K; Ghosh, S K; Mashburn, N A; Chaikin, M A; Shaw, G M; Benveniste, E N

    1994-01-01

    Infection by human immunodeficiency virus type 1 (HIV-1) is often complicated by a variety of neurological abnormalities. The most common clinical syndrome, termed acquired immunodeficiency syndrome (AIDS) dementia complex, presents as a subcortical dementia with cognitive, motor, and behavioral disturbances and is unique to HIV-1 infection. The pathogenesis of this syndrome is poorly understood but is believed to involve interactions among virally infected macrophages/microglia, astrocytes, and neurons. In this study, we show that exposure of primary rat and human astrocytes to heat-activated HIV-1 virions, or to eukaryotically expressed HIV-1 and HIV-2 envelope glycoproteins (gp120) stimulates amiloride-sensitive Na+/H+ antiport, potassium conductance, and glutamate efflux. These effects are blocked specifically by amiloride, an inhibitor of Na+/H+ antiport and by the selective removal of gp120 with immobilized monoclonal antibody. As a result of modulation of astrocytic function by gp120, the ensuing neuronal depolarization and glutamate exposure could activate both voltage-gated and N-methyl-D-aspartate-regulated Ca2+ channels, leading to increases in intraneuronal Ca2+ and neuronal death. These findings implicate the astrocyte directly in the pathogenesis of AIDS dementia complex. PMID:8290553

  19. Multiscale Modeling Indicates That Temperature Dependent [Ca2+]i Spiking in Astrocytes Is Quantitatively Consistent with Modulated SERCA Activity

    PubMed Central

    Komin, Niko; Moein, Mahsa; Ellisman, Mark H.; Skupin, Alexander

    2015-01-01

    Changes in the cytosolic Ca2+ concentration ([Ca2+]i) are the most predominant active signaling mechanism in astrocytes that can modulate neuronal activity and is assumed to influence neuronal plasticity. Although Ca2+ signaling in astrocytes has been intensively studied in the past, our understanding of the signaling mechanism and its impact on tissue level is still incomplete. Here we revisit our previously published data on the strong temperature dependence of Ca2+ signals in both cultured primary astrocytes and astrocytes in acute brain slices of mice. We apply multiscale modeling to test the hypothesis that the temperature dependent [Ca2+]i spiking is mainly caused by the increased activity of the sarcoendoplasmic reticulum ATPases (SERCAs) that remove Ca2+ from the cytosol into the endoplasmic reticulum. Quantitative comparison of experimental data with multiscale simulations supports the SERCA activity hypothesis. Further analysis of multiscale modeling and traditional rate equations indicates that the experimental observations are a spatial phenomenon where increasing pump strength leads to a decoupling of Ca2+ release sites and subsequently to vanishing [Ca2+]i spikes. PMID:26347125

  20. A "Hit and Run" Approach to Inducible Direct Reprogramming of Astrocytes to Neural Stem Cells.

    PubMed

    Poulou, Maria; Mandalos, Nikolaos P; Karnavas, Theodoros; Saridaki, Marannia; McKay, Ronald D G; Remboutsika, Eumorphia

    2016-01-01

    Temporal and spatial control of gene expression can be achieved using an inducible system as a fundamental tool for regulated transcription in basic, applied and eventually in clinical research. We describe a novel "hit and run" inducible direct reprogramming approach. In a single step, 2 days post-transfection, transiently transfected Sox2(FLAG) under the Leu3p-αIPM inducible control (iSox2) triggers the activation of endogenous Sox2, redirecting primary astrocytes into abundant distinct nestin-positive radial glia cells. This technique introduces a unique novel tool for safe, rapid and efficient reprogramming amendable to regenerative medicine. PMID:27148066

  1. Radiotherapy and temozolomide for anaplastic astrocytic gliomas

    PubMed Central

    Nayak, Lakshmi; Panageas, Katherine S.; Reiner, Anne S.; Huse, Jason T.; Pentsova, Elena; Braunthal, Stephanie G.; Abrey, Lauren E.; DeAngelis, Lisa M.

    2015-01-01

    We previously reported results of a phase II non-comparative trial that randomized patients with glioblastoma following radiotherapy to one of two different temozolomide schedules, followed by 13-cis-retinoic acid (RA) maintenance. Here we report the results of an exploratory cohort of patients accrued with anaplastic astrocytic tumors. Patients with newly diagnosed anaplastic astrocytoma (AA) or anaplastic oligo-astrocytoma (AOA) were treated with concurrent radiotherapy (60 Gy over 6 weeks) and temozolomide (75 mg/m2), and six adjuvant 28-day cycles of either dose-dense (150 mg/m2, days 1–7, 15–21) or metronomic (50 mg/m2, days 1–28) temozolomide. Subsequently, maintenance RA (100 mg/m2, days 1–21/28) was administered until disease progression. All outcome measures were descriptive without intention to compare between treatment arms. Survival was measured by the Kaplan–Meier method. There were 31 patients (21 men, 10 women) with median age 48 years (range 28–74), median KPS 90 (range 60–100). Extent of resection was gross-total in 35 %, subtotal 23 %, and biopsy 42 %. Histology was AA in 90 %, and AOA in 10 %. MGMT promoter methylation was methylated in 20 %, unmethylated in 50 %, and uninformative in 30 % of 30 tested. Median progression-free survival was 2.1 years (95 % CI 0.95–Not Reached), and overall survival 2.9 years (95 % CI 2.0–Not Reached). We report outcomes among a homogeneously treated population with anaplastic astrocytic tumors. Survival was unexpectedly short compared to other reports. These data may be useful as a contemporary historic control for other ongoing or future randomized trials. PMID:25920709

  2. Radiotherapy and temozolomide for anaplastic astrocytic gliomas.

    PubMed

    Nayak, Lakshmi; Panageas, Katherine S; Reiner, Anne S; Huse, Jason T; Pentsova, Elena; Braunthal, Stephanie G; Abrey, Lauren E; DeAngelis, Lisa M; Lassman, Andrew B

    2015-05-01

    We previously reported results of a phase II non-comparative trial that randomized patients with glioblastoma following radiotherapy to one of two different temozolomide schedules, followed by 13-cis-retinoic acid (RA) maintenance. Here we report the results of an exploratory cohort of patients accrued with anaplastic astrocytic tumors. Patients with newly diagnosed anaplastic astrocytoma (AA) or anaplastic oligo-astrocytoma (AOA) were treated with concurrent radiotherapy (60 Gy over 6 weeks) and temozolomide (75 mg/m(2)), and six adjuvant 28-day cycles of either dose-dense (150 mg/m(2), days 1-7, 15-21) or metronomic (50 mg/m(2), days 1-28) temozolomide. Subsequently, maintenance RA (100 mg/m(2), days 1-21/28) was administered until disease progression. All outcome measures were descriptive without intention to compare between treatment arms. Survival was measured by the Kaplan-Meier method. There were 31 patients (21 men, 10 women) with median age 48 years (range 28-74), median KPS 90 (range 60-100). Extent of resection was gross-total in 35%, subtotal 23%, and biopsy 42%. Histology was AA in 90%, and AOA in 10%. MGMT promoter methylation was methylated in 20%, unmethylated in 50%, and uninformative in 30% of 30 tested. Median progression-free survival was 2.1 years (95% CI 0.95-Not Reached), and overall survival 2.9 years (95 % CI 2.0-Not Reached). We report outcomes among a homogeneously treated population with anaplastic astrocytic tumors. Survival was unexpectedly short compared to other reports. These data may be useful as a contemporary historic control for other ongoing or future randomized trials. PMID:25920709

  3. Transfer of mitochondria from astrocytes to neurons after stroke.

    PubMed

    Hayakawa, Kazuhide; Esposito, Elga; Wang, Xiaohua; Terasaki, Yasukazu; Liu, Yi; Xing, Changhong; Ji, Xunming; Lo, Eng H

    2016-07-28

    Neurons can release damaged mitochondria and transfer them to astrocytes for disposal and recycling. This ability to exchange mitochondria may represent a potential mode of cell-to-cell signalling in the central nervous system. Here we show that astrocytes in mice can also release functional mitochondria that enter neurons. Astrocytic release of extracellular mitochondrial particles was mediated by a calcium-dependent mechanism involving CD38 and cyclic ADP ribose signalling. Transient focal cerebral ischaemia in mice induced entry of astrocytic mitochondria into adjacent neurons, and this entry amplified cell survival signals. Suppression of CD38 signalling by short interfering RNA reduced extracellular mitochondria transfer and worsened neurological outcomes. These findings suggest a new mitochondrial mechanism of neuroglial crosstalk that may contribute to endogenous neuroprotective and neurorecovery mechanisms after stroke. PMID:27466127

  4. Monitoring local synaptic activity with astrocytic patch pipettes

    PubMed Central

    Henneberger, Christian; Rusakov, Dmitri A

    2013-01-01

    Rapid signal exchange between astroglia and neurons has emerged as a key player in neural communication in the brain. To understand the mechanisms involved, it is often important to have access to individual astrocytes while monitoring the activity of nearby synapses. Achieving this with standard electrophysiological tools is not always feasible. The protocol presented here enables the monitoring of synaptic activity using whole-cell current-clamp recordings from a local astrocyte. This approach takes advantage of the fact that the low input resistance of electrically passive astroglia allows extracellular currents to pass through the astrocytic membrane with relatively little attenuation. Once the slice preparation is ready, it takes ~30 min to several hours to implement this protocol, depending on the experimental design, which is similar to other patch-clamp techniques. The technique presented here can be used to directly access the intracellular medium of individual astrocytes while examining synapses functioning in their immediate proximity. PMID:23196973

  5. Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways

    NASA Astrophysics Data System (ADS)

    Chung, Won-Suk; Clarke, Laura E.; Wang, Gordon X.; Stafford, Benjamin K.; Sher, Alexander; Chakraborty, Chandrani; Joung, Julia; Foo, Lynette C.; Thompson, Andrew; Chen, Chinfei; Smith, Stephen J.; Barres, Ben A.

    2013-12-01

    To achieve its precise neural connectivity, the developing mammalian nervous system undergoes extensive activity-dependent synapse remodelling. Recently, microglial cells have been shown to be responsible for a portion of synaptic pruning, but the remaining mechanisms remain unknown. Here we report a new role for astrocytes in actively engulfing central nervous system synapses. This process helps to mediate synapse elimination, requires the MEGF10 and MERTK phagocytic pathways, and is strongly dependent on neuronal activity. Developing mice deficient in both astrocyte pathways fail to refine their retinogeniculate connections normally and retain excess functional synapses. Finally, we show that in the adult mouse brain, astrocytes continuously engulf both excitatory and inhibitory synapses. These studies reveal a novel role for astrocytes in mediating synapse elimination in the developing and adult brain, identify MEGF10 and MERTK as critical proteins in the synapse remodelling underlying neural circuit refinement, and have important implications for understanding learning and memory as well as neurological disease processes.

  6. Astrocytes in Multiple Sclerosis: A Product of their Environment

    PubMed Central

    Nair, Aji; Frederick, Terra J.; Miller, Stephen D.

    2010-01-01

    It has long been thought that astrocytes, like other glial cells, simply provide a support mechanism for neuronal function in the healthy and inflamed central nervous system (CNS). However, recent evidence suggests that astrocytes play an active and dual role in CNS inflammatory diseases such as multiple sclerosis (MS). Astrocytes not only have the ability to enhance immune responses and inhibit myelin repair, but they can also be protective and limit CNS inflammation while supporting oligodendrocyte and axonal regeneration. The particular impact of these cells on the pathogenesis and repair of an inflammatory demyelinating process is dependent upon a number of factors, including the stage of the disease, the type and microenvironment of the lesion, and the interactions with other cell types and factors that influence their activation. In this review, we summarize recent data supporting the idea that astrocytes play a complex role in the regulation of CNS autoimmunity. PMID:18516496

  7. mGlu3 receptor and astrocytes: partners in neuroprotection.

    PubMed

    Durand, Daniela; Carniglia, Lila; Caruso, Carla; Lasaga, Mercedes

    2013-03-01

    Astrocytes are currently studied intensively because of their now highlighted relevance as key players with neurons that modulate a wide range of central functions, from synaptic plasticity and synaptogenesis to regulation of metabolic and neuroinflammatory processes. Since the discovery of mGlu3 receptors on astrocytes, accumulating evidence supports a role of these receptors not only in maintaining synaptic homeostasis and treating psychiatric disorders but also in promoting astrocyte survival in several pathologic conditions. This review focuses on providing up-to-date knowledge regarding effects of activating astroglial mGlu3 receptors on psychiatric disorders, astrocyte and neuronal survival, and neurodegenerative diseases. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'. PMID:22564439

  8. Aquaporin-4 expression in cultured astrocytes after fluid percussion injury.

    PubMed

    Rao, Kakulavarapu V Rama; Reddy, Pichili V B; Curtis, Kevin M; Norenberg, Michael D

    2011-03-01

    The development of cytotoxic brain edema resulting in increased intracranial pressure is a major cause of death occurring in the early phase of traumatic brain injury (TBI). Such edema predominantly develops as a consequence of astrocyte swelling. We recently documented that fluid percussion injury (FPI) to cultured astrocytes causes cell swelling. Since aquaporin-4 (AQP4) has been strongly implicated in the development of brain edema/astrocyte swelling in various neurological conditions, this study examined the effect of in vitro trauma on AQP4 protein expression in cultured astrocytes. Exposure of astrocytes to FPI resulted in a significant upregulation of AQP4 protein in the plasma membrane due to neosynthesis, as cycloheximide blocked the trauma-induced AQP4 upregulation. Silencing the aqp4 gene by siRNA resulted in a significant reduction in trauma-induced astrocyte swelling, indicating a critical role of AQP4 in this process. We recently documented that oxidative/nitrative stress (ONS), the mitochondrial permeability transition (mPT), and activation of mitogen-activated protein kinases (MAPKs), contribute to trauma-induced astrocyte swelling in culture. We now show that inhibition of these factors reduces the upregulation of AQP4 following trauma. Since TBI has been shown to activate nuclear factor-kappa B (NF-κB), as well as the Na(+),K(+),Cl(-) co-transporter (NKCC), both of which are implicated in brain edema/astrocyte swelling in other conditions, we also examined the effect of BAY 11-7082 and bumetanide, inhibitors of NF-κB and NKCC, respectively, and found that these agents also significantly inhibited the trauma-induced AQP4 upregulation. Our findings show that in vitro trauma upregulates AQP4, and that ONS, MAPKs, mPT, NF-κB, and NKCC are involved in its upregulation.

  9. Astrocyte structural reactivity and plasticity in models of retinal detachment.

    PubMed

    Luna, Gabriel; Keeley, Patrick W; Reese, Benjamin E; Linberg, Kenneth A; Lewis, Geoffrey P; Fisher, Steven K

    2016-09-01

    Although retinal neurodegenerative conditions such as age-related macular degeneration, glaucoma, diabetic retinopathy, retinitis pigmentosa, and retinal detachment have different etiologies and pathological characteristics, they also have many responses in common at the cellular level, including neural and glial remodeling. Structural changes in Müller cells, the large radial glia of the retina in retinal disease and injury have been well described, that of the retinal astrocytes remains less so. Using modern imaging technology to describe the structural remodeling of retinal astrocytes after retinal detachment is the focus of this paper. We present both a review of critical literature as well as novel work focusing on the responses of astrocytes following rhegmatogenous and serous retinal detachment. The mouse presents a convenient model system in which to study astrocyte reactivity since the Mϋller cell response is muted in comparison to other species thereby allowing better visualization of the astrocytes. We also show data from rat, cat, squirrel, and human retina demonstrating similarities and differences across species. Our data from immunolabeling and dye-filling experiments demonstrate previously undescribed morphological characteristics of normal astrocytes and changes induced by detachment. Astrocytes not only upregulate GFAP, but structurally remodel, becoming increasingly irregular in appearance, and often penetrating deep into neural retina. Understanding these responses, their consequences, and what drives them may prove to be an important component in improving visual outcome in a variety of therapeutic situations. Our data further supports the concept that astrocytes are important players in the retina's overall response to injury and disease. PMID:27060374

  10. Astrocyte structural reactivity and plasticity in models of retinal detachment.

    PubMed

    Luna, Gabriel; Keeley, Patrick W; Reese, Benjamin E; Linberg, Kenneth A; Lewis, Geoffrey P; Fisher, Steven K

    2016-09-01

    Although retinal neurodegenerative conditions such as age-related macular degeneration, glaucoma, diabetic retinopathy, retinitis pigmentosa, and retinal detachment have different etiologies and pathological characteristics, they also have many responses in common at the cellular level, including neural and glial remodeling. Structural changes in Müller cells, the large radial glia of the retina in retinal disease and injury have been well described, that of the retinal astrocytes remains less so. Using modern imaging technology to describe the structural remodeling of retinal astrocytes after retinal detachment is the focus of this paper. We present both a review of critical literature as well as novel work focusing on the responses of astrocytes following rhegmatogenous and serous retinal detachment. The mouse presents a convenient model system in which to study astrocyte reactivity since the Mϋller cell response is muted in comparison to other species thereby allowing better visualization of the astrocytes. We also show data from rat, cat, squirrel, and human retina demonstrating similarities and differences across species. Our data from immunolabeling and dye-filling experiments demonstrate previously undescribed morphological characteristics of normal astrocytes and changes induced by detachment. Astrocytes not only upregulate GFAP, but structurally remodel, becoming increasingly irregular in appearance, and often penetrating deep into neural retina. Understanding these responses, their consequences, and what drives them may prove to be an important component in improving visual outcome in a variety of therapeutic situations. Our data further supports the concept that astrocytes are important players in the retina's overall response to injury and disease.

  11. Leptin signaling in astrocytes regulates hypothalamic neuronal circuits and feeding.

    PubMed

    Kim, Jae Geun; Suyama, Shigetomo; Koch, Marco; Jin, Sungho; Argente-Arizon, Pilar; Argente, Jesús; Liu, Zhong-Wu; Zimmer, Marcelo R; Jeong, Jin Kwon; Szigeti-Buck, Klara; Gao, Yuanqing; Garcia-Caceres, Cristina; Yi, Chun-Xia; Salmaso, Natalina; Vaccarino, Flora M; Chowen, Julie; Diano, Sabrina; Dietrich, Marcelo O; Tschöp, Matthias H; Horvath, Tamas L

    2014-07-01

    We found that leptin receptors were expressed in hypothalamic astrocytes and that their conditional deletion led to altered glial morphology and synaptic inputs onto hypothalamic neurons involved in feeding control. Leptin-regulated feeding was diminished, whereas feeding after fasting or ghrelin administration was elevated in mice with astrocyte-specific leptin receptor deficiency. These data reveal an active role of glial cells in hypothalamic synaptic remodeling and control of feeding by leptin.

  12. Group B Streptococcal Infection and Activation of Human Astrocytes

    PubMed Central

    Stoner, Terri D.; Weston, Thomas A.; Trejo, JoAnn; Doran, Kelly S.

    2015-01-01

    Background Streptococcus agalactiae (Group B Streptococcus, GBS) is the leading cause of life-threatening meningitis in human newborns in industrialized countries. Meningitis results from neonatal infection that occurs when GBS leaves the bloodstream (bacteremia), crosses the blood-brain barrier (BBB), and enters the central nervous system (CNS), where the bacteria contact the meninges. Although GBS is known to invade the BBB, subsequent interaction with astrocytes that physically associate with brain endothelium has not been well studied. Methodology/Principal Findings We hypothesize that human astrocytes play a unique role in GBS infection and contribute to the development of meningitis. To address this, we used a well- characterized human fetal astrocyte cell line, SVG-A, and examined GBS infection in vitro. We observed that all GBS strains of representative clinically dominant serotypes (Ia, Ib, III, and V) were able to adhere to and invade astrocytes. Cellular invasion was dependent on host actin cytoskeleton rearrangements, and was specific to GBS as Streptococcus gordonii failed to enter astrocytes. Analysis of isogenic mutant GBS strains deficient in various cell surface organelles showed that anchored LTA, serine-rich repeat protein (Srr1) and fibronectin binding (SfbA) proteins all contribute to host cell internalization. Wild-type GBS also displayed an ability to persist and survive within an intracellular compartment for at least 12 h following invasion. Moreover, GBS infection resulted in increased astrocyte transcription of interleukin (IL)-1β, IL-6 and VEGF. Conclusions/Significance This study has further characterized the interaction of GBS with human astrocytes, and has identified the importance of specific virulence factors in these interactions. Understanding the role of astrocytes during GBS infection will provide important information regarding BBB disruption and the development of neonatal meningitis. PMID:26030618

  13. Coronavirus induction of class I major histocompatibility complex expression in murine astrocytes is virus strain specific

    PubMed Central

    1994-01-01

    Neurotropic strains of mouse hepatitis viruses (MHV) such as MHV-A59 (A59) and MHV-4 (JHMV) cause acute and chronic encephalomyelitis and demyelination in susceptible strains of mice and rats. They are widely used as models of human demyelinating diseases such as multiple sclerosis (MS), in which immune mechanisms are thought to participate in the development of lesions in the central nervous system (CNS). The effects of MHV infection on target cell functions in the CNS are not well understood, but A59 has been shown to induce the expression of MHC class I molecules in glial cells after in vivo and in vitro infection. Changes in class I expression in infected cells may contribute to the immunopathogenesis of MHV infection in the CNS. In this communication, a large panel of MHV strains was tested for their ability to stimulate class I expression in primary astrocytes in vitro. The data show that the more hepatotropic strains, such as MHV-A59, MHV-1, MHV-2, MHV-3, MHV-D, MHV-K, and MHV-NuU, were potent inducers of class I expression in astrocytes during acute infection, measured by radioimmunoassay. The Kb molecule was preferentially expressed over Db. By contrast, JHMV and several viral strains derived from it did not stimulate the expression of class I molecules. Assays of virus infectivity indicated that the class I-inducing activity did not correlate with the ability of the individual viral strain to replicate in astrocytes. However, exposure of the viruses or the supernatants from infected astrocytes to ultraviolet light abolished the class I-inducing activity, indicating that infectious virus is required for class I expression. These data also suggest that class I expression was induced directly by virus infection, and not by the secretion of a soluble substance into the medium by infected astrocytes. Finally, analyses of A59/JHMV recombinant viral strains suggest that class I-inducing activity resides in one of the A59 structural genes. PMID:8064222

  14. Histamine induces the production of matrix metalloproteinase-9 in human astrocytic cultures via H1-receptor subtype.

    PubMed

    Patel, Aarti; Vasanthan, Vishnu; Fu, Wen; Fahlman, Richard P; MacTavish, David; Jhamandas, Jack H

    2016-05-01

    Accumulation of β-amyloid (Aβ) protein within the brain is a neuropathological hallmark of Alzheimer's disease (AD). One strategy to facilitate Aβ clearance from the brain is to promote Aβ catabolism. Matrix metalloproteinase-9 (MMP-9), a member of the family of Zn(+2)-containing endoproteases, known to be expressed and secreted by astrocytes, is capable of degrading Aβ. Histamine, a major aminergic brain neurotransmitter, stimulates the production of MMP-9 in keratinocytes through the histamine H1 receptor (H1R). In the present study, we show that histamine evokes a concentration- and calcium-dependent release of MMP-9 from human astrocytic U373 cells and primary cultures of human and rat astrocytes through the H1R subtype. Activation of H1R on astrocytes elevated intracellular levels of Ca(2+) that was accompanied by time-dependent increases in MAP kinase p44/p42 and PKC. In-cell western blots revealed dose-dependent increases in both enzymes, confirming involvement of these signal transduction pathways. We next investigated the extent of recombinant human MMP-9 (rhMMP-9) proteolytic activity on soluble oligomeric Aβ (soAβ). Mass spectrometry demonstrated time-dependent cleavage of soAβ (20 μM), but not another amyloidogenic protein amylin, upon incubation with rhMMP-9 (100 nM) at 1, 4 and 17 h. Furthermore, Western blots showed a shift in soAβ equilibrium toward lower order, less toxic monomeric species. In conclusion, both MAPK p44/p42 and PKC pathways appear to be involved in histamine-upregulated MMP-9 release via H1Rs in astrocytes. Furthermore, MMP-9 appears to cleave soAβ into less toxic monomeric species. Given the key role of histamine in MMP-9 release, this neurotransmitter may serve as a potential therapeutic target for AD.

  15. Eicosanoid receptor subtype-mediated opposing regulation of TLR-stimulated expression of astrocyte glial-derived neurotrophic factor

    PubMed Central

    Li, Xianwu; Cudaback, Eiron; Breyer, Richard M.; Montine, Kathleen S.; Keene, C. Dirk; Montine, Thomas J.

    2012-01-01

    A major therapeutic target for Parkinson's disease (PD) is providing increased glial-derived neurotrophic factor (GDNF) to dopaminergic neurons. We tested the hypothesis that innate immune activation increases astrocyte GDNF production and that this is regulated by specific eicosanoid receptors. Innate immune-activated primary murine astrocytes were assayed for GDNF expression and secretion. Controls were agent vehicle exposure and wild-type mice. Rank order for up to 10-fold selectively increased GDNF expression was activators of TLR3 > TLR2 or TLR4 > TLR9. TLR3 activator-stimulated GDNF expression was selectively JNK-dependent, followed cyclooxygenase (COX)-2, was coincident with membranous PGE2 synthase, and was not significantly altered by a nonspecific COX- or a COX-2-selective inhibitor. Specific eicosanoid receptors had opposing effects on TLR3 activator-induced GDNF expression: ∼60% enhancement by blocking or ablating of PGE2 receptor subtype 1 (EP1), ∼30% enhancement by activating PGF2α receptor or thromboxane receptor, or ∼15% enhancement by activating EP4. These results demonstrate functionally antagonistic eicosanoid receptor subtype regulation of innate immunity-induced astrocyte GDNF expression and suggest that selective inhibition of EP1 signaling might be a means to augment astrocyte GDNF secretion in the context of innate immune activation in diseased regions of brain in PD.—Li, X., Cudaback, E., Breyer, R. M., Montine, K. S., Keene, C. D., Montine, T. J. Eicosanoid receptor subtype-mediated opposing regulation of Toll-like receptor-stimulated expression of astrocyte glial-derived neurotrophic factor. PMID:22499581

  16. Both Neurons and Astrocytes Exhibited Tetrodotoxin-Resistant Metabotropic Glutamate Receptor-Dependent Spontaneous Slow Ca2+ Oscillations in Striatum

    PubMed Central

    Tamura, Atsushi; Yamada, Naohiro; Yaguchi, Yuichi; Machida, Yoshio; Mori, Issei; Osanai, Makoto

    2014-01-01

    The striatum plays an important role in linking cortical activity to basal ganglia outputs. Group I metabotropic glutamate receptors (mGluRs) are densely expressed in the medium spiny projection neurons and may be a therapeutic target for Parkinson's disease. The group I mGluRs are known to modulate the intracellular Ca2+ signaling. To characterize Ca2+ signaling in striatal cells, spontaneous cytoplasmic Ca2+ transients were examined in acute slice preparations from transgenic mice expressing green fluorescent protein (GFP) in the astrocytes. In both the GFP-negative cells (putative-neurons) and astrocytes of the striatum, spontaneous slow and long-lasting intracellular Ca2+ transients (referred to as slow Ca2+ oscillations), which lasted up to approximately 200 s, were found. Neither the inhibition of action potentials nor ionotropic glutamate receptors blocked the slow Ca2+ oscillation. Depletion of the intracellular Ca2+ store and the blockade of inositol 1,4,5-trisphosphate receptors greatly reduced the transient rate of the slow Ca2+ oscillation, and the application of an antagonist against mGluR5 also blocked the slow Ca2+ oscillation in both putative-neurons and astrocytes. Thus, the mGluR5-inositol 1,4,5-trisphosphate signal cascade is the primary contributor to the slow Ca2+ oscillation in both putative-neurons and astrocytes. The slow Ca2+ oscillation features multicellular synchrony, and both putative-neurons and astrocytes participate in the synchronous activity. Therefore, the mGluR5-dependent slow Ca2+ oscillation may involve in the neuron-glia interaction in the striatum. PMID:24454845

  17. Effect of u18666a on beta-glucosidase, sphingomyelinase, and beta-galactosidase activities in astrocytes of young rats.

    PubMed

    Santos, Daniela Copetti; da Silva Garcia, Cristina; de Andrade, Carla Vieira; Daitx, Vanessa Vitcoski; da Costa Moraes, Vitória; Rohden, Francieli; Coelho, Janice Carneiro

    2015-04-01

    Niemann-Pick type C disease (NPC) is a neurodegenerative genetic disorder caused by accumulation of lipids, especially cholesterol, in the perinuclear space. U18666A is a cholesterol transport-inhibiting agent, being used to mimic NPC, mainly in fibroblasts. The objective of this study was to observe the effect of the drug U18666A, which causes the accumulation of cholesterol in the cytoplasm of astrocytes from newborn rats, on some lysosomal hydrolase activities. Filipin staining and fluorescence microscopy, through CellM software, were used for visualization and quantification of cholesterol. The dose of U18666A that provided the greatest accumulation of cholesterol was that of 0.25 µg/mL in incubation for 48 h. Primary rat astrocytes incubated with the drug (NPC) showed a significantly higher amount of cholesterol than those without U18666A (controls). The measurement of activity of enzymes sphingomyelinase and beta-glucosidase in astrocytes of rats with NPC was significantly lower than that of control astrocytes, which is consistent with the disease in humans. The activity of the enzyme beta-galactosidase showed no significant difference between both groups. We concluded that U18666A appears to be an excellent intracellular cholesterol transport-inhibiting agent affecting some metabolic pathways in astrocytes of young rats, which mimics NPC in these animals. Just like the change in the activity of lysosomal enzymes has been demonstrated, other biochemical parameters of the cell can be tested with this animal model, thus contributing to a better understanding of the disease.

  18. Two-pore Domain Potassium Channels in Astrocytes

    PubMed Central

    Ryoo, Kanghyun

    2016-01-01

    Two-pore domain potassium (K2P) channels have a distinct structure and channel properties, and are involved in a background K+ current. The 15 members of the K2P channels are identified and classified into six subfamilies on the basis of their sequence similarities. The activity of the channels is dynamically regulated by various physical, chemical, and biological effectors. The channels are expressed in a wide variety of tissues in mammals in an isoform specific manner, and play various roles in many physiological and pathophysiological conditions. To function as channels, the K2P channels form dimers, and some isoforms form heterodimers that provide diversity in channel properties. In the brain, TWIK1, TREK1, TREK2, TRAAK, TASK1, and TASK3 are predominantly expressed in various regions, including the cerebral cortex, dentate gyrus, CA1-CA3, and granular layer of the cerebellum. TWIK1, TREK1, and TASK1 are highly expressed in astrocytes, where they play specific cellular roles. Astrocytes keep leak K+ conductance, called the passive conductance, which mainly involves TWIK1-TREK1 heterodimeric channel. TWIK1 and TREK1 also mediate glutamate release from astrocytes in an exocytosis-independent manner. The expression of TREK1 and TREK2 in astrocytes increases under ischemic conditions, that enhance neuroprotection from ischemia. Accumulated evidence has indicated that astrocytes, together with neurons, are involved in brain function, with the K2P channels playing critical role in these astrocytes. PMID:27790056

  19. Role of astrocytes in memory and psychiatric disorders.

    PubMed

    Moraga-Amaro, R; Jerez-Baraona, J M; Simon, F; Stehberg, J

    2014-01-01

    Over the past decade, the traditional description of astrocytes as being merely accessories to brain function has shifted to one in which their role has been pushed into the forefront of importance. Current views suggest that astrocytes:(1) are excitable through calcium fluctuations and respond to neurotransmitters released at synapses; (2) communicate with each other via calcium waves and release their own gliotransmitters which are essential for synaptic plasticity; (3) activate hundreds of synapses at once, thereby synchronizing neuronal activity and activating or inhibiting complete neuronal networks; (4) release vasoactive substances to the smooth muscle surrounding blood vessels enabling the coupling of circulation (blood flow) to local brain activity; and (5) release lactate in an activity-dependent manner in order to supply neuronal metabolic demand. In consequence, the role of astrocytes and astrocytic gliotransmitters is now believed to be critical for higher brain function and recently, evidence begins to gather suggesting that astrocytes are pivotal for learning and memory. All of the above are reviewed here while focusing on the role of astrocytes in memory and psychiatric disorders.

  20. Astrocytic modulation of blood brain barrier: perspectives on Parkinson's disease.

    PubMed

    Cabezas, Ricardo; Avila, Marcos; Gonzalez, Janneth; El-Bachá, Ramon Santos; Báez, Eliana; García-Segura, Luis Miguel; Jurado Coronel, Juan Camilo; Capani, Francisco; Cardona-Gomez, Gloria Patricia; Barreto, George E

    2014-01-01

    The blood-brain barrier (BBB) is a tightly regulated interface in the Central Nervous System (CNS) that regulates the exchange of molecules in and out from the brain thus maintaining the CNS homeostasis. It is mainly composed of endothelial cells (ECs), pericytes and astrocytes that create a neurovascular unit (NVU) with the adjacent neurons. Astrocytes are essential for the formation and maintenance of the BBB by providing secreted factors that lead to the adequate association between the cells of the BBB and the formation of strong tight junctions. Under neurological disorders, such as chronic cerebral ischemia, brain trauma, Epilepsy, Alzheimer and Parkinson's Diseases, a disruption of the BBB takes place, involving a lost in the permeability of the barrier and phenotypical changes in both the ECs and astrocytes. In this aspect, it has been established that the process of reactive gliosis is a common feature of astrocytes during BBB disruption, which has a detrimental effect on the barrier function and a subsequent damage in neuronal survival. In this review we discuss the implications of astrocyte functions in the protection of the BBB, and in the development of Parkinson's disease (PD) and related disorders. Additionally, we highlight the current and future strategies in astrocyte protection aimed at the development of restorative therapies for the BBB in pathological conditions. PMID:25136294

  1. Glucose Tightly Controls Morphological and Functional Properties of Astrocytes.

    PubMed

    Lee, Chun-Yao; Dallérac, Glenn; Ezan, Pascal; Anderova, Miroslava; Rouach, Nathalie

    2016-01-01

    The main energy source powering the brain is glucose. Strong energy needs of our nervous system are fulfilled by conveying this essential metabolite through blood via an extensive vascular network. Glucose then reaches brain tissues by cell uptake, diffusion and metabolization, processes primarily undertaken by astrocytes. Deprivation of glucose can however occur in various circumstances. In particular, ageing is associated with cognitive disturbances that are partly attributable to metabolic deficiency leading to brain glycopenia. Despite the crucial role of glucose and its metabolites in sustaining neuronal activity, little is known about its moment-to-moment contribution to astroglial physiology. We thus here investigated the early structural and functional alterations induced in astrocytes by a transient metabolic challenge consisting in glucose deprivation. Electrophysiological recordings of hippocampal astroglial cells of the stratum radiatum in situ revealed that shortage of glucose specifically increases astrocyte membrane capacitance, whilst it has no impact on other passive membrane properties. Consistent with this change, morphometric analysis unraveled a prompt increase in astrocyte volume upon glucose deprivation. Furthermore, characteristic functional properties of astrocytes are also affected by transient glucose deficiency. We indeed found that glucoprivation decreases their gap junction-mediated coupling, while it progressively and reversibly increases their intracellular calcium levels during the slow depression of synaptic transmission occurring simultaneously, as assessed by dual electrophysiological and calcium imaging recordings. Together, these data indicate that astrocytes rapidly respond to metabolic dysfunctions, and are therefore central to the neuroglial dialog at play in brain adaptation to glycopenia. PMID:27148048

  2. Astrocytes, Synapses and Brain Function: A Computational Approach

    NASA Astrophysics Data System (ADS)

    Nadkarni, Suhita

    2006-03-01

    Modulation of synaptic reliability is one of the leading mechanisms involved in long- term potentiation (LTP) and long-term depression (LTD) and therefore has implications in information processing in the brain. A recently discovered mechanism for modulating synaptic reliability critically involves recruitments of astrocytes - star- shaped cells that outnumber the neurons in most parts of the central nervous system. Astrocytes until recently were thought to be subordinate cells merely participating in supporting neuronal functions. New evidence, however, made available by advances in imaging technology has changed the way we envision the role of these cells in synaptic transmission and as modulator of neuronal excitability. We put forward a novel mathematical framework based on the biophysics of the bidirectional neuron-astrocyte interactions that quantitatively accounts for two distinct experimental manifestation of recruitment of astrocytes in synaptic transmission: a) transformation of a low fidelity synapse transforms into a high fidelity synapse and b) enhanced postsynaptic spontaneous currents when astrocytes are activated. Such a framework is not only useful for modeling neuronal dynamics in a realistic environment but also provides a conceptual basis for interpreting experiments. Based on this modeling framework, we explore the role of astrocytes for neuronal network behavior such as synchrony and correlations and compare with experimental data from cultured networks.

  3. Glucose Tightly Controls Morphological and Functional Properties of Astrocytes

    PubMed Central

    Lee, Chun-Yao; Dallérac, Glenn; Ezan, Pascal; Anderova, Miroslava; Rouach, Nathalie

    2016-01-01

    The main energy source powering the brain is glucose. Strong energy needs of our nervous system are fulfilled by conveying this essential metabolite through blood via an extensive vascular network. Glucose then reaches brain tissues by cell uptake, diffusion and metabolization, processes primarily undertaken by astrocytes. Deprivation of glucose can however occur in various circumstances. In particular, ageing is associated with cognitive disturbances that are partly attributable to metabolic deficiency leading to brain glycopenia. Despite the crucial role of glucose and its metabolites in sustaining neuronal activity, little is known about its moment-to-moment contribution to astroglial physiology. We thus here investigated the early structural and functional alterations induced in astrocytes by a transient metabolic challenge consisting in glucose deprivation. Electrophysiological recordings of hippocampal astroglial cells of the stratum radiatum in situ revealed that shortage of glucose specifically increases astrocyte membrane capacitance, whilst it has no impact on other passive membrane properties. Consistent with this change, morphometric analysis unraveled a prompt increase in astrocyte volume upon glucose deprivation. Furthermore, characteristic functional properties of astrocytes are also affected by transient glucose deficiency. We indeed found that glucoprivation decreases their gap junction-mediated coupling, while it progressively and reversibly increases their intracellular calcium levels during the slow depression of synaptic transmission occurring simultaneously, as assessed by dual electrophysiological and calcium imaging recordings. Together, these data indicate that astrocytes rapidly respond to metabolic dysfunctions, and are therefore central to the neuroglial dialog at play in brain adaptation to glycopenia. PMID:27148048

  4. Unveiling astrocytic control of cerebral blood flow with optogenetics.

    PubMed

    Masamoto, Kazuto; Unekawa, Miyuki; Watanabe, Tatsushi; Toriumi, Haruki; Takuwa, Hiroyuki; Kawaguchi, Hiroshi; Kanno, Iwao; Matsui, Ko; Tanaka, Kenji F; Tomita, Yutaka; Suzuki, Norihiro

    2015-06-16

    Cortical neural activities lead to changes in the cerebral blood flow (CBF), which involves astrocytic control of cerebrovascular tone. However, the manner in which astrocytic activity specifically leads to vasodilation or vasoconstriction is difficult to determine. Here, cortical astrocytes genetically expressing a light-sensitive cation channel, channelrhodopsin-2 (ChR2), were transcranially activated with a blue laser while the spatiotemporal changes in CBF were noninvasively monitored with laser speckle flowgraphy in the anesthetised mouse cortex. A brief photostimulation induced a fast transient increase in CBF. The average response onset time was 0.7 ± 0.7 sec at the activation foci, and this CBF increase spread widely from the irradiation spot with an apparent propagation speed of 0.8-1.1 mm/sec. The broad increase in the CBF could be due to a propagation of diffusible vasoactive signals derived from the stimulated astrocytes. Pharmacological manipulation showed that topical administration of a K(+) channel inhibitor (BaCl2; 0.1-0.5 mM) significantly reduced the photostimulation-induced CBF responses, which indicates that the ChR2-evoked astrocytic activity involves K(+) signalling to the vascular smooth muscle cells. These findings demonstrate a unique model for exploring the role of the astrocytes in gliovascular coupling using non-invasive, time-controlled, cell-type specific perturbations.

  5. A1R-A2AR heteromers coupled to Gs and G i/0 proteins modulate GABA transport into astrocytes.

    PubMed

    Cristóvão-Ferreira, Sofia; Navarro, Gemma; Brugarolas, Marc; Pérez-Capote, Kamil; Vaz, Sandra H; Fattorini, Giorgia; Conti, Fiorenzo; Lluis, Carmen; Ribeiro, Joaquim A; McCormick, Peter J; Casadó, Vicent; Franco, Rafael; Sebastião, Ana M

    2013-09-01

    Astrocytes play a key role in modulating synaptic transmission by controlling extracellular gamma-aminobutyric acid (GABA) levels via GAT-1 and GAT-3 GABA transporters (GATs). Using primary cultures of rat astrocytes, we show here that a further level of regulation of GABA uptake occurs via modulation of the GATs by the adenosine A1 (A1R) and A2A (A2AR) receptors. This regulation occurs through A1R-A2AR heteromers that signal via two different G proteins, Gs and Gi/0, and either enhances (A2AR) or inhibits (A1R) GABA uptake. These results provide novel mechanistic insight into how GPCR heteromers signal. Furthermore, we uncover a previously unknown mechanism where adenosine, in a concentration-dependent manner, acts via a heterocomplex of adenosine receptors in astrocytes to significantly contribute to neurotransmission at the tripartite (neuron-glia-neuron) synapse.

  6. Ca(2+) -dependent endoplasmic reticulum stress correlates with astrogliosis in oligomeric amyloid β-treated astrocytes and in a model of Alzheimer's disease.

    PubMed

    Alberdi, Elena; Wyssenbach, Ane; Alberdi, María; Sánchez-Gómez, M V; Cavaliere, Fabio; Rodríguez, José J; Verkhratsky, Alexei; Matute, Carlos

    2013-04-01

    Neurotoxic effects of amyloid β peptides are mediated through deregulation of intracellular Ca(2+) homeostasis and signaling, but relatively little is known about amyloid β modulation of Ca(2+) homeostasis and its pathological influence on glia. Here, we found that amyloid β oligomers caused a cytoplasmic Ca(2+) increase in cultured astrocytes, which was reduced by inhibitors of PLC and ER Ca(2+) release. Furthermore, amyloid β peptides triggered increased expression of glial fibrillary acidic protein (GFAP), as well as oxidative and ER stress, as indicated by eIF2α phosphorylation and overexpression of chaperone GRP78. These effects were decreased by ryanodine and 2APB, inhibitors of ryanodine receptors and InsP3 receptors, respectively, in both primary cultured astrocytes and organotypic cultures of hippocampus and entorhinal cortex. Importantly, intracerebroventricular injection of amyloid β oligomers triggered overexpression of GFAP and GRP78 in astrocytes of the hippocampal dentate gyrus. These data were validated in a triple-transgenic mouse model of Alzheimer's disease (AD). Overexpression of GFAP and GRP78 in the hippocampal astrocytes correlated with the amyloid β oligomer load in 12-month-old mice, suggesting that this parameter drives astrocytic ER stress and astrogliosis in vivo. Together, these results provide evidence that amyloid β oligomers disrupt ER Ca(2+) homeostasis, which induces ER stress that leads to astrogliosis; this mechanism may be relevant to AD pathophysiology.

  7. Endogenous IL-6 of mesenchymal stem cell improves behavioral outcome of hypoxic-ischemic brain damage neonatal rats by supressing apoptosis in astrocyte

    PubMed Central

    Gu, Yan; He, Mulan; Zhou, Xiaoqin; Liu, Jinngjing; Hou, Nali; Bin, Tan; Zhang, Yun; Li, Tingyu; Chen, Jie

    2016-01-01

    Mesenchymal stem cell (MSC) transplantation reduces the neurological impairment caused by hypoxic-ischemic brain damage (HIBD) via immunomodulation. In the current study, we found that MSC transplantation improved learning and memory function and enhanced long-term potentiation in neonatal rats subjected to HIBD and the amount of IL-6 released from MSCs was far greater than that of other cytokines. However, the neuroprotective effect of MSCs infected with siIL-6-transduced recombinant lentivirus (siIL-6 MSCs) was significantly weakened in the behavioural tests and electrophysiological analysis. Meanwhile, the hippocampal IL-6 levels were decreased following siIL-6 MSC transplantation. In vitro, the levels of IL-6 release and the levels of IL-6R and STAT3 expression were increased in both primary neurons and astrocytes subjected to oxygen and glucose deprivation (OGD) following MSCs co-culture. The anti-apoptotic protein Bcl-2 was upregulated and the pro-apoptotic protein Bax was downregulated in OGD-injured astrocytes co-cultured with MSCs. However, the siIL-6 MSCs suppressed ratio of Bcl-2/Bax in the injured astrocytes and induced apoptosis number of the injured astrocytes. Taken together, these data suggest that the neuroprotective effect of MSC transplantation in neonatal HIBD rats is partly mediated by IL-6 to enhance anti-apoptosis of injured astrocytes via the IL-6/STAT3 signaling pathway. PMID:26766745

  8. Ceftriaxone reverses ketamine-induced lasting EEG and astrocyte alterations in juvenile mice

    PubMed Central

    Dodman, K.; Featherstone, R.E.; Bang, J.; Liang, Y.; Siegel, S.J.

    2015-01-01

    Background Ketamine, an N-Methyl-D-aspartate receptor antagonist, is used as a pediatric anesthetic because of its favorable safety profile. It is also being investigated as an antidepressant. Unfortunately, ketamine causes adverse reactions including hallucinations and is associated with a high prevalence of abuse among adolescents. Although chronic ketamine use has been shown to produce cognitive impairments even years following cessation, little is known about its long-term consequences on adolescents. The beta-lactam ceftriaxone has been shown to attenuate alcohol withdrawal, and alleviate early brain injury and memory impairments following subarachnoid hemorrhage. However, its ability to reverse the effects of adolescent ketamine exposure is not known. Previous data indicate that ketamine causes a reduction in the number of Excitatory Amino Acid Transporter Type 2 (EAAT2)-containing astrocytes. Additionally, the beta lactam antibiotic ceftriaxone increased expression of EAAT2. As EAAT2 is a principal mechanism of glutamate clearance from the synapse, the current study tests the hypothesis that ceftriaxone may reverse functional consequences of ketamine exposure. Methods We examined the effects of chronic ketamine in juvenile mice as well as reversal by ceftriaxone using electroencephalography (EEG). Subsequently, we assessed the effects of these treatments on markers of astrocyte proliferation, using Glial Fibrillary Acidic Protein (GFAP), and function, as evidenced by EAAT2. Results Juvenile mice exposed to chronic ketamine showed lasting alterations in EEG measurements as well as markers of astrocyte number and function. These alterations were reversed by ceftriaxone. Conclusions Data suggest that ceftriaxone may be able to ameliorate ketamine-induced long-term disturbances in adolescent brains. PMID:26442907

  9. Neighborly interactions of metabolically-activated astrocytes in vivo.

    PubMed

    Dienel, Gerald A; Cruz, Nancy F

    2003-01-01

    Metabolic responses of brain cells to a stimulus are governed, in part, by their enzymatic specialization and interrelationships with neighboring cells, and local shifts in functional metabolism during brain activation are likely to be influenced by the neurotransmitter system, subcellular compartmentation, and anatomical structure. Selected examples of functional activation illustrate the complexity of metabolic interactions in working brain and of interpretation of changes in brain lactate levels. The major focus of this article is the disproportionately higher metabolism of glucose compared to oxygen in normoxic brain, a phenomenon that occurs during activation in humans and animals. The glucose utilized in excess of oxygen is not fully explained by accumulation of glucose, lactate, or glycogen in brain or by lactate efflux from brain to blood. Thus, any lactate derived from the excess glucose could not have been stoichiometrically exported to and metabolized by neighboring neurons because oxygen consumption would have otherwise increased and matched that of glucose. Metabolic labeling of tricarboxylic acid cycle-derived amino acids increased during brief sensory stimulation, reflecting a rise in oxidative metabolism. Brain glycogen is mainly in astrocytes, and its level falls throughout the stimulus and early post-activation interval. Glycogenolysis cannot be accounted for by lactate accumulation or oxidation; there must be rapid product clearance. Glycogen restoration is slow and diversion of glucose from oxidative pathways for its re-synthesis could reduce the global O(2)/glucose uptake ratio; astrocytes could downshift this ratio for up to an hour after 5 min stimulus. Morphological studies of astrocytes reveal a paucity of cytoplasm and organelles in the fine processes that surround synapses and form gap junction connections with neighboring astrocytes. Specialized regions of astrocytes, e.g. their endfeet and thin peripheral lamellae, are likely to have

  10. Electrodiffusive Model for Astrocytic and Neuronal Ion Concentration Dynamics

    PubMed Central

    Halnes, Geir; Østby, Ivar; Pettersen, Klas H.; Omholt, Stig W.; Einevoll, Gaute T.

    2013-01-01

    The cable equation is a proper framework for modeling electrical neural signalling that takes place at a timescale at which the ionic concentrations vary little. However, in neural tissue there are also key dynamic processes that occur at longer timescales. For example, endured periods of intense neural signaling may cause the local extracellular K+-concentration to increase by several millimolars. The clearance of this excess K+ depends partly on diffusion in the extracellular space, partly on local uptake by astrocytes, and partly on intracellular transport (spatial buffering) within astrocytes. These processes, that take place at the time scale of seconds, demand a mathematical description able to account for the spatiotemporal variations in ion concentrations as well as the subsequent effects of these variations on the membrane potential. Here, we present a general electrodiffusive formalism for modeling of ion concentration dynamics in a one-dimensional geometry, including both the intra- and extracellular domains. Based on the Nernst-Planck equations, this formalism ensures that the membrane potential and ion concentrations are in consistency, it ensures global particle/charge conservation and it accounts for diffusion and concentration dependent variations in resistivity. We apply the formalism to a model of astrocytes exchanging ions with the extracellular space. The simulations show that K+-removal from high-concentration regions is driven by a local depolarization of the astrocyte membrane, which concertedly (i) increases the local astrocytic uptake of K+, (ii) suppresses extracellular transport of K+, (iii) increases axial transport of K+ within astrocytes, and (iv) facilitates astrocytic relase of K+ in regions where the extracellular concentration is low. Together, these mechanisms seem to provide a robust regulatory scheme for shielding the extracellular space from excess K+. PMID:24367247

  11. Astrocyte sodium signaling and the regulation of neurotransmission.

    PubMed

    Kirischuk, Sergei; Héja, László; Kardos, Julianna; Billups, Brian

    2016-10-01

    The transmembrane Na(+) concentration gradient is an important source of energy required not only to enable the generation of action potentials in excitable cells, but also for various transmembrane transporters both in excitable and non-excitable cells, like astrocytes. One of the vital functions of astrocytes in the central nervous system (CNS) is to regulate neurotransmitter concentrations in the extracellular space. Most neurotransmitters in the CNS are removed from the extracellular space by Na(+) -dependent neurotransmitter transporters (NeuTs) expressed both in neurons and astrocytes. Neuronal NeuTs control mainly phasic synaptic transmission, i.e., synaptically induced transient postsynaptic potentials, while astrocytic NeuTs contribute to the termination of phasic neurotransmission and modulate the tonic tone, i.e., the long-lasting activation of extrasynaptic receptors by neurotransmitter that has diffused out of the synaptic cleft. Consequently, local intracellular Na(+) ([Na(+) ]i ) transients occurring in astrocytes, for example via the activation of ionotropic neurotransmitter receptors, can affect the driving force for neurotransmitter uptake, in turn modulating the spatio-temporal profiles of neurotransmitter levels in the extracellular space. As some NeuTs are close to thermodynamic equilibrium under resting conditions, an increase in astrocytic [Na(+) ]i can stimulate the direct release of neurotransmitter via NeuT reversal. In this review we discuss the role of astrocytic [Na(+) ]i changes in the regulation of uptake/release of neurotransmitters. It is emphasized that an activation of one neurotransmitter system, including either its ionotropic receptor or Na(+) -coupled co-transporter, can strongly influence, or even reverse, other Na(+) -dependent NeuTs, with potentially significant consequences for neuronal communication. GLIA 2016;64:1655-1666. PMID:26566753

  12. Effects of aspartame metabolites on astrocytes and neurons.

    PubMed

    Rycerz, Karol; Jaworska-Adamu, Jadwiga Elżbieta

    2013-01-01

    Aspartame, a widespread sweetener used in many food products, is considered as a highly hazardous compound. Aspartame was discovered in 1965 and raises a lot of controversy up to date. Astrocytes are glial cells, the presence and functions of which are closely connected with the central nervous system (CNS). The aim of this article is to demonstrate the direct and indirect role of astrocytes participating in the harmful effects of aspartame metabolites on neurons. The artificial sweetener is broken down into phenylalanine (50%), aspartic acid (40%) and methanol (10%) during metabolism in the body. The excess of phenylalanine blocks the transport of important amino acids to the brain contributing to reduced levels of dopamine and serotonin. Astrocytes directly affect the transport of this amino acid and also indirectly by modulation of carriers in the endothelium. Aspartic acid at high concentrations is a toxin that causes hyperexcitability of neurons and is also a precursor of other excitatory amino acid - glutamates. Their excess in quantity and lack of astrocytic uptake induces excitotoxicity and leads to the degeneration of astrocytes and neurons. The methanol metabolites cause CNS depression, vision disorders and other symptoms leading ultimately to metabolic acidosis and coma. Astrocytes do not play a significant role in methanol poisoning due to a permanent consumption of large amounts of aspartame. Despite intense speculations about the carcinogenicity of aspartame, the latest studies show that its metabolite - diketopiperazine - is cancirogenic in the CNS. It contributes to the formation of tumors in the CNS such as gliomas, medulloblastomas and meningiomas. Glial cells are the main source of tumors, which can be caused inter alia by the sweetener in the brain. On the one hand the action of astrocytes during aspartame poisoning may be advantageous for neuro-protection while on the other it may intensify the destruction of neurons. The role of the glia in

  13. Astrocyte sodium signaling and the regulation of neurotransmission.

    PubMed

    Kirischuk, Sergei; Héja, László; Kardos, Julianna; Billups, Brian

    2016-10-01

    The transmembrane Na(+) concentration gradient is an important source of energy required not only to enable the generation of action potentials in excitable cells, but also for various transmembrane transporters both in excitable and non-excitable cells, like astrocytes. One of the vital functions of astrocytes in the central nervous system (CNS) is to regulate neurotransmitter concentrations in the extracellular space. Most neurotransmitters in the CNS are removed from the extracellular space by Na(+) -dependent neurotransmitter transporters (NeuTs) expressed both in neurons and astrocytes. Neuronal NeuTs control mainly phasic synaptic transmission, i.e., synaptically induced transient postsynaptic potentials, while astrocytic NeuTs contribute to the termination of phasic neurotransmission and modulate the tonic tone, i.e., the long-lasting activation of extrasynaptic receptors by neurotransmitter that has diffused out of the synaptic cleft. Consequently, local intracellular Na(+) ([Na(+) ]i ) transients occurring in astrocytes, for example via the activation of ionotropic neurotransmitter receptors, can affect the driving force for neurotransmitter uptake, in turn modulating the spatio-temporal profiles of neurotransmitter levels in the extracellular space. As some NeuTs are close to thermodynamic equilibrium under resting conditions, an increase in astrocytic [Na(+) ]i can stimulate the direct release of neurotransmitter via NeuT reversal. In this review we discuss the role of astrocytic [Na(+) ]i changes in the regulation of uptake/release of neurotransmitters. It is emphasized that an activation of one neurotransmitter system, including either its ionotropic receptor or Na(+) -coupled co-transporter, can strongly influence, or even reverse, other Na(+) -dependent NeuTs, with potentially significant consequences for neuronal communication. GLIA 2016;64:1655-1666.

  14. Dysfunctional TCA-Cycle Metabolism in Glutamate Dehydrogenase Deficient Astrocytes.

    PubMed

    Nissen, Jakob D; Pajęcka, Kamilla; Stridh, Malin H; Skytt, Dorte M; Waagepetersen, Helle S

    2015-12-01

    Astrocytes take up glutamate in the synaptic area subsequent to glutamatergic transmission by the aid of high affinity glutamate transporters. Glutamate is converted to glutamine or metabolized to support intermediary metabolism and energy production. Glutamate dehydrogenase (GDH) and aspartate aminotransferase (AAT) catalyze the reversible reaction between glutamate and α-ketoglutarate, which is the initial step for glutamate to enter TCA cycle metabolism. In contrast to GDH, AAT requires a concomitant interconversion of oxaloacetate and aspartate. We have investigated the role of GDH in astrocyte glutamate and glucose metabolism employing siRNA mediated knock down (KD) of GDH in cultured astrocytes using stable and radioactive isotopes for metabolic mapping. An increased level of aspartate was observed upon exposure to [U-(13) C]glutamate in astrocytes exhibiting reduced GDH activity. (13) C Labeling of aspartate and TCA cycle intermediates confirmed that the increased amount of aspartate is associated with elevated TCA cycle flux from α-ketoglutarate to oxaloacetate, i.e. truncated TCA cycle. (13) C Glucose metabolism was elevated in GDH deficient astrocytes as observed by increased de novo synthesis of aspartate via pyruvate carboxylation. In the absence of glucose, lactate production from glutamate via malic enzyme was lower in GDH deficient astrocytes. In conclusions, our studies reveal that metabolism via GDH serves an important anaplerotic role by adding net carbon to the TCA cycle. A reduction in GDH activity seems to cause the astrocytes to up-regulate activity in pathways involved in maintaining the amount of TCA cycle intermediates such as pyruvate carboxylation as well as utilization of alternate substrates such as branched chain amino acids.

  15. The Role of Astrocytes in Multiple Sclerosis Progression

    PubMed Central

    Correale, Jorge; Farez, Mauricio F.

    2015-01-01

    Multiple sclerosis (MS) is an inflammatory disorder causing central nervous system (CNS) demyelination and axonal injury. Although its etiology remains elusive, several lines of evidence support the concept that autoimmunity plays a major role in disease pathogenesis. The course of MS is highly variable; nevertheless, the majority of patients initially present a relapsing–remitting clinical course. After 10–15 years of disease, this pattern becomes progressive in up to 50% of untreated patients, during which time clinical symptoms slowly cause constant deterioration over a period of many years. In about 15% of MS patients, however, disease progression is relentless from disease onset. Published evidence supports the concept that progressive MS reflects a poorly understood mechanism of insidious axonal degeneration and neuronal loss. Recently, the type of microglial cell and of astrocyte activation and proliferation observed has suggested contribution of resident CNS cells may play a critical role in disease progression. Astrocytes could contribute to this process through several mechanisms: (a) as part of the innate immune system, (b) as a source of cytotoxic factors, (c) inhibiting remyelination and axonal regeneration by forming a glial scar, and (d) contributing to axonal mitochondrial dysfunction. Furthermore, regulatory mechanisms mediated by astrocytes can be affected by aging. Notably, astrocytes might also limit the detrimental effects of pro-inflammatory factors, while providing support and protection for oligodendrocytes and neurons. Because of the dichotomy observed in astrocytic effects, the design of therapeutic strategies targeting astrocytes becomes a challenging endeavor. Better knowledge of molecular and functional properties of astrocytes, therefore, should promote understanding of their specific role in MS pathophysiology, and consequently lead to development of novel and more successful therapeutic approaches. PMID:26347709

  16. Electrodiffusive model for astrocytic and neuronal ion concentration dynamics.

    PubMed

    Halnes, Geir; Ostby, Ivar; Pettersen, Klas H; Omholt, Stig W; Einevoll, Gaute T

    2013-01-01

    The cable equation is a proper framework for modeling electrical neural signalling that takes place at a timescale at which the ionic concentrations vary little. However, in neural tissue there are also key dynamic processes that occur at longer timescales. For example, endured periods of intense neural signaling may cause the local extracellular K(+)-concentration to increase by several millimolars. The clearance of this excess K(+) depends partly on diffusion in the extracellular space, partly on local uptake by astrocytes, and partly on intracellular transport (spatial buffering) within astrocytes. These processes, that take place at the time scale of seconds, demand a mathematical description able to account for the spatiotemporal variations in ion concentrations as well as the subsequent effects of these variations on the membrane potential. Here, we present a general electrodiffusive formalism for modeling of ion concentration dynamics in a one-dimensional geometry, including both the intra- and extracellular domains. Based on the Nernst-Planck equations, this formalism ensures that the membrane potential and ion concentrations are in consistency, it ensures global particle/charge conservation and it accounts for diffusion and concentration dependent variations in resistivity. We apply the formalism to a model of astrocytes exchanging ions with the extracellular space. The simulations show that K(+)-removal from high-concentration regions is driven by a local depolarization of the astrocyte membrane, which concertedly (i) increases the local astrocytic uptake of K(+), (ii) suppresses extracellular transport of K(+), (iii) increases axial transport of K(+) within astrocytes, and (iv) facilitates astrocytic relase of K(+) in regions where the extracellular concentration is low. Together, these mechanisms seem to provide a robust regulatory scheme for shielding the extracellular space from excess K(+). PMID:24367247

  17. Voluntary Exercise Induces Astrocytic Structural Plasticity in the Globus Pallidus

    PubMed Central

    Tatsumi, Kouko; Okuda, Hiroaki; Morita-Takemura, Shoko; Tanaka, Tatsuhide; Isonishi, Ayami; Shinjo, Takeaki; Terada, Yuki; Wanaka, Akio

    2016-01-01

    Changes in astrocyte morphology are primarily attributed to the fine processes where intimate connections with neurons form the tripartite synapse and participate in neurotransmission. Recent evidence has shown that neurotransmission induces dynamic synaptic remodeling, suggesting that astrocytic fine processes may adapt their morphologies to the activity in their environment. To illustrate such a neuron-glia relationship in morphological detail, we employed a double transgenic Olig2CreER/WT; ROSA26-GAP43-EGFP mice, in which Olig2-lineage cells can be visualized and traced with membrane-targeted GFP. Although Olig2-lineage cells in the adult brain usually become mature oligodendrocytes or oligodendrocyte precursor cells with NG2-proteoglycan expression, we found a population of Olig2-lineage astrocytes with bushy morphology in several brain regions. The globus pallidus (GP) preferentially contains Olig2-lineage astrocytes. Since the GP exerts pivotal motor functions in the indirect pathway of the basal ganglionic circuit, we subjected the double transgenic mice to voluntary wheel running to activate the GP and examined morphological changes of Olig2-lineage astrocytes at both the light and electron microscopic levels. The double transgenic mice were divided into three groups: control group mice were kept in a cage with a locked running wheel for 3 weeks, Runner group were allowed free access to a running wheel for 3 weeks, and the Runner-Rest group took a sedentary 3-week rest after a 3-week running period. GFP immunofluorescence analysis and immunoelectron microscopy revealed that astrocytic fine processes elaborated complex arborization in the Runner mice, and reverted to simple morphology comparable to that of the Control group in the Runner-Rest group. Our results indicated that the fine processes of the Olig2-lineage astrocytes underwent plastic changes that correlated with overall running activities, suggesting that they actively participate in motor

  18. Voluntary Exercise Induces Astrocytic Structural Plasticity in the Globus Pallidus.

    PubMed

    Tatsumi, Kouko; Okuda, Hiroaki; Morita-Takemura, Shoko; Tanaka, Tatsuhide; Isonishi, Ayami; Shinjo, Takeaki; Terada, Yuki; Wanaka, Akio

    2016-01-01

    Changes in astrocyte morphology are primarily attributed to the fine processes where intimate connections with neurons form the tripartite synapse and participate in neurotransmission. Recent evidence has shown that neurotransmission induces dynamic synaptic remodeling, suggesting that astrocytic fine processes may adapt their morphologies to the activity in their environment. To illustrate such a neuron-glia relationship in morphological detail, we employed a double transgenic Olig2(CreER/WT); ROSA26-GAP43-EGFP mice, in which Olig2-lineage cells can be visualized and traced with membrane-targeted GFP. Although Olig2-lineage cells in the adult brain usually become mature oligodendrocytes or oligodendrocyte precursor cells with NG2-proteoglycan expression, we found a population of Olig2-lineage astrocytes with bushy morphology in several brain regions. The globus pallidus (GP) preferentially contains Olig2-lineage astrocytes. Since the GP exerts pivotal motor functions in the indirect pathway of the basal ganglionic circuit, we subjected the double transgenic mice to voluntary wheel running to activate the GP and examined morphological changes of Olig2-lineage astrocytes at both the light and electron microscopic levels. The double transgenic mice were divided into three groups: control group mice were kept in a cage with a locked running wheel for 3 weeks, Runner group were allowed free access to a running wheel for 3 weeks, and the Runner-Rest group took a sedentary 3-week rest after a 3-week running period. GFP immunofluorescence analysis and immunoelectron microscopy revealed that astrocytic fine processes elaborated complex arborization in the Runner mice, and reverted to simple morphology comparable to that of the Control group in the Runner-Rest group. Our results indicated that the fine processes of the Olig2-lineage astrocytes underwent plastic changes that correlated with overall running activities, suggesting that they actively participate in motor

  19. Silencing of Abcd1 and Abcd2 genes sensitizes astrocytes for inflammation: implication for X-adrenoleukodystrophy*

    PubMed Central

    Singh, Jaspreet; Khan, Mushfiquddin; Singh, Inderjit

    2009-01-01

    X-linked adrenoleukodystrophy is a metabolic disorder arising from a mutation/deletion in the ABCD1 gene, leading to a defect in the peroxisomal adrenoleukodystrophy protein (ALDP), which inhibits the oxidation of very long chain fatty acids (VLCFAs). Thus, these VLCFAs accumulate. In a cerebral form of ALD (cALD), VLCFA accumulation induces neuroinflammation that leads to loss of oligodendrocytes and myelin, which ultimately shortens the lifespan. To establish a relationship between the metabolic disease and inflammatory disease induction, we document that small interfering RNA (siRNA)-mediated silencing of Abcd1 (ALDP) and Abcd2 [adrenoleukodystrophy-related protein (ALDRP)] genes in mice primary astrocyte cultures resulted in accumulation of VLCFA and induction of an inflammatory response characteristic of human cALD. Correction of the metabolic defect using monoenoic FAs in Abcd1/Abcd2-silenced cultured astrocytes decreased inducible nitric oxide synthase and inflammatory cytokine expression, suggesting a link between VLCFA accumulation and inflammation. The inflammatory response was found to be mediated by transcription factors NF-κB, AP-1, and C/EBP in Abcd1/Abcd2-silenced mouse primary astrocytes. Although mechanisms of VLCFA-mediated induction of the inflammatory response have been investigated here in vitro, the in vivo mediators remain elusive. Our data represent the first study to suggest a direct link between the accumulation of VLCFA and the induction of inflammatory mediators. PMID:18723473

  20. Astrocytes as Gatekeepers of GABAB Receptor Function

    PubMed Central

    Beenhakker, Mark P.; Huguenard, John R.

    2010-01-01

    The long-lasting actions of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) result from the activation of metabotropic GABAB receptors. Enhanced GABAB-mediated inhibitory postsynaptic currents (IPSCs) are critical for the generation of generalized thalamocortical seizures. Here, we demonstrate that GABAB-mediated inhibitory postsynaptic currents (IPSCs) recorded in the thalamus are largely defined by GABA diffusion and activation of distal extrasynaptic receptors potentially up to tens of μm from synapses. We also show that this diffusion is differentially regulated by two astrocytic GABA transporters, GAT1 and GAT3, which are localized near and far from synapses, respectively. A biologically constrained model of GABA diffusion and uptake shows how the two GATs differentially modulate amplitude and duration of GABAB IPSCs. Specifically, the perisynaptic expression of GAT1 enables it to regulate GABA levels near synapses and selectively modulate peak IPSC amplitude, which is largely dependent on perisynaptic receptor occupancy. GAT3 expression, on the other hand, is broader and includes distal extrasynaptic regions. As such, GAT3 acts as a gatekeeper to prevent diffusion of GABA away from synapses towards extrasynaptic regions that contain a potentially enormous pool of GABAB receptors. Targeting this gatekeeper function may provide new pharmacotherapeutic opportunities to prevent the excessive GABAB receptor activation that appears necessary for thalamic seizure generation. PMID:21068331

  1. Pro-inflammatory cytokines and HIV-1 synergistically enhance CXCL10 expression in human astrocytes

    PubMed Central

    Williams, Rachel; Dhillon, Navneet K.; Hegde, Sonia T.; Yao, Honghong; Peng, Fuwang; Callen, Shannon; Chebloune, Yahia; Davis, Randall L.; Buch, Shilpa J.

    2009-01-01

    HIV encephalitis (HIVE), the pathologic correlate of HIV-associated dementia (HAD) is characterized by astrogliosis, cytokine/chemokine dysregulation and neuronal degeneration. Increasing evidence suggests that inflammation is actively involved in the pathogenesis of HAD. In fact, the severity of HAD/HIVE correlates more closely with the presence of activated glial cells than with the presence and amount of HIV-infected cells in the brain. Astrocytes, the most numerous cell type within the brain, provide an important reservoir for the generation of inflammatory mediators, including interferon-γ inducible peptide-10 (CXCL10), a neurotoxin and a chemoattractant, implicated in the pathophysiology of HAD. Additionally, the pro-inflammatory cytokines, IFN-γ and TNF-α, are also markedly increased in CNS tissues during HIV-1 infection. In the present study we hypothesized that the interplay of host cytokines and HIV-1 could lead to enhanced expression of the toxic chemokine, CXCL10. Our findings demonstrate a synergistic induction of CXCL10 mRNA and protein in human astrocytes exposed to HIV-1 and the pro-inflammatory cytokines. Signaling molecules, including JAK, STATs, MAPK (via activation of Erk1/2, AKT, and p38), and NF-κB were identified as instrumental in the synergistic induction of CXCL10. Understanding the mechanisms involved in HIV-1 and cytokine mediated up-regulation of CXCL10 could aid in the development of therapeutic modalities for HAD. PMID:18985732

  2. Medium-chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte-neuron lactate and ketone body shuttle systems.

    PubMed

    Thevenet, Jonathan; De Marchi, Umberto; Domingo, Jaime Santo; Christinat, Nicolas; Bultot, Laurent; Lefebvre, Gregory; Sakamoto, Kei; Descombes, Patrick; Masoodi, Mojgan; Wiederkehr, Andreas

    2016-05-01

    Medium-chain triglycerides have been used as part of a ketogenic diet effective in reducing epileptic episodes. The health benefits of the derived medium-chain fatty acids (MCFAs) are thought to result from the stimulation of liver ketogenesis providing fuel for the brain. We tested whether MCFAs have direct effects on energy metabolism in induced pluripotent stem cell-derived human astrocytes and neurons. Using single-cell imaging, we observed an acute pronounced reduction of the mitochondrial electrical potential and a concomitant drop of the NAD(P)H signal in astrocytes, but not in neurons. Despite the observed effects on mitochondrial function, MCFAs did not lower intracellular ATP levels or activate the energy sensor AMP-activated protein kinase. ATP concentrations in astrocytes were unaltered, even when blocking the respiratory chain, suggesting compensation through accelerated glycolysis. The MCFA decanoic acid (300 μM) promoted glycolysis and augmented lactate formation by 49.6%. The shorter fatty acid octanoic acid (300 μM) did not affect glycolysis but increased the rates of astrocyte ketogenesis 2.17-fold compared with that of control cells. MCFAs may have brain health benefits through the modulation of astrocyte metabolism leading to activation of shuttle systems that provide fuel to neighboring neurons in the form of lactate and ketone bodies.-Thevenet, J., De Marchi, U., Santo Domingo, J., Christinat, N., Bultot, L., Lefebvre, G., Sakamoto, K., Descombes, P., Masoodi, M., Wiederkehr, A. Medium-chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte-neuron lactate and ketone body shuttle systems.

  3. Medium-chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte-neuron lactate and ketone body shuttle systems.

    PubMed

    Thevenet, Jonathan; De Marchi, Umberto; Domingo, Jaime Santo; Christinat, Nicolas; Bultot, Laurent; Lefebvre, Gregory; Sakamoto, Kei; Descombes, Patrick; Masoodi, Mojgan; Wiederkehr, Andreas

    2016-05-01

    Medium-chain triglycerides have been used as part of a ketogenic diet effective in reducing epileptic episodes. The health benefits of the derived medium-chain fatty acids (MCFAs) are thought to result from the stimulation of liver ketogenesis providing fuel for the brain. We tested whether MCFAs have direct effects on energy metabolism in induced pluripotent stem cell-derived human astrocytes and neurons. Using single-cell imaging, we observed an acute pronounced reduction of the mitochondrial electrical potential and a concomitant drop of the NAD(P)H signal in astrocytes, but not in neurons. Despite the observed effects on mitochondrial function, MCFAs did not lower intracellular ATP levels or activate the energy sensor AMP-activated protein kinase. ATP concentrations in astrocytes were unaltered, even when blocking the respiratory chain, suggesting compensation through accelerated glycolysis. The MCFA decanoic acid (300 μM) promoted glycolysis and augmented lactate formation by 49.6%. The shorter fatty acid octanoic acid (300 μM) did not affect glycolysis but increased the rates of astrocyte ketogenesis 2.17-fold compared with that of control cells. MCFAs may have brain health benefits through the modulation of astrocyte metabolism leading to activation of shuttle systems that provide fuel to neighboring neurons in the form of lactate and ketone bodies.-Thevenet, J., De Marchi, U., Santo Domingo, J., Christinat, N., Bultot, L., Lefebvre, G., Sakamoto, K., Descombes, P., Masoodi, M., Wiederkehr, A. Medium-chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte-neuron lactate and ketone body shuttle systems. PMID:26839375

  4. Window contamination on Expose-R

    NASA Astrophysics Data System (ADS)

    Demets, R.; Bertrand, M.; Bolkhovitinov, A.; Bryson, K.; Colas, C.; Cottin, H.; Dettmann, J.; Ehrenfreund, P.; Elsaesser, A.; Jaramillo, E.; Lebert, M.; van Papendrecht, G.; Pereira, C.; Rohr, T.; Saiagh, K.

    2015-01-01

    Expose is a multi-user instrument for astrobiological and astrochemical experiments in space. Installed at the outer surface of the International Space Station, it enables investigators to study the impact of the open space environment on biological and biochemical test samples. Two Expose missions have been completed so far, designated as Expose-E (Rabbow et al. 2012) and Expose-R (Rabbow et al. this issue). One of the space-unique environmental factors offered by Expose is full-spectrum, ultraviolet (UV)-rich electromagnetic radiation from the Sun. This paper describes and analyses how on Expose-R, access of the test samples to Solar radiation degraded during space exposure in an unpredicted way. Several windows in front of the Sun-exposed test samples acquired a brown shade, resulting in a reduced transparency in visible light, UV and vacuum UV (VUV). Post-flight investigations revealed the discolouration to be caused by a homogenous film of cross-linked organic polymers at the inside of the windows. The chemical signature varied per sample carrier. No such films were found on windows from sealed, pressurized compartments, or on windows that had been kept out of the Sun. This suggests that volatile compounds originating from the interior of the Expose facility were cross-linked and photo-fixed by Solar irradiation at the rear side of the windows. The origin of the volatiles was not fully identified; most probably there was a variety of sources involved including the biological test samples, adhesives, plastics and printed circuit boards. The outer surface of the windows (pointing into space) was chemically impacted as well, with a probable effect on the transparency in VUV. The reported analysis of the window contamination on Expose-R is expected to help the interpretation of the scientific results and offers possibilities to mitigate this problem on future missions - in particular Expose-R2, the direct successor of Expose-R.

  5. In vitro synchronization of mammalian astrocytic cultures by serum deprivation.

    PubMed

    Chou, Richard C; Langan, Thomas J

    2003-07-01

    The study of the regulation of cell division cycle in vitro requires cell cultures growing in the same phase of the cycle. The procedure by which cells are arrested in specific phases of the cell cycle is termed synchronization. Synchronization is particularly important in the study of astrocyte biology, as its application allows astrocytes to re-enter the cell cycle from a state of quiescence (G(0)), and, under carefully defined experimental conditions, move together into subsequent phases such as the G(1) and S phases. A number of methods have been established to synchronize mammalian cell cultures, including centrifugal elutriation, mitotic shake-off, and chemically induced cell cycle arrest. Yet there are intrinsic limitations associated with these methods. In the present protocol, we describe a simple, reliable, and reversible procedure to synchronize astrocytic cultures from newborn rat brains by serum deprivation. This protocol consists essentially of two parts: (1) proliferation of astrocytes under optimal conditions in vitro until reaching desired confluence; and (2) synchronization of cultures by serum down-shift and arrested in the G(0) phase of the cell cycle. This procedure has recently been extended toward the study of cell cycle control in astroglioma cells and astrocytes from injured adult brains. Since it was also employed in recent precursor cloning studies in developmental biology, this procedure will certainly find increasing use in future research.

  6. Simultaneous neuron- and astrocyte-specific fluorescent marking.

    PubMed

    Schulze, Wiebke; Hayata-Takano, Atsuko; Kamo, Toshihiko; Nakazawa, Takanobu; Nagayasu, Kazuki; Kasai, Atsushi; Seiriki, Kaoru; Shintani, Norihito; Ago, Yukio; Farfan, Camille; Hashimoto, Ryota; Baba, Akemichi; Hashimoto, Hitoshi

    2015-03-27

    Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignment of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains.

  7. Targeting astrocytes in brain injuries: A translational research approach.

    PubMed

    Barreto, George E

    2016-09-01

    In the brain, the astrocentric view has increasingly changed in the past few years. The classical and old view of astrocytes as "just supporting cells" has assigned these cells some functions to help neurons maintain their homeostasis. This neuronal supportive function of astrocytes includes maintenance of ion and extracellular pH equilibrium, neuroendocrine signaling, metabolic support, clearance of glutamate and other neurotransmitters, and antioxidant protection. However, recent findings have shed some light on the new roles, some controversial though, performed by astrocytes that might change our view about the central nervous system functioning. Since astrocytes are important for neuronal survival, it is a potential approach to favor astrocytic functions in order to improve the outcome. Such translational strategies may include the use of genetically targeted proteins, and/or pharmacological therapies by administering androgens and estrogens, which have shown promising results in vitro and in vivo models. It is noteworthy that successful strategies reviewed in here shall be extrapolated to human subjects, and this is probably the next step we should move on.

  8. Study of curcumin immunomodulatory effects on reactive astrocyte cell function.

    PubMed

    Seyedzadeh, Mir Hadi; Safari, Zohreh; Zare, Ahad; Gholizadeh Navashenaq, Jamshid; Razavi, Seyed Alireza; Kardar, Gholam Ali; Khorramizadeh, Mohammad Reza

    2014-09-01

    Multiple sclerosis (MS) is considered an inflammatory and neurodegenerative disease of the central nervous system (CNS) which most often presents as relapsing-remitting episodes. Recent evidence suggests that activated astrocytes play a dual functional role in CNS inflammatory disorders such as MS. In this study, we tried to induce anti-inflammatory functions of astrocytes by curcumin. The effects of curcumin were examined on human a astrocyte cell line (U373-MG) induced by lipopolysaccharide (LPS) in vitro. Matrix metalloproteinase (MMP)-9 activity was assessed by gelatin zymography. Cytokine levels were evaluated by quantitative ELISA method and mRNA expression was measured by real-time PCR. We found that curcumin decreased the release of IL-6 and reduced MMP-9 enzyme activity. It down-regulated MCP-1 mRNA expression too. However, curcumin did not have significant effects on the expression of neurotrophin (NT)-3 and insulin-like growth factor (IGF)-1 mRNAs. Results suggest that curcumin might beneficially affect astrocyte population in CNS neuroinflammatory environment lean to anti-inflammatory response and help to components in respects of CNS repair. Our findings offer curcumin as a new therapeutic agent with the potential of regulating astrocyte-mediated inflammatory diseases in the CNS. PMID:24998635

  9. Astrocytes and NG2-glia: what's in a name?

    PubMed Central

    Nishiyama, Akiko; Yang, Zhongshu; Butt, Arthur

    2005-01-01

    Classic studies recognize two functionally segregated macroglial cell types in the central nervous system (CNS), namely astrocytes and oligodendrocytes. A third macroglial cell type has now been identified by its specific expression of the NG2 chondroitin sulphate proteoglycan (NG2-glia). These NG2-glia exist abundantly in both grey and white matter of the mature CNS and are almost as numerous as astrocytes. It is well established that NG2-glia give rise to oligodendrocytes. However, the majority of NG2-glia in the adult CNS proliferate very slowly and are non-motile. Both astrocytes and NG2-glia display a stellate morphology and express ion channels and receptors to neurotransmitters used by neurons. Both types of glia make intimate contacts with neurons in grey and white matter, and their functional differences and similarities are only beginning to be unravelled. Recent observations emphasize the need to examine the relationship between astrocytes and NG2-glia, and address the question of whether they represent overlapping or two distinct glial cell populations. To be of any relevance, this classification must relate to specific functions in the neural network. At present, the balance of evidence is that NG2-glia and astrocytes are functionally segregated populations. PMID:16367796

  10. Targeting astrocytes in brain injuries: A translational research approach.

    PubMed

    Barreto, George E

    2016-09-01

    In the brain, the astrocentric view has increasingly changed in the past few years. The classical and old view of astrocytes as "just supporting cells" has assigned these cells some functions to help neurons maintain their homeostasis. This neuronal supportive function of astrocytes includes maintenance of ion and extracellular pH equilibrium, neuroendocrine signaling, metabolic support, clearance of glutamate and other neurotransmitters, and antioxidant protection. However, recent findings have shed some light on the new roles, some controversial though, performed by astrocytes that might change our view about the central nervous system functioning. Since astrocytes are important for neuronal survival, it is a potential approach to favor astrocytic functions in order to improve the outcome. Such translational strategies may include the use of genetically targeted proteins, and/or pharmacological therapies by administering androgens and estrogens, which have shown promising results in vitro and in vivo models. It is noteworthy that successful strategies reviewed in here shall be extrapolated to human subjects, and this is probably the next step we should move on. PMID:27659056

  11. Interferon-Gamma Promotes Infection of Astrocytes by Trypanosoma cruzi

    PubMed Central

    Silva, Rafael Rodrigues; Mariante, Rafael M.; Silva, Andrea Alice; dos Santos, Ana Luiza Barbosa; Roffê, Ester; Santiago, Helton; Gazzinelli, Ricardo Tostes; Lannes-Vieira, Joseli

    2015-01-01

    The inflammatory cytokine interferon-gamma (IFNγ) is crucial for immunity against intracellular pathogens such as the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (CD). IFNγ is a pleiotropic cytokine which regulates activation of immune and non-immune cells; however, the effect of IFNγ in the central nervous system (CNS) and astrocytes during CD is unknown. Here we show that parasite persists in the CNS of C3H/He mice chronically infected with the Colombian T. cruzi strain despite the increased expression of IFNγ mRNA. Furthermore, most of the T. cruzi-bearing cells were astrocytes located near IFNγ+ cells. Surprisingly, in vitro experiments revealed that pretreatment with IFNγ promoted the infection of astrocytes by T. cruzi increasing uptake and proliferation of intracellular forms, despite inducing increased production of nitric oxide (NO). Importantly, the effect of IFNγ on T. cruzi uptake and growth is completely blocked by the anti-tumor necrosis factor (TNF) antibody Infliximab and partially blocked by the inhibitor of nitric oxide synthesis L-NAME. These data support that IFNγ fuels astrocyte infection by T. cruzi and critically implicate IFNγ-stimulated T. cruzi-infected astrocytes as sources of TNF and NO, which may contribute to parasite persistence and CNS pathology in CD. PMID:25695249

  12. Calcium Signaling and Gliotransmission in Normal vs. Reactive Astrocytes

    PubMed Central

    Agulhon, Cendra; Sun, Min-Yu; Murphy, Thomas; Myers, Timothy; Lauderdale, Kelli; Fiacco, Todd A.

    2012-01-01

    A prominent area of neuroscience research over the past 20 years has been the acute modulation of neuronal synaptic activity by Ca2+-dependent release of the transmitters ATP, D-serine, and glutamate (called gliotransmitters) by astrocytes. Although the physiological relevance of this mechanism is under debate, emerging evidence suggests that there are critical factors in addition to Ca2+ that are required for gliotransmitters to be released from astrocytes. Interestingly, these factors include activated microglia and the proinflammatory cytokine Tumor Necrosis Factor α (TNFα), chemotactic cytokine Stromal cell-Derived Factor-1α (SDF-1α), and inflammatory mediator prostaglandin E2 (PGE2). Of note, microglial activation and release of inflammatory molecules from activated microglia and reactive astrocytes can occur within minutes of a triggering stimulus. Therefore, activation of astrocytes by inflammatory molecules combined with Ca2+ elevations may lead to gliotransmitter release, and be an important step in the early sequence of events contributing to hyperexcitability, excitotoxicity, and neurodegeneration in the damaged or diseased brain. In this review, we will first examine evidence questioning Ca2+-dependent gliotransmitter release from astrocytes in healthy brain tissue, followed by a close examination of recent work suggesting that Ca2+-dependent gliotransmitter release occurs as an early event in the development of neurological disorders and neuroinflammatory and neurodegenerative diseases. PMID:22811669

  13. Interferon-gamma promotes infection of astrocytes by Trypanosoma cruzi.

    PubMed

    Silva, Rafael Rodrigues; Mariante, Rafael M; Silva, Andrea Alice; dos Santos, Ana Luiza Barbosa; Roffê, Ester; Santiago, Helton; Gazzinelli, Ricardo Tostes; Lannes-Vieira, Joseli

    2015-01-01

    The inflammatory cytokine interferon-gamma (IFNγ) is crucial for immunity against intracellular pathogens such as the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (CD). IFNγ is a pleiotropic cytokine which regulates activation of immune and non-immune cells; however, the effect of IFNγ in the central nervous system (CNS) and astrocytes during CD is unknown. Here we show that parasite persists in the CNS of C3H/He mice chronically infected with the Colombian T. cruzi strain despite the increased expression of IFNγ mRNA. Furthermore, most of the T. cruzi-bearing cells were astrocytes located near IFNγ+ cells. Surprisingly, in vitro experiments revealed that pretreatment with IFNγ promoted the infection of astrocytes by T. cruzi increasing uptake and proliferation of intracellular forms, despite inducing increased production of nitric oxide (NO). Importantly, the effect of IFNγ on T. cruzi uptake and growth is completely blocked by the anti-tumor necrosis factor (TNF) antibody Infliximab and partially blocked by the inhibitor of nitric oxide synthesis L-NAME. These data support that IFNγ fuels astrocyte infection by T. cruzi and critically implicate IFNγ-stimulated T. cruzi-infected astrocytes as sources of TNF and NO, which may contribute to parasite persistence and CNS pathology in CD. PMID:25695249

  14. Glucocorticoids decrease astrocyte numbers by reducing glucocorticoid receptor expression in vitro and in vivo.

    PubMed

    Unemura, Kazuhiro; Kume, Toshiaki; Kondo, Minami; Maeda, Yuki; Izumi, Yasuhiko; Akaike, Akinori

    2012-01-01

    Glucocorticoids are stress hormones released from the adrenal cortex and their concentration is controlled by the hypothalamic-pituitary-adrenal axis. In this study, we investigated the effect of glucocorticoids on the number of astrocytes and glucocorticoid receptor (GR) expression in vitro and in vivo. Proliferation of cultured astrocytes was reduced following treatment with corticosterone and dexamethasone for 72 h. Corticosterone and dexamethasone also reduced GR expression in astrocytes. RU486, a GR antagonist, inhibited the reduction in both astrocyte proliferation and GR expression. Furthermore, GR knockdown by siRNA inhibited astrocyte proliferation. We also examined the effect of excessive glucocorticoid release on GR expression and the number of astrocytes in vivo by administering adrenocorticotropic hormone to rats for 14 days. GR expression was reduced in the prefrontal cortex and hippocampus and the number of astrocytes was reduced in the frontal cortex. Overall, our results suggest that glucocorticoids decrease the number of astrocytes by reducing GR expression.

  15. Isolation of intact astrocytes from the optic nerve head of adult mice

    PubMed Central

    Choi, Hee Joo; Sun, Daniel; Jakobs, Tatjana C.

    2015-01-01

    The astrocytes of the optic nerve head are a specialized subtype of white matter astrocytes that form the direct cellular environment of the unmyelinated ganglion cell axons. Due to their potential involvement in glaucoma, these astrocytes have become a target of research. Due to the heterogeneity of the optic nerve tissue, which also contains other cell types, in some cases it may be desirable to conduct gene expression studies on small numbers of well-characterized astrocytes or even individual cells. Here, we describe a simple method to isolate individual astrocytes. This method permits obtaining astrocytes with intact morphology from the adult mouse optic nerve and reduces contamination of the isolated astrocytes by other cell types. Individual astrocytes can be recognized by their morphology and collected under microscopic control. The whole procedure can be completed in 2-3 hours. We also discuss downstream applications like multiplex single-cell PCR and quantitative PCR (qPCR). PMID:26093274

  16. Astrocytic and vascular remodeling in the injured adult rat spinal cord after chondroitinase ABC treatment.

    PubMed

    Milbreta, Ulla; von Boxberg, Ysander; Mailly, Philippe; Nothias, Fatiha; Soares, Sylvia

    2014-05-01

    Upregulation of extracellular chondroitin sulfate proteoglycans (CSPG) is a primary cause for the failure of axons to regenerate after spinal cord injury (SCI), and the beneficial effect of their degradation by chondroitinase ABC (ChABC) is widely documented. Little is known, however, about the effect of ChABC treatment on astrogliosis and revascularization, two important factors influencing axon regrowth. This was investigated in the present study. Immediately after a spinal cord hemisection at thoracic level 8-9, we injected ChABC intrathecally at the sacral level, repeated three times until 10 days post-injury. Our results show an effective cleavage of CSPG glycosaminoglycan chains and stimulation of axonal remodeling within the injury site, accompanied by an extended period of astrocyte remodeling (up to 4 weeks). Interestingly, ChABC treatment favored an orientation of astrocytic processes directed toward the injury, in close association with axons at the lesion entry zone, suggesting a correlation between axon and astrocyte remodeling. Further, during the first weeks post-injury, ChABC treatment affected the morphology of laminin-positive blood vessel basement membranes and vessel-independent laminin deposits: hypertrophied blood vessels with detached or duplicated basement membrane were more numerous than in lesioned untreated animals. In contrast, at later time points, laminin expression increased and became more directly associated with newly formed blood vessels, the size of which tended to be closer to that found in intact tissue. Our data reinforce the idea that ChABC injection in combination with other synergistic treatments is a promising therapeutic strategy for SCI repair.

  17. Induction of glial fibrillary acidic protein expression in astrocytes by nitric oxide.

    PubMed

    Brahmachari, Saurav; Fung, Yiu K; Pahan, Kalipada

    2006-05-01

    Increased expression of glial fibrillary acidic protein (GFAP) represents astroglial activation and gliosis during neurodegeneration. However, the molecular mechanism behind increased expression of GFAP in astrocytes is poorly understood. The present study was undertaken to explore the role of nitric oxide (NO) in the expression of GFAP. Bacterial lipopolysachharides (LPSs) induced the production of NO and the expression of GFAP in mouse primary astrocytes. Either a scavenger of NO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)] or an inhibitor of inducible nitric oxide synthase [l-N6-(I-iminoethyl)-lysine hydrochloride] blocked this induction of GFAP expression. Similarly, other inducers of NO production such as interferon-gamma, interleukin-1beta, human immunodeficiency virus type 1 gp120, fibrillar amyloid beta peptides, and double-stranded RNA (polyinosinic-polycytidilic acid) also induced the expression of GFAP through NO. The role of NO in the expression of GFAP was supported further by increased expression of GFAP by S-nitroso glutathione (GSNO), an NO donor. Interestingly, inhibition of nuclear factor kappaB (NF-kappaB) suppressed LPS- but not GSNO-induced expression of GFAP, suggesting that NO does not require NF-kappaB to induce GFAP and that NF-kappaB functions upstream of NO production. However, inhibition of LPS- and GSNO-induced expression of GFAP either by NS-2028 [a specific inhibitor of guanylate cyclase (GC)] or by KT5823 [a specific inhibitor of cGMP-activated protein kinase (PKG)], and induction of GFAP expression by either 8-Br cGMP (a cell-permeable cGMP analog) or MY-5445 (a specific inhibitor of cGMP phosphodiesterase) suggests that NO induces GFAP via GC-cGMP-PKG. This study illustrates a novel biological role of NO in regulating the expression of GFAP in astrocytes through the GC-cGMP-PKG pathway that may participate in the pathogenesis of neurodegenerative disorders.

  18. Expression Analysis of Genes Involved in the RB/E2F Pathway in Astrocytic Tumors

    PubMed Central

    Ferreira, Wallax Augusto Silva; Araújo, Mariana Diniz; de Oliveira, Edivaldo Herculano Correa; Brito, José Reginaldo Nascimento; Burbano, Rommel Rodriguez; Harada, Maria Lúcia; Borges, Bárbara do Nascimento

    2015-01-01

    Astrocytic gliomas, which are derived from glial cells, are considered the most common primary neoplasias of the central nervous system (CNS) and are histologically classified as low grade (I and II) or high grade (III and IV). Recent studies have shown that astrocytoma formation is the result of the deregulation of several pathways, including the RB/E2F pathway, which is commonly deregulated in various human cancers via genetic or epigenetic mechanisms. On the basis of the assumption that the study of the mechanisms controlling the INK4/ARF locus can help elucidate the molecular pathogenesis of astrocytic tumors, identify diagnostic and prognostic markers, and help select appropriate clinical treatments, the present study aimed to evaluate and compare methylation patterns using bisulfite sequencing PCR and evaluate the gene expression profile using real-time PCR in the genes CDKN2A, CDKN2B, CDC6, Bmi-1, CCND1, and RB1 in astrocytic tumors. Our results indicate that all the evaluated genes are not methylated independent of the tumor grade. However, the real-time PCR results indicate that these genes undergo progressive deregulation as a function of the tumor grade. In addition, the genes CDKN2A, CDKN2B, and RB1 were underexpressed, whereas CDC6, Bmi-1, and CCND1 were overexpressed; the increase in gene expression was significantly associated with decreased patient survival. Therefore, we propose that the evaluation of the expression levels of the genes involved in the RB/E2F pathway can be used in the monitoring of patients with astrocytomas in clinical practice and for the prognostic indication of disease progression. PMID:26317630

  19. Expression Analysis of Genes Involved in the RB/E2F Pathway in Astrocytic Tumors.

    PubMed

    Ferreira, Wallax Augusto Silva; Araújo, Mariana Diniz; Anselmo, Nilson Praia; de Oliveira, Edivaldo Herculano Correa; Brito, José Reginaldo Nascimento; Burbano, Rommel Rodriguez; Harada, Maria Lúcia; Borges, Bárbara do Nascimento

    2015-01-01

    Astrocytic gliomas, which are derived from glial cells, are considered the most common primary neoplasias of the central nervous system (CNS) and are histologically classified as low grade (I and II) or high grade (III and IV). Recent studies have shown that astrocytoma formation is the result of the deregulation of several pathways, including the RB/E2F pathway, which is commonly deregulated in various human cancers via genetic or epigenetic mechanisms. On the basis of the assumption that the study of the mechanisms controlling the INK4/ARF locus can help elucidate the molecular pathogenesis of astrocytic tumors, identify diagnostic and prognostic markers, and help select appropriate clinical treatments, the present study aimed to evaluate and compare methylation patterns using bisulfite sequencing PCR and evaluate the gene expression profile using real-time PCR in the genes CDKN2A, CDKN2B, CDC6, Bmi-1, CCND1, and RB1 in astrocytic tumors. Our results indicate that all the evaluated genes are not methylated independent of the tumor grade. However, the real-time PCR results indicate that these genes undergo progressive deregulation as a function of the tumor grade. In addition, the genes CDKN2A, CDKN2B, and RB1 were underexpressed, whereas CDC6, Bmi-1, and CCND1 were overexpressed; the increase in gene expression was significantly associated with decreased patient survival. Therefore, we propose that the evaluation of the expression levels of the genes involved in the RB/E2F pathway can be used in the monitoring of patients with astrocytomas in clinical practice and for the prognostic indication of disease progression. PMID:26317630

  20. Rare individual amyloid-β oligomers act on astrocytes to initiate neuronal damage.

    PubMed

    Narayan, Priyanka; Holmström, Kira M; Kim, Dong-Hyun; Whitcomb, Daniel J; Wilson, Mark R; St George-Hyslop, Peter; Wood, Nicholas W; Dobson, Christopher M; Cho, Kwangwook; Abramov, Andrey Y; Klenerman, David

    2014-04-22

    Oligomers of the amyloid-β (Aβ) peptide have been implicated in the neurotoxicity associated with Alzheimer's disease. We have used single-molecule techniques to examine quantitatively the cellular effects of adding well characterized Aβ oligomers to primary hippocampal cells and hence determine the initial pathway of damage. We found that even picomolar concentrations of Aβ (1-40) and Aβ (1-42) oligomers can, within minutes of addition, increase the levels of intracellular calcium in astrocytes but not in neurons, and this effect is saturated at a concentration of about 10 nM of oligomers. Both Aβ (1-40) and Aβ (1-42) oligomers have comparable effects. The rise in intracellular calcium is followed by an increase in the rate of ROS production by NADPH oxidase in both neurons and astrocytes. The increase in ROS production then triggers caspase-3 activation resulting in the inhibition of long-term potentiation. Our quantitative approach also reveals that only a small fraction of the oligomers are damaging and that an individual rare oligomer binding to an astrocyte can initiate the aforementioned cascade of responses, making it unlikely to be due to any specific interaction. Preincubating the Aβ oligomers with an extracellular chaperone, clusterin, sequesters the oligomers in long-lived complexes and inhibits all of the physiological damage, even at a ratio of 100:1, total Aβ to clusterin. To explain how Aβ oligomers are so damaging but that it takes decades to develop Alzheimer's disease, we suggest a model for disease progression where small amounts of neuronal damage from individual unsequestered oligomers can accumulate over time leading to widespread tissue-level dysfunction. PMID:24717093

  1. Antofine-induced connexin43 gap junction disassembly in rat astrocytes involves protein kinase Cβ.

    PubMed

    Huang, Yu-Fang; Liao, Chih-Kai; Lin, Jau-Chen; Jow, Guey-Mei; Wang, Hwai-Shi; Wu, Jiahn-Chun

    2013-03-01

    Antofine, a phenanthroindolizidine alkaloid derived from Cryptocaryachinensis and Ficusseptica in the Asclepiadaceae milkweed family, is cytotoxic for various cancer cell lines. In this study, we demonstrated that treatment of rat primary astrocytes with antofine induced dose-dependent inhibition of gap junction intercellular communication (GJIC), as assessed by scrape-loading 6-carboxyfluorescein dye transfer. Levels of Cx43 protein were also decreased in a dose- and time-dependent manner following antofine treatment. Double-labeling immunofluorescence microscopy showed that antofine (10ng/ml) induced endocytosis of surface gap junctions into the cytoplasm, where Cx43 was co-localized with the early endosome marker EEA1. Inhibition of lysosomes or proteasomes by co-treatment with antofine and their respective specific inhibitors, NH4Cl or MG132, partially inhibited the antofine-induced decrease in Cx43 protein levels, but did not inhibit the antofine-induced inhibition of GJIC. After 30min of treatment, antofine induced a rapid increase in the intracellular Ca(2+) concentration and activation of protein kinase C (PKC)α/βII, which was maintained for at least 6h. Co-treatment of astrocytes with antofine and the intracellular Ca(2+) chelator BAPTA-AM prevented downregulation of Cx43 and inhibition of GJIC. Moreover, co-treatment with antofine and a specific PKCβ inhibitor prevented endocytosis of gap junctions, downregulation of Cx43, and inhibition of GJIC. Taken together, these findings indicate that antofine induces Cx43 gap junction disassembly by the PKCβ signaling pathway. Inhibition of GJIC by antofine may undermine the neuroprotective effect of astrocytes in CNS. PMID:23403203

  2. Cytotoxicity of the diterpene 14-O-methyl-ryanodanol from Erythroxylum passerinum in an astrocytic cells model.

    PubMed

    Menezes-Filho, Noélio de Jesus; Souza, Cleide dos Santos; Costa, Tereza Cristina Silva; Da Silva, Victor Diógenes Amaral; Ribeiro, Cátia Suse de Oliveira; Barreiros, Marizeth Liborio; Costa, Jose Fernando Oliveira; David, Jorge Mauricio; David, Juceni P L; Costa, Silvia Lima

    2014-09-01

    Plant secondary metabolites, such as, specifically, alkaloids and terpenes, may present psychoactive properties that modify the function of the central nervous system (CNS) and induce neurotoxicity. Neurotoxicity involves the response of glial cells, mainly astrocytes, which play a fundamental role in the control of homeostasis of the CNS. Some Erythroxylum species are indigenous to the state of Bahia in Brazil. This study investigated the cytotoxic activity of the diterpene AEP-1, extracted from the fruit of E. passerinum in a GL-15 cell line, astrocytic, glial cells model. The effects on cell viability, analyzed by the MTT assay, demonstrated a dose-dependent cytotoxic effect, with maximum effect at 500 μg/mL of AEP-1, and with a reduction of about 40 and 47% on cellular viability after 24 h and 72 h treatment, respectively. Evidence for induction of apoptosis by AEP-1 was first obtained when GL-15 glial cells were incubated with 250 μg/mL AEP-1 causing reniform and/or pyknotic nuclei and apoptotic bodies revealed by chromatin staining with Hoechst 33258. Increase in DNA fragmentation was also observed by comet assays in cells incubated with 500 μg/mL of AEP-1. Moreover, cells exposed to a sub toxic dose of AEP-1 (250 μg/mL) showed significant changes in morphology--contraction of the cytoplasm and expansion of cellular projections--signifying the presence of astrocytic cytoskeletal protein and glial fibrillary acidic protein (GFAP). These findings indicated astrocytic cells as the target for terpene AEP-1 and suggest the involvement of glial cells with psychoactive symptoms observed in humans and animals after consumption of fruits of plants of the genus Erythroxylum. PMID:25918783

  3. Form follows function: astrocyte morphology and immune dysfunction in SIV neuroAIDS.

    PubMed

    Lee, Kim M; Chiu, Kevin B; Renner, Nicole A; Sansing, Hope A; Didier, Peter J; MacLean, Andrew G

    2014-10-01

    Cortical function is disrupted in neuroinflammatory disorders, including HIV-associated neurocognitive disorders (HAND). Astrocyte dysfunction includes retraction of foot processes from the blood-brain barrier and decreased removal of neurotransmitters from synaptic clefts. Mechanisms of astrocyte activation, including innate immune function and the fine neuroanatomy of astrocytes, however, remain to be investigated. We quantified the number of glial fibrillary acidic protein (GFAP)-labeled astrocytes per square millimeter and the proportion of astrocytes immunopositive for Toll-like receptor 2 (TLR2) to examine innate immune activation in astrocytes. We also performed detailed morphometric analyses of gray and white matter astrocytes in the frontal and parietal lobes of rhesus macaques infected with simian immunodeficiency virus (SIV), both with and without encephalitis, an established model of AIDS neuropathogenesis. Protoplasmic astrocytes (gray matter) and fibrous astrocytes (deep white matter) were imaged, and morphometric features were analyzed using Neurolucida. Gray matter and white matter astrocytes showed no change in cell body size in animals infected with SIV regardless of encephalitic status. In SIV-infected macaques, both gray and white matter astrocytes had shorter, less ramified processes, resulting in decreased cell arbor compared with controls. SIV-infected macaques with encephalitis showed decreases in arbor length in white matter astrocytes and reduced complexity in gray matter astrocytes compared to controls. These results provide the first evidence that innate immune activation of astrocytes is linked to altered cortical astrocyte morphology in SIV/HIV infection. Here, we demonstrate that astrocyte remodeling is correlated with infection. Perturbed neuron-glia signaling may be a driving factor in the development of HAND.

  4. Calcium Signaling Involvement in Cadmium-Induced Astrocyte Cytotoxicity and Cell Death Through Activation of MAPK and PI3K/Akt Signaling Pathways.

    PubMed

    Jiang, Jiao Hua; Ge, Guo; Gao, Kai; Pang, Ying; Chai, Rui Chao; Jia, Xi Hua; Kong, Jin Ge; Yu, Albert Cheung-Hoi

    2015-09-01

    Cadmium (Cd), a highly ubiquitous toxic heavy metal, can contaminate the environment, including agricultural soil, water and air, via industrial runoff and other sources of pollution. Cd accumulated in the body via direct exposure or through the food chain results in neurodegeneration and many other diseases. Previous studies on its toxicity in the central nervous system (CNS) focused mainly on neurons. To obtain a more comprehensive understanding of Cd toxicity for the CNS, we investigated how astrocytes respond to acute and chronic Cd exposure and its toxic molecular mechanisms. When primary cultures of cerebral cortical astrocytes incubated with 1-300 μM CdCl2, morphological changes, LDH release and cell death were observed in a time and dose-dependent manner. Further studies demonstrated that acute and chronic Cd treatment phosphorylated JNK, p38 and Akt to different degrees, while ERK1/2 was only phosphorylated under low doses of Cd (10 μM) exposure. Inhibition of JNK and PI3K/Akt, but not of p38, could partially protect astrocyte from cytotoxicity in chronic and acute Cd exposure. Moreover, Cd also induced a strong calcium signal, while BAPTA, a specific intracellular calcium (Ca(2+)) chelator, prevented Cd-induced intracellular increase of calcium levels in astrocytes; inhibited the Cd-induced activation of ERK1/2, JNK, p38 and Akt; and also significantly reduced astrocyte cell death. All of these results suggested that the Cd-Ca(2+)-MAPK and PI3K/Akt signaling pathways were involved in Cd-induced toxicity in astrocytes. This toxicity involvement indicates that these pathways may be exploited as a target for the prevention of Cd-induced neurodegenerative diseases. PMID:26248512

  5. Cell type-specific dependency on the PI3K/Akt signaling pathway for the endogenous Epo and VEGF induction by baicalein in neurons versus astrocytes.

    PubMed

    Sun, Yu-Yo; Lin, Shang-Hsuan; Lin, Hung-Cheng; Hung, Chia-Chi; Wang, Chen-Yu; Lin, Yen-Chu; Hung, Kuo-Sheng; Lien, Cheng-Chang; Kuan, Chia-Yi; Lee, Yi-Hsuan

    2013-01-01

    The neuroprotective effect of baicalein is generally attributed to inhibition of 12/15-lipoxygenase (12/15-LOX) and suppression of oxidative stress, but recent studies showed that baicalein also activates hypoxia-inducible factor-α (HIF1α) through inhibition of prolyl hydrolase 2 (PHD2) and activation of the phosphatidylinositide-3 kinase (PI3K)/Akt signaling pathway. Yet, the significance and regulation of prosurvival cytokines erythropoietin (Epo) and vascular endothelial growth factor (VEGF), two transcriptional targets of HIF1α, in baicalein-mediated neuroprotection in neurons and astrocytes remains unknown. Here we investigated the causal relationship between the PI3K/Akt signaling pathway and Epo/VEGF expression in baicalein-mediated neuroprotection in primary rat cortical neurons and astrocytes. Our results show that baicalein induced Epo and VEGF expression in a HIF1α- and PI3K/Akt-dependent manner in neurons. Baicalein also protected neurons against excitotoxicity in a PI3K- and Epo/VEGF-dependent manner without affecting neuronal excitability. In contrast, at least a 10-fold higher concentration of baicalein was needed to induce Epo/VEGF production and PI3K/Akt activity in astrocytes for protection of neurons. Moreover, only baicalein-induced astrocytic VEGF, but not Epo expression requires HIF1α, while PI3K/Akt signaling had little role in baicalein-induced astrocytic Epo/VEGF expression. These results suggest distinct mechanisms of baicalein-mediated Epo/VEGF production in neurons and astrocytes for neuroprotection, and provide new insights into the mechanisms and potential of baicalein in treating brain injury in vivo. PMID:23904909

  6. Tumor necrosis factor-alpha mediates activation of NF-κB and JNK signaling cascades in retinal ganglion cells and astrocytes in opposite ways

    PubMed Central

    Dvoriantchikova, Galina; Ivanov, Dmitry

    2014-01-01

    Tumor necrosis factor-alpha (TNF) is an important mediator of the innate immune response in the retina. TNF can activate various signaling cascades, including NF-κB, nuclear factor kappa B (NF-κB) and c-Jun N-terminal kinase (JNK) pathways. The harmful role of these pathways, as well as of TNF, has previously been shown in several retinal neurodegenerative conditions including glaucoma and retinal ischemia. However, TNF and TNF-regulated signaling cascades are capable not only of mediating neurotoxicity, but of being protective. We performed this study to delineate the beneficial and detrimental effects of TNF signaling in the retina. To this end, we used TNF-treated primary retinal ganglion cell (RGC) and astrocyte cultures. Levels of expression of NF-κB subunits in RGCs and astrocytes were evaluated by quantitative RT-PCR (qRT-PCR) and Western blot (WB) analysis. NF-κB and JNK activity in TNF-treated cells was determined in a time-dependent manner using ELISA and WB. Gene expression in TNF-treated astrocytes was measured by qRT-PCR. We found that NF-κB family members were present in RGCs and astrocytes at the mRNA and protein levels. RGCs failed to activate NF-κB in the presence of TNF, a phenomenon that was associated with sustained JNK activation and RGC death. However, TNF initiated the activation of NF-κB and mediated transient JNK activation in astrocytes. These events were associated with glial survival and increased expression of neurotoxic pro-inflammatory factors. Our findings suggest that, in the presence of TNF, NF-κB and JNK signaling cascades are activated in opposite ways in RGCs and astrocytes. These events can directly and indirectly facilitate RGC death. PMID:25160799

  7. Melatonin modulates intercellular communication among cultured chick astrocytes.

    PubMed

    Peters, Jennifer L; Cassone, Vincent M; Zoran, Mark J

    2005-01-01

    Melatonin, a pineal neurohormone, mediates circadian and seasonal processes in birds and mammals. Diencephalic astrocytes are sites of action, at least in birds, since they express melatonin receptors and melatonin affects their metabolism. We tested whether astrocytic calcium waves are also modulated by melatonin. Calcium waves, which we found to be regulated in cultured chick glial cells by an IP(3)-dependent mechanism, were potentiated by physiological concentrations of melatonin. Melatonin also increased resting calcium levels and reduced gap junctional coupling among astrocytes, at concentrations that facilitated calcium waves. These modulatory effects were diminished by melatonin receptor blockade and pertussis toxin (PTX). Thus, melatonin induced a functional shift in the mode of intercellular communication, between junctional coupling and calcium waves, among glial cells. We suggest a mechanism where neuroglial physiology, involving GTP-binding protein signaling pathways, links rhythmic circadian outputs to pervasive neurobehavioral states. PMID:15621008

  8. Neuron–astrocyte interactions in neurodegenerative diseases: Role of neuroinflammation

    PubMed Central

    Rama Rao, Kakulavarapu V.; Kielian, Tammy

    2015-01-01

    Selective neuron loss in discrete brain regions is a hallmark of various neurodegenerative disorders, although the mechanisms responsible for this regional vulnerability of neurons remain largely unknown. Earlier studies attributed neuron dysfunction and eventual loss during neurodegenerative diseases as exclusively cell autonomous. Although cell-intrinsic factors are one critical aspect in dictating neuron death, recent evidence also supports the involvement of other central nervous system cell types in propagating non-cell autonomous neuronal injury during neurodegenerative diseases. One such example is astrocytes, which support neuronal and synaptic function, but can also contribute to neuroinflammatory processes through robust chemokine secretion. Indeed, aberrations in astrocyte function have been shown to negatively impact neuronal integrity in several neurological diseases. The present review focuses on neuroinflammatory paradigms influenced by neuron–astrocyte cross-talk in the context of select neurodegenerative diseases. PMID:26543505

  9. Diverse subtypes of astrocytes and their development during corticogenesis

    PubMed Central

    Tabata, Hidenori

    2015-01-01

    Astrocytes are one of the most abundant cell types in the mammalian central nervous system, and are known to have a wide variety of physiological functions, including maintenance of neurons, formation of the blood brain barrier, and regulation of synapse functions. Although the migration and positioning of neurons has been extensively studied over the last several decades and many aspects have been uncovered, the process underlying glial development was largely unknown until recently due to the existence of multiple subtypes of glia and the sustained proliferative ability of these cells through adulthood. To overcome these difficulties, new gene transfer techniques and genetically modified mice were developed, and have been gradually revealing when and how astrocytes develop during corticogenesis. In this paper, I review the diversity of astrocytes and summarize our knowledge about their production and migration. PMID:25904839

  10. In vivo astrocytic Ca2+ signaling in health and brain disorders

    PubMed Central

    Ding, Shinghua

    2013-01-01

    Astrocytes are the predominant glial cell type in the CNS. Although astrocytes are electrically nonexcitable, their excitability is manifested by their Ca2+ signaling, which serves as a mediator of neuron–glia bidirectional interactions via tripartite synapses. Studies from in vivo two-photon imaging indicate that in healthy animals, the properties of spontaneous astrocytic Ca2+ signaling are affected by animal species, age, wakefulness and the location of astrocytes in the brain. Intercellular Ca2+ waves in astrocytes can be evoked by a variety of stimulations. In animal models of some brain disorders, astrocytes can exhibit enhanced Ca2+ excitability featured as regenerative intercellular Ca2+ waves. This review first briefly summarizes the astrocytic Ca2+ signaling pathway and the procedure of in vivo two-photon Ca2+ imaging of astrocytes. It subsequently summarizes in vivo astrocytic Ca2+ signaling in health and brain disorders from experimental studies of animal models, and discusses the possible mechanisms and therapeutic implications underlying the enhanced Ca2+ excitability in astrocytes in brain disorders. Finally, this review summarizes molecular genetic approaches used to selectively manipulate astrocyte function in vivo and their applications to study the role of astrocytes in synaptic plasticity and brain disorders. PMID:24376372

  11. Contributions of Glycogen to Astrocytic Energetics during Brain Activation

    PubMed Central

    Dienel, Gerald A.; Cruz, Nancy F.

    2014-01-01

    Glycogen is the major store of glucose in brain and is mainly in astrocytes. Brain glycogen levels in unstimulated, carefully-handled rats are 10-12 mol/g, and assuming that astrocytes account for half the brain mass, astrocytic glycogen content is twice as high. Glycogen turnover is slow under basal conditions, but it is mobilized during activation. There is no net increase in incorporation of label from glucose during activation, whereas label release from pre-labeled glycogen exceeds net glycogen consumption, which increases during stronger stimuli. Because glycogen level is restored by non-oxidative metabolism, astrocytes can influence the global ratio of oxygen to glucose utilization. Compensatory increases in utilization of blood glucose during inhibition of glycogen phosphorylase are large and approximate glycogenolysis rates during sensory stimulation. In contrast, glycogenolysis rates during hypoglycemia are low due to continued glucose delivery and oxidation of endogenous substrates; rates that preserve neuronal function in the absence of glucose are also low, probably due to metabolite oxidation. Modeling studies predict that glycogenolysis maintains a high level of glucose-6-phosphate in astrocytes to maintain feedback inhibition of hexokinase, thereby diverting glucose for use by neurons. The fate of glycogen carbon in vivo is not known, but lactate efflux from brain best accounts for the major metabolic characteristics during activation of living brain. Substantial shuttling coupled with oxidation of glycogen-derived lactate is inconsistent with available evidence. Glycogen has important roles in astrocytic energetics, including glucose sparing, control of extracellular K+ level, oxidative stress management, and memory consolidation; it is a multi-functional compound. PMID:24515302

  12. Astrocytes in Oligodendrocyte Lineage Development and White Matter Pathology

    PubMed Central

    Li, Jiasi; Zhang, Lei; Chu, Yongxin; Namaka, Michael; Deng, Benqiang; Kong, Jiming; Bi, Xiaoying

    2016-01-01

    White matter is primarily composed of myelin and myelinated axons. Structural and functional completeness of myelin is critical for the reliable and efficient transmission of information. White matter injury has been associated with the development of many demyelinating diseases. Despite a variety of scientific advances aimed at promoting re-myelination, their benefit has proven at best to be marginal. Research suggests that the failure of the re-myelination process may be the result of an unfavorable microenvironment. Astrocytes, are the most ample and diverse type of glial cells in central nervous system (CNS) which display multiple functions for the cells of the oligodendrocytes lineage. As such, much attention has recently been drawn to astrocyte function in terms of white matter myelin repair. They are different in white matter from those in gray matter in specific regards to development, morphology, location, protein expression and other supportive functions. During the process of demyelination and re-myelination, the functions of astrocytes are dynamic in that they are able to change functions in accordance to different time points, triggers or reactive pathways resulting in vastly different biologic effects. They have pivotal effects on oligodendrocytes and other cell types in the oligodendrocyte lineage by serving as an energy supplier, a participant of immunological and inflammatory functions, a source of trophic factors and iron and a sustainer of homeostasis. Astrocytic impairment has been shown to be directly linked to the development of neuromyelities optica (NMO). In addition, astroctyes have also been implicated in other white matter conditions such as psychiatric disorders and neurodegenerative diseases such as Alzheimer’s disease (AD), multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Inhibiting specifically detrimental signaling pathways in astrocytes while preserving their beneficial functions may be a promising approach for

  13. Astrocytes in Oligodendrocyte Lineage Development and White Matter Pathology.

    PubMed

    Li, Jiasi; Zhang, Lei; Chu, Yongxin; Namaka, Michael; Deng, Benqiang; Kong, Jiming; Bi, Xiaoying

    2016-01-01

    White matter is primarily composed of myelin and myelinated axons. Structural and functional completeness of myelin is critical for the reliable and efficient transmission of information. White matter injury has been associated with the development of many demyelinating diseases. Despite a variety of scientific advances aimed at promoting re-myelination, their benefit has proven at best to be marginal. Research suggests that the failure of the re-myelination process may be the result of an unfavorable microenvironment. Astrocytes, are the most ample and diverse type of glial cells in central nervous system (CNS) which display multiple functions for the cells of the oligodendrocytes lineage. As such, much attention has recently been drawn to astrocyte function in terms of white matter myelin repair. They are different in white matter from those in gray matter in specific regards to development, morphology, location, protein expression and other supportive functions. During the process of demyelination and re-myelination, the functions of astrocytes are dynamic in that they are able to change functions in accordance to different time points, triggers or reactive pathways resulting in vastly different biologic effects. They have pivotal effects on oligodendrocytes and other cell types in the oligodendrocyte lineage by serving as an energy supplier, a participant of immunological and inflammatory functions, a source of trophic factors and iron and a sustainer of homeostasis. Astrocytic impairment has been shown to be directly linked to the development of neuromyelities optica (NMO). In addition, astroctyes have also been implicated in other white matter conditions such as psychiatric disorders and neurodegenerative diseases such as Alzheimer's disease (AD), multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Inhibiting specifically detrimental signaling pathways in astrocytes while preserving their beneficial functions may be a promising approach for

  14. Contributions of glycogen to astrocytic energetics during brain activation.

    PubMed

    Dienel, Gerald A; Cruz, Nancy F

    2015-02-01

    Glycogen is the major store of glucose in brain and is mainly in astrocytes. Brain glycogen levels in unstimulated, carefully-handled rats are 10-12 μmol/g, and assuming that astrocytes account for half the brain mass, astrocytic glycogen content is twice as high. Glycogen turnover is slow under basal conditions, but it is mobilized during activation. There is no net increase in incorporation of label from glucose during activation, whereas label release from pre-labeled glycogen exceeds net glycogen consumption, which increases during stronger stimuli. Because glycogen level is restored by non-oxidative metabolism, astrocytes can influence the global ratio of oxygen to glucose utilization. Compensatory increases in utilization of blood glucose during inhibition of glycogen phosphorylase are large and approximate glycogenolysis rates during sensory stimulation. In contrast, glycogenolysis rates during hypoglycemia are low due to continued glucose delivery and oxidation of endogenous substrates; rates that preserve neuronal function in the absence of glucose are also low, probably due to metabolite oxidation. Modeling studies predict that glycogenolysis maintains a high level of glucose-6-phosphate in astrocytes to maintain feedback inhibition of hexokinase, thereby diverting glucose for use by neurons. The fate of glycogen carbon in vivo is not known, but lactate efflux from brain best accounts for the major metabolic characteristics during activation of living brain. Substantial shuttling coupled with oxidation of glycogen-derived lactate is inconsistent with available evidence. Glycogen has important roles in astrocytic energetics, including glucose sparing, control of extracellular K(+) level, oxidative stress management, and memory consolidation; it is a multi-functional compound.

  15. Astrocytes in Oligodendrocyte Lineage Development and White Matter Pathology.

    PubMed

    Li, Jiasi; Zhang, Lei; Chu, Yongxin; Namaka, Michael; Deng, Benqiang; Kong, Jiming; Bi, Xiaoying

    2016-01-01

    White matter is primarily composed of myelin and myelinated axons. Structural and functional completeness of myelin is critical for the reliable and efficient transmission of information. White matter injury has been associated with the development of many demyelinating diseases. Despite a variety of scientific advances aimed at promoting re-myelination, their benefit has proven at best to be marginal. Research suggests that the failure of the re-myelination process may be the result of an unfavorable microenvironment. Astrocytes, are the most ample and diverse type of glial cells in central nervous system (CNS) which display multiple functions for the cells of the oligodendrocytes lineage. As such, much attention has recently been drawn to astrocyte function in terms of white matter myelin repair. They are different in white matter from those in gray matter in specific regards to development, morphology, location, protein expression and other supportive functions. During the process of demyelination and re-myelination, the functions of astrocytes are dynamic in that they are able to change functions in accordance to different time points, triggers or reactive pathways resulting in vastly different biologic effects. They have pivotal effects on oligodendrocytes and other cell types in the oligodendrocyte lineage by serving as an energy supplier, a participant of immunological and inflammatory functions, a source of trophic factors and iron and a sustainer of homeostasis. Astrocytic impairment has been shown to be directly linked to the development of neuromyelities optica (NMO). In addition, astroctyes have also been implicated in other white matter conditions such as psychiatric disorders and neurodegenerative diseases such as Alzheimer's disease (AD), multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Inhibiting specifically detrimental signaling pathways in astrocytes while preserving their beneficial functions may be a promising approach for

  16. Theophylline potentiates lipopolysaccharide-induced NO production in cultured astrocytes.

    PubMed

    Ogawa, Mizue; Takano, Katsura; Kawabe, Kenji; Moriyama, Mitsuaki; Ihara, Hideshi; Nakamura, Yoichi

    2014-01-01

    Elucidation of the functions of astrocytes is important for understanding of the pathogenic mechanism of various neurodegenerative diseases. Theophylline is a common drug for bronchial asthma and occasionally develops side-effects, such as acute encephalopathy; although the pathogenic mechanism of the side-effects is unknown. The lipopolysaccharide (LPS)-induced nitricoxide (NO) production is generally used for an index of the activation of astrocyte in vitro. In this study, in order to elucidate the effect of theophylline on the astrocytic functions, we examined the LPS-induced NO production and the expression of iNOS in cultured rat cortex astrocytes.Theophylline alone could not induce the NO production; however, NO production induced by LPS was enhanced by theophylline in a dose-dependent manner; and by isobutylmethylxanthine, a phosphodiesterase inhibitor. The theophylline enhancement of LPS-induced NO production was further increased by dibutyryl cyclic AMP, a membrane-permeable cAMP analog; and by forskolin, an adenylate cyclase activator. When the cells were preincubated with Rp-8-Br-cAMP, an inhibitor of protein kinase A, the theophylline enhancement of LPS-induced NO production was decreased. The extent of iNOS protein expression induced by LPS was also enhanced by theophylline.It is likely that phosphodiesterase inhibition is a major action mechanism for the theophylline enhancement of LPS-induced NO production in astrocytes. Theophylline-induced acute encephalopathy might be due to the hyper-activation of astrocytes via cAMP signaling to produce excess amount of NO.

  17. Dual role of astrocytes in perinatal asphyxia injury and neuroprotection.

    PubMed

    Romero, J; Muñiz, J; Logica Tornatore, T; Holubiec, M; González, J; Barreto, G E; Guelman, L; Lillig, C H; Blanco, E; Capani, F

    2014-04-17

    Perinatal asphyxia represents an important cause of severe neurological deficits including delayed mental and motor development, epilepsy, major cognitive deficits and blindness. However, at the moment, most of the therapeutic strategies were not well targeted toward the processes that induced the brain injury during perinatal asphyxia. Traditionally, experimental research focused on neurons, whereas astrocytes have been more related with the damage mechanisms of perinatal asphyxia. In this work, we propose to review possible protective as well as deleterious roles of astrocytes in the asphyctic brain with the aim to stimulate further research in this area of perinatal asphyxia still not well studied. PMID:24172702

  18. Dual role of astrocytes in perinatal asphyxia injury and neuroprotection.

    PubMed

    Romero, J; Muñiz, J; Logica Tornatore, T; Holubiec, M; González, J; Barreto, G E; Guelman, L; Lillig, C H; Blanco, E; Capani, F

    2014-04-17

    Perinatal asphyxia represents an important cause of severe neurological deficits including delayed mental and motor development, epilepsy, major cognitive deficits and blindness. However, at the moment, most of the therapeutic strategies were not well targeted toward the processes that induced the brain injury during perinatal asphyxia. Traditionally, experimental research focused on neurons, whereas astrocytes have been more related with the damage mechanisms of perinatal asphyxia. In this work, we propose to review possible protective as well as deleterious roles of astrocytes in the asphyctic brain with the aim to stimulate further research in this area of perinatal asphyxia still not well studied.

  19. Unregulated brain iron deposition in transgenic mice over-expressing HMOX1 in the astrocytic compartment.

    PubMed

    Song, Wei; Zukor, Hillel; Lin, Shih-Hsiung; Liberman, Adrienne; Tavitian, Ayda; Mui, Jeannie; Vali, Hojatollah; Fillebeen, Carine; Pantopoulos, Kostas; Wu, Ting-Di; Guerquin-Kern, Jean-Luc; Schipper, Hyman M

    2012-10-01

    The mechanisms responsible for pathological iron deposition in the aging and degenerating mammalian CNS remain poorly understood. The stress protein, HO-1 mediates the degradation of cellular heme to biliverdin/bilirubin, free iron, and CO and is up-regulated in the brains of persons with Alzheimer's disease and Parkinson's disease. HO-1 induction in primary astroglial cultures promotes deposition of non-transferrin iron, mitochondrial damage and macroautophagy, and predisposes cocultured neuronal elements to oxidative injury. To gain a better appreciation of the role of glial HO-1 in vivo, we probed for aberrant brain iron deposition using Perls' method and dynamic secondary ion mass spectrometry in novel, conditional GFAP.HMOX1 transgenic mice that selectively over-express human HO-1 in the astrocytic compartment. At 48 weeks, the GFAP.HMOX1 mice exhibited increased deposits of glial iron in hippocampus and other subcortical regions without overt changes in iron-regulatory and iron-binding proteins relative to age-matched wild-type animals. Dynamic secondary ion mass spectrometry revealed abundant FeO⁻ signals in the transgenic, but not wild-type, mouse brain that colocalized to degenerate mitochondria and osmiophilic cytoplasmic inclusions (macroautophagy) documented by TEM. Sustained up-regulation of HO-1 in astrocytes promotes pathological brain iron deposition and oxidative mitochondrial damage characteristic of Alzheimer's disease-affected neural tissues. Curtailment of glial HO-1 hyperactivity may limit iron-mediated cytotoxicity in aging and degenerating neural tissues.

  20. Mutant SOD1-expressing astrocytes release toxic factors that trigger motoneuron death by inducing hyperexcitability.

    PubMed

    Fritz, Elsa; Izaurieta, Pamela; Weiss, Alexandra; Mir, Franco R; Rojas, Patricio; Gonzalez, David; Rojas, Fabiola; Brown, Robert H; Madrid, Rodolfo; van Zundert, Brigitte

    2013-06-01

    Amyotrophic lateral sclerosis (ALS) is a devastating paralytic disorder caused by dysfunction and degeneration of motoneurons starting in adulthood. Recent studies using cell or animal models document that astrocytes expressing disease-causing mutations of human superoxide dismutase 1 (hSOD1) contribute to the pathogenesis of ALS by releasing a neurotoxic factor(s). Neither the mechanism by which this neurotoxic factor induces motoneuron death nor its cellular site of action has been elucidated. Here we show that acute exposure of primary wild-type spinal cord cultures to conditioned medium derived from astrocytes expressing mutant SOD1 (ACM-hSOD1(G93A)) increases persistent sodium inward currents (PC(Na)), repetitive firing, and intracellular calcium transients, leading to specific motoneuron death days later. In contrast to TTX, which paradoxically increased twofold the amplitude of calcium transients and killed motoneurons, reduction of hyperexcitab