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Sample records for human primary astrocytes

  1. Primary cultures of astrocytes

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    Lange, Sofie C; Bak, Lasse Kristoffer; Waagepetersen, Helle S;

    2012-01-01

    During the past few decades of astrocyte research it has become increasingly clear that astrocytes have taken a central position in all central nervous system activities. Much of our new understanding of astrocytes has been derived from studies conducted with primary cultures of astrocytes. Such ...

  2. Insulin promotes glycogen storage and cell proliferation in primary human astrocytes.

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    Martin Heni

    Full Text Available INTRODUCTION: In the human brain, there are at least as many astrocytes as neurons. Astrocytes are known to modulate neuronal function in several ways. Thus, they may also contribute to cerebral insulin actions. Therefore, we examined whether primary human astrocytes are insulin-responsive and whether their metabolic functions are affected by the hormone. METHODS: Commercially available Normal Human Astrocytes were grown in the recommended medium. Major players in the insulin signaling pathway were detected by real-time RT-PCR and Western blotting. Phosphorylation events were detected by phospho-specific antibodies. Glucose uptake and glycogen synthesis were assessed using radio-labeled glucose. Glycogen content was assessed by histochemistry. Lactate levels were measured enzymatically. Cell proliferation was assessed by WST-1 assay. RESULTS: We detected expression of key proteins for insulin signaling, such as insulin receptor β-subunit, insulin receptor substrat-1, Akt/protein kinase B and glycogen synthase kinase 3, in human astrocytes. Akt was phosphorylated and PI-3 kinase activity increased following insulin stimulation in a dose-dependent manner. Neither increased glucose uptake nor lactate secretion after insulin stimulation could be evidenced in this cell type. However, we found increased insulin-dependent glucose incorporation into glycogen. Furthermore, cell numbers increased dose-dependently upon insulin treatment. DISCUSSION: This study demonstrated that human astrocytes are insulin-responsive at the molecular level. We identified glycogen synthesis and cell proliferation as biological responses of insulin signaling in these brain cells. Hence, this cell type may contribute to the effects of insulin in the human brain.

  3. Could astrocytes be the primary target of an offending agent causing the primary degenerative diseases of the human central nervous system? A hypothesis.

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    Sica, Roberto E

    2015-05-01

    Most of the named primary degenerative diseases of the human central nervous system have been attributed to a direct, primary damage of some particular population of neurons. Within the spectrum of these illnesses there are disorders like amyotrophic lateral sclerosis, fronto-temporal dementia, Alzheimer's dementia, Parkinson's disease, Huntington's dementia and cerebellar ataxias affecting exclusively the human species. In the last years it has been shown that non-neural cells, mainly astrocytes, have a crucial role in the starting and development of these diseases. We suggest that the causative agent of these illnesses gets home first within the astrocytes, rather than the neurons, making them sick by modifying the structure of some proteins; from these cells the abnormal process would start a trip to other astrocytes having the same genetic, metabolic, structural and functional profiles that the originally affected astrocytes have, going through the gap junctions which connect that particular population devoted to a particular set of neurons. This appears to be a likely hypothesis because the astrocytes related to a defined population of neurons have their own, private properties and characteristics needed to support one particular set of neurons performing a defined function, making them a different and unique population, a fact which would limit the spreading of the disease to those astrocytes, sparing other astrocyte populations which do not share those characteristics. If this were the mechanism underlying these illnesses, the neurons, which their health depends on those astrocytes, would be deprived of their patronage and would start all the changes that characterizes a programmed cell death, and the clinical manifestations of a defined pathology would consequently appear. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Identification of gene products suppressed by human immunodeficiency virus type 1 infection or gp120 exposure of primary human astrocytes by rapid subtraction hybridization.

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    Su, Zao-Zhong; Kang, Dong-Chul; Chen, Yinming; Pekarskaya, Olga; Chao, Wei; Volsky, David J; Fisher, Paul B

    2003-06-01

    Neurodegeneration and human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) are the major disease manifestations of HIV-1 colonization of the central nervous system (CNS). In the brain, HIV-1 replicates in microglial cells and infiltrating macrophages and it persists in a low-productive, noncytolytic state in astrocytes. Astrocytes play critical roles in the maintenance of the brain microenvironment, responses to injury, and in neuronal signal transmission, and disruption of these functions by HIV-1 could contribute to HAD. To better understand the potential effects of HIV-1 on astrocyte biology, the authors investigated changes in gene expression using an efficient and sensitive rapid subtraction hybridization approach, RaSH. Primary human astrocytes were isolated from abortus brain tissue, low-passage cells were infected with HIV-1 or mock infected, and total cellular RNAs were isolated at multiple time points over a period of 1 week. This approach is designed to identify gene products modulated early and late after HIV-1 infection and limits the cloning of genes displaying normal cell-cycle fluctuations in astrocytes. By subtracting temporal cDNAs derived from HIV-1-infected astrocytes from temporal cDNAs made from uninfected cells, 10 genes displaying reduced expression in infected cells, termed astrocyte suppressed genes (ASGs), were identified and their suppression was confirmed by Northern blot hybridization. Both known and novel ASGs, not reported in current DNA databases, that are down-regulated by HIV-1 infection are described. Northern blotting confirms suppression of the same panel of ASGs by treatment of astrocytes with recombinant HIV-1 envelope glycoprotein, gp120. These results extend our previous analysis of astrocyte genes induced or enhanced by HIV-1 infection and together they suggest that HIV-1 and viral proteins have profound effects on astrocyte physiology, which may influence their function in the CNS.

  5. Repression of GW/P body components and the RNAi microprocessor impacts primary ciliogenesis in human astrocytes

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    Rattner Jerome B

    2011-08-01

    Full Text Available Abstract Background In most cells, the centriolar component of the centrosome can function as a basal body supporting the formation of a primary cilium, a non-motile sensory organelle that monitors information from the extracellular matrix and relays stimuli into the cell via associated signaling pathways. Defects in the formation and function of primary cilia underlie multiple human diseases and are hallmarks of malignancy. The RNA silencing pathway is involved in the post-transcriptional silencing of > 50% of mRNA that occurs within GW/P bodies. GW/P bodies are found throughout the cytoplasm and previously published live cell imaging data suggested that in a malignant cell type (U2OS, two GW/P bodies reside at the centrosome during interphase. This led us to investigate if a similar relationship exists in primary cells and if the inhibition of the miRNA pathway impairs primary cilium formation. Results Two GW/P bodies as marked by GW182 and hAgo2 colocalized to the basal body of primary human astrocytes as well as human synoviocytes during interphase and specifically with the distal end of the basal body in the pericentriolar region. Since it is technically challenging to examine the two centrosomal GW/P bodies in isolation, we investigated the potential relationship between the global population of GW/P bodies and primary ciliogenesis. Astrocytes were transfected with siRNA directed to GW182 and hAgo2 and unlike control astrocytes, a primary cilium was no longer associated with the centrosome as detected in indirect immunofluorescence assays. Ultrastructural analysis of siRNA transfected astrocytes revealed that knock down of GW182, hAgo2, Drosha and DGCR8 mRNA did not affect the appearance of the earliest stage of ciliogenesis but did prevent the formation and elongation of the ciliary axoneme. Conclusions This study confirms and extends a previously published report that GW/P bodies reside at the centrosome in U2OS cells and documents that

  6. Effects of Kynurenine Pathway Metabolites on Intracellular NAD+ Synthesis and Cell Death in Human Primary Astrocytes and Neurons

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    Nady Braidy

    2009-01-01

    Full Text Available The kynurenine pathway (KP is a major route of L-tryptophan catabolism resulting in the production of the essential pyridine nucleotide nicotinamide adenine dinucleotide, (NAD+. Up-regulation of the KP during inflammation leads to the release of a number of biologically active metabolites into the brain. We hypothesised that while some of the extracellular KP metabolites may be beneficial for intracellular NAD+ synthesis and cell survival at physiological concentrations, they may contribute to neuronal and astroglial dysfunction and cell death at pathophysiological concentrations. In this study, we found that treatment of human primary neurons and astrocytes with 3-hydroxyanthranilic acid (3-HAA, 3-hydroxykynurenine (3-HK, quinolinic acid (QUIN, and picolinic acid (PIC at concentrations below 100 nM significantly increased intracellular NAD+ levels compared to non-treated cells. However, a dose dependent decrease in intracellular NAD+ levels and increased extracellular LDH activity was observed in human astrocytes and neurons treated with 3-HAA, 3-HK, QUIN and PIC at concentrations 100 nM and kynurenine (KYN, at concentrations above 1 μM. Intracellular NAD+ levels were unchanged in the presence of the neuroprotectant, kynurenic acid (KYNA, and a dose dependent increase in intracellular NAD+ levels was observed for TRP up to 1 mM. While anthranilic acid (AA increased intracellular NAD+ levels at concentration below 10 μM in astrocytes. NAD+ depletion and cell death was observed in AA treated neurons at concentrations above 500 nM. Therefore, the differing responses of astrocytes and neurons to an increase in KP metabolites should be considered when assessing KP toxicity during neuroinflammation.

  7. Comparison of the gene expression profiles of human fetal cortical astrocytes with pluripotent stem cell derived neural stem cells identifies human astrocyte markers and signaling pathways and transcription factors active in human astrocytes.

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    Malik, Nasir; Wang, Xiantao; Shah, Sonia; Efthymiou, Anastasia G; Yan, Bin; Heman-Ackah, Sabrina; Zhan, Ming; Rao, Mahendra

    2014-01-01

    Astrocytes are the most abundant cell type in the central nervous system (CNS) and have a multitude of functions that include maintenance of CNS homeostasis, trophic support of neurons, detoxification, and immune surveillance. It has only recently been appreciated that astrocyte dysfunction is a primary cause of many neurological disorders. Despite their importance in disease very little is known about global gene expression for human astrocytes. We have performed a microarray expression analysis of human fetal astrocytes to identify genes and signaling pathways that are important for astrocyte development and maintenance. Our analysis confirmed that the fetal astrocytes express high levels of the core astrocyte marker GFAP and the transcription factors from the NFI family which have been shown to play important roles in astrocyte development. A group of novel markers were identified that distinguish fetal astrocytes from pluripotent stem cell-derived neural stem cells (NSCs) and NSC-derived neurons. As in murine astrocytes, the Notch signaling pathway appears to be particularly important for cell fate decisions between the astrocyte and neuronal lineages in human astrocytes. These findings unveil the repertoire of genes expressed in human astrocytes and serve as a basis for further studies to better understand astrocyte biology, especially as it relates to disease.

  8. The microRNA and messengerRNA profile of the RNA-induced silencing complex in human primary astrocyte and astrocytoma cells.

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    Joanna J Moser

    Full Text Available BACKGROUND: GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA-mediated messenger RNA (mRNA silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC. To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells. METHODOLOGY/PRINCIPAL FINDINGS: RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1 miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2 astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3 miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4 the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells. CONCLUSIONS/SIGNIFICANCE: The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and

  9. Characterisation of the expression of NMDA receptors in human astrocytes.

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    Ming-Chak Lee

    Full Text Available Astrocytes have long been perceived only as structural and supporting cells within the central nervous system (CNS. However, the discovery that these glial cells may potentially express receptors capable of responding to endogenous neurotransmitters has resulted in the need to reassess astrocytic physiology. The aim of the current study was to characterise the expression of NMDA receptors (NMDARs in primary human astrocytes, and investigate their response to physiological and excitotoxic concentrations of the known endogenous NMDAR agonists, glutamate and quinolinic acid (QUIN. Primary cultures of human astrocytes were used to examine expression of these receptors at the mRNA level using RT-PCR and qPCR, and at the protein level using immunocytochemistry. The functionality role of the receptors was assessed using intracellular calcium influx experiments and measuring extracellular lactate dehydrogenase (LDH activity in primary cultures of human astrocytes treated with glutamate and QUIN. We found that all seven currently known NMDAR subunits (NR1, NR2A, NR2B, NR2C, NR2D, NR3A and NR3B are expressed in astrocytes, but at different levels. Calcium influx studies revealed that both glutamate and QUIN could activate astrocytic NMDARs, which stimulates Ca2+ influx into the cell and can result in dysfunction and death of astrocytes. Our data also show that the NMDAR ion channel blockers, MK801, and memantine can attenuate glutamate and QUIN mediated cell excitotoxicity. This suggests that the mechanism of glutamate and QUIN gliotoxicity is at least partially mediated by excessive stimulation of NMDARs. The present study is the first to provide definitive evidence for the existence of functional NMDAR expression in human primary astrocytes. This discovery has significant implications for redefining the cellular interaction between glia and neurons in both physiological processes and pathological conditions.

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

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    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.

  11. Superantigen presenting capacity of human astrocytes

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    Hassan-Zahraee, M; Ladiwala, U; Lavoie, P M

    2000-01-01

    We found that human fetal astrocytes (HFA) are able to support superantigen (SAG) staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 (TSST-1)-induced activation of immediately ex vivo allogenic human CD4 T cells. Using radiolabelled toxins, we demonstrate that both SEB and TSST-1...... bind with high affinity to MHC class II antigen expressing astrocytes; binding is displaceable with excess cold toxin. Competition experiments further indicate that TSST-1 and SEB at least partially compete with each other for binding to astrocytes suggesting they bind to the same HLA-DR region...

  12. Influence of rat substrain and growth conditions on the characteristics of primary cultures of adult rat spinal cord astrocytes.

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    Codeluppi, Simone; Gregory, Ebba Norsted; Kjell, Jacob; Wigerblad, Gustaf; Olson, Lars; Svensson, Camilla I

    2011-04-15

    Primary astrocyte cell cultures have become a valuable tool for studies of signaling pathways that regulate astrocyte physiology, reactivity, and function; however, differences in culture preparation affect data reproducibility. The aim of this work was to define optimal conditions for obtaining primary astrocytes from adult rat spinal cord with an expression profile most similar to adult human spinal cord astrocytes. Hence, we examined whether different Sprague-Dawley substrains and culture conditions affect astrocyte culture quality. Medium supplemented with fetal bovine serum from three sources (Sigma, Gibco, Hyclone) or a medium with defined composition (AM medium) was used to culture astrocytes isolated from spinal cords of adult Harlan and Charles River Spraque-Dawley rats. Purity was significantly different between cultures established in media with different sera. No microglia were detected in AM or Hyclone cultures. Gene expression was also affected, with AM cultures expressing the highest level of glutamine synthetase, connexin-43, and glutamate transporter-1. Interestingly, cell response to starvation was substrain dependent. Charles River-derived cultures responded the least, while astrocytes derived from Harlan rats showed a greater decrease in Gfap and glutamine synthetase, suggesting a more quiescent phenotype. Human and Harlan astrocytes cultured in AM media responded similarly to starvation. Taken together, this study shows that rat substrain and growth medium composition affect purity, expression profile and response to starvation of primary astrocytes suggesting that cultures of Harlan rats in AM media have optimal astrocyte characteristics, purity, and similarity to human astrocytes.

  13. Superantigen presenting capacity of human astrocytes

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    Hassan-Zahraee, M; Ladiwala, U; Lavoie, P M;

    2000-01-01

    We found that human fetal astrocytes (HFA) are able to support superantigen (SAG) staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 (TSST-1)-induced activation of immediately ex vivo allogenic human CD4 T cells. Using radiolabelled toxins, we demonstrate that both SEB and TSST-1...

  14. Apoptosis and Proinflammatory Cytokine Responses of Primary Mouse Microglia and Astrocytes Induced by Human H1N1 and Avian H5N1 Influenza Viruses

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    Gefei Wang; Kangsheng Li; Juan Zhang; Weizhong Li; Gang Xin; Yun Su; Yuanli Gao; Heng Zhang; Guimei Lin; Xiaoyang Jiao

    2008-01-01

    Patients with an influenza virus infection can be complicated by acute encephalopathy and encephalitis. To investigate the immune reactions involved in the neurocomplication, mouse microglia and astrocytes were isolated,infected with human H1N1 and avian H5N1 influenza viruses, and examined for their immune responses. We observed homogeneously distributed viral receptors, sialic acid (SA)-α2,3-Galactose (Gal) and SA-α2,6-Gal, on microglia and astrocytes. Both viruses were replicative and productive in microglia and astrocytes. Virus-induced apoptosis and cytopathy in infected cells were observed at 24 h post-infection (p.i.). Expression of IL-1β, IL-6 and TNF-α mRNA examined at 6 h and 24 h p.i. Was up-regulated, and their expression levels were considerably higher in H5N1 infection. The amounts of secreted proinflammatory IL-1β, IL-6 and TNF-α at 6 h and 24 h p.i. Were also induced, with greater induction by H5N1 infection. This study is the first demonstration that both human H1N1 and avian H5N1 influenza viruses can infect mouse microglia and astrocytes and induce apoptosis, cytopathy, and proinflammatory cytokine production in them in vitro. Our results suggest that the direct cellular damage and the consequences of immunopathological injury in the CNS contribute to the influenza viral pathogenesis.

  15. Decreased expression of glutamate transporters in astrocytes after human traumatic brain injury.

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    van Landeghem, Frank K H; Weiss, Thorsten; Oehmichen, Manfred; von Deimling, Andreas

    2006-10-01

    The primary mechanism for eliminating synaptically released glutamate is uptake by astrocytes. In the present study, we examined whether traumatic brain injury (TBI) affects the cellular expression of glutamate transporters EAAT1 and EAAT2. Morphometrical immunohistochemical analysis demonstrated a predominant expression of EAAT1 and EAAT2 in astrocytes of normal human neocortex (n = 10). Following traumatic injury of human brain (n = 55), the number of EAAT2-positive cells was decreased for a prolonged survival period within the traumatized neocortex and the pericontusional region. GFAP-positive astrocytes decreased in number within the first 24 h. Thereafter, the number of GFAP-positive astrocytes increased again, indicating formation of reactive gliosis. Double immunofluorescence examinations revealed a reduction in absolute numbers of GFAP-positive astrocytes coexpressing EAAT1 or EAAT2 at survival times up to 7 days. In addition, the relative fractions of astrocytes coexpressing glutamate transporters decreased following TBI. We conclude that the posttraumatic reduction in cellular EAAT 1 and EAAT2 expression is predominantly due to degeneration of astrocytes and to downregulation in surviving astrocytes. Our results support the view that reduced glutamate uptake by astrocytes contributes to posttraumatic elevation of extracellular glutamate in humans.

  16. Is amyotrophic lateral sclerosis a primary astrocytic disease?

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    Sica, Roberto E

    2012-12-01

    Amyotrophic lateral sclerosis (ALS) is thought to be due to primary involvement of motor neurons. Pathogenic mechanisms underlying its appearance are relatively well known and include inflammation, excitotoxicity, oxidative stress, endoplasmic reticulum stress, protein damage, genetic abnormalities and type of neuronal death. Although these processes have been investigated in detail in the past two decades none of them appear to be the cause of the illness. In addition several possible environmental agents have been investigated but the results, in every case, were conflicting and therefore inconclusive. However, since the motor neurons display the features of apoptosis in this illness, the possibility remains that the motor neurons die because of a hostile environment, one that is unable to sustain their health, rather than being directly targeted themselves. The above considerations lead to an examination of astrocytes, for these cells play a key role in controlling the environment of neurons. It is known that astrocytes are exquisitely plastic, adapting their metabolism and behaviour to the needs of the neurons they contact. Each population of astrocytes is therefore unique and, were one to be adversely affected at the start of a disease process, the consequences would extend to the neurons that it normally chaperoned. The disturbed relationship might involve inappropriate production and secretion of astrocytic neurotransmitters, defective transport of glutamate and impaired trophic and metabolic support of the motor neurons. In order to explain the spread of weakness and pyramidal signs in ALS patients, which is very often from one group of muscles to a neighbouring one, it is postulated that, within the spinal cord, the brainstem and the motor cortex, the disease-causing process is also spreading-in this case, from one group of astrocytes to its neighbours. A misfolded protein, possibly a prion-like protein, would be a candidate for this type of transmission

  17. Modulation of interleukin-1beta mediated inflammatory response in human astrocytes by flavonoids: implications in neuroprotection.

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    Sharma, Vivek; Mishra, Mamata; Ghosh, Soumya; Tewari, Richa; Basu, Anirban; Seth, Pankaj; Sen, Ellora

    2007-06-15

    The proinflammatory cytokine interleukin-1beta (IL-1beta) contributes to inflammation and neuronal death in CNS injuries and neurodegenerative pathologies, and astrocytes have been implicated as the primary mediators of IL-1beta induced neuronal death. As astrocytes play an important role in supporting the survival and functions of neurons, we investigated the effect of plant flavonoids quercetin and luteolin, with known anti-inflammatory properties in modulating the response of human astrocytes to IL-1beta for therapeutic intervention. Flavonoids significantly decreased the release of reactive oxygen species (ROS) from astrocytes stimulated with IL-1beta. This decrease was accompanied by an increase in expression of superoxide dismutase (SOD-1) and thioredoxin (TRX1)-mediators associated with protection against oxidative stress. Flavonoids not only modulated the expression of astrocytes specific molecules such as glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), and ceruloplasmin (CP) both in the presence and absence of IL-1beta but also decreased the elevated levels of proinflammatory cytokine interleukin-6 (IL-6) and chemokines interleukin-8 (IL-8), interferon-inducible protein (IP-10), monocyte-chemoattractant protein-1 (MCP-1), and RANTES from IL-1beta activated astrocytes. Significant decrease in neuronal apoptosis was observed in neurons cultured in conditioned medium obtained from astrocytes treated with a combination of IL-1beta and flavonoids as compared to that treated with IL-1beta alone. Our result suggests that by (i) enhancing the potential of activated astrocytes to detoxify free radical, (ii) reducing the expression of proinflammatory cytokines and chemokines, and (iii) modulating expression of mediators associated with enhanced physiological activity of astrocyte in response to injury, flavonoids confer (iv) protection against IL-1beta induced astrocyte mediated neuronal damage.

  18. Human astrocytes: secretome profiles of cytokines and chemokines.

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    Sung S Choi

    Full Text Available Astrocytes play a key role in maintenance of neuronal functions in the central nervous system by producing various cytokines, chemokines, and growth factors, which act as a molecular coordinator of neuron-glia communication. At the site of neuroinflammation, astrocyte-derived cytokines and chemokines play both neuroprotective and neurotoxic roles in brain lesions of human neurological diseases. At present, the comprehensive profile of human astrocyte-derived cytokines and chemokines during inflammation remains to be fully characterized. We investigated the cytokine secretome profile of highly purified human astrocytes by using a protein microarray. Non-stimulated human astrocytes in culture expressed eight cytokines, including G-CSF, GM-CSF, GROα (CXCL1, IL-6, IL-8 (CXCL8, MCP-1 (CCL2, MIF and Serpin E1. Following stimulation with IL-1β and TNF-α, activated astrocytes newly produced IL-1β, IL-1ra, TNF-α, IP-10 (CXCL10, MIP-1α (CCL3 and RANTES (CCL5, in addition to the induction of sICAM-1 and complement component 5. Database search indicated that most of cytokines and chemokines produced by non-stimulated and activated astrocytes are direct targets of the transcription factor NF-kB. These results indicated that cultured human astrocytes express a distinct set of NF-kB-target cytokines and chemokines in resting and activated conditions, suggesting that the NF-kB signaling pathway differentially regulates gene expression of cytokines and chemokines in human astrocytes under physiological and inflammatory conditions.

  19. Transcriptomic analyses of primary astrocytes under TNFα treatment

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    Cindy Birck

    2016-03-01

    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.

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

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    Wan, Zhongxiao; Mah, Dorrian; Simtchouk, Svetlana [School of Health and Exercise Sciences, University of British Columbia Okanagan, Kelowna, BC (Canada); Klegeris, Andis [Department of Biology, University of British Columbia Okanagan, Kelowna, BC (Canada); Little, Jonathan P., E-mail: jonathan.little@ubc.ca [School of Health and Exercise Sciences, University of British Columbia Okanagan, Kelowna, BC (Canada)

    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.

  1. Methamphetamine inhibits the glucose uptake by human neurons and astrocytes: stabilization by acetyl-L-carnitine.

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    P M Abdul Muneer

    Full Text Available 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.

  2. Quinolinic acid selectively induces apoptosis of human astrocytes: potential role in AIDS dementia complex

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    Wang Lily

    2005-07-01

    Full Text Available Abstract There is evidence that the kynurenine pathway (KP and particularly one of its end products, quinolinic acid (QUIN play a role in the pathogenesis of several major neuroinflammatory diseases, and more particularly AIDS dementia complex (ADC. We hypothesized that QUIN may be involved in astrocyte apoptosis because: 1 apoptotic astrocytes have been observed in the brains of ADC patients, 2 ADC patients have elevated cerebrospinal fluid QUIN concentrations, and 3 QUIN can induce astrocyte death. Primary cultures of human fetal astrocytes were treated with three pathophysiological concentrations of QUIN. Numeration of apoptotic cells was assessed using double immunocytochemistry for expression of active caspase 3 and for nucleus condensation. We found that treatment of human astrocytes with QUIN induced morphological (cell body shrinking and biochemical changes (nucleus condensation and over-expression of active caspase 3 of apoptosis. After 24 hours of treatment with QUIN 500 nM and 1200 nM respectively 10 and 14% of astrocytes were undergoing apoptosis. This would be expected to lead to a relative lack of trophic support factors with consequent neuronal dysfunction and possibly death. Astroglial apoptosis induced by QUIN provides another potential mechanism for the neurotoxicity of QUIN during ADC.

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

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    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

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

    Science.gov (United States)

    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.

  5. Extensive astrocyte infection is prominent in human immunodeficiency virus-associated dementia.

    Science.gov (United States)

    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.

  6. Effect of quinolinic acid on human astrocytes morphology and functions: implications in Alzheimer's disease

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    Brew Bruce J

    2009-12-01

    Full Text Available Abstract The excitotoxin quinolinic acid (QUIN is synthesized through the kynurenine pathway (KP by activated monocyte lineage cells. QUIN is likely to play a role in the pathogenesis of several major neuroinflammatory diseases including Alzheimer's disease (AD. The presence of reactive astrocytes, astrogliosis, increased oxidative stress and inflammatory cytokines are important pathological hallmarks of AD. We assessed the stimulatory effects of QUIN at low physiological to high excitotoxic concentrations in comparison with the cytokines commonly associated with AD including IFN-γ and TNF-α on primary human astrocytes. We found that QUIN induces IL-1β expression, a key mediator in AD pathogenesis, in human astrocytes. We also explored the effect of QUIN on astrocyte morphology and functions. At low concentrations, QUIN treatment induced concomitantly a marked increase in glial fibrillary acid protein levels and reduction in vimentin levels compared to controls; features consistent with astrogliosis. At pathophysiological concentrations QUIN induced a switch between structural protein expressions in a dose dependent manner, increasing VIM and concomitantly decreasing GFAP expression. Glutamine synthetase (GS activity was used as a functional metabolic test for astrocytes. We found a significant dose-dependent reduction in GS activity following QUIN treatment. All together, this study showed that QUIN is an important factor for astroglial activation, dysregulation and cell death with potential relevance to AD and other neuroinflammatory diseases.

  7. The Antidiabetic Drug Metformin Stimulates Glycolytic Lactate Production in Cultured Primary Rat Astrocytes.

    Science.gov (United States)

    Westhaus, Adrian; Blumrich, Eva Maria; Dringen, Ralf

    2017-01-01

    Metformin is the most frequently used drug for the treatment of type 2 diabetes in humans. However, only little is known about effects of metformin on brain metabolism. To investigate potential metabolic consequences of an exposure of brain cells to metformin, we incubated rat astrocyte-rich primary cultures with this compound. Metformin in concentrations of up to 30 mM did not acutely compromise the viability of astrocytes, but caused a time- and concentration-dependent increase in cellular glucose consumption and lactate production. For acute incubations in the hour range, the presence of 10 mM metformin doubled the glycolytic flux, while already 1 mM metformin doubled glycolytic flux during incubation for 24 h. In addition to metformin, also other guanidino compounds increased astrocytic lactate production. After 4 h of incubation, half-maximal stimulation of glycolysis was observed for metformin, guanidine and phenformin at concentrations of around 3 mM, 3 mM and 30 µM, respectively. The acute stimulation of glycolytic lactate production by metformin was persistent after removal of extracellular metformin and was also observed, if glucose was absent from the incubation medium or replaced by other hexoses. The metformin-induced stimulation of glycolytic flux was not prevented by compound C, an inhibitor of AMP-dependent protein kinase, nor was it additive to the stimulation of glycolytic flux caused by respiratory chain inhibitors. These data demonstrate that the antidiabetic drug metformin has the potential to strongly activate glycolytic lactate production in brain astrocytes.

  8. Glucose-coated gold nanoparticles transfer across human brain endothelium and enter astrocytes in vitro.

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    Radka Gromnicova

    Full Text Available The blood-brain barrier prevents the entry of many therapeutic agents into the brain. Various nanocarriers have been developed to help agents to cross this barrier, but they all have limitations, with regard to tissue-selectivity and their ability to cross the endothelium. This study investigated the potential for 4 nm coated gold nanoparticles to act as selective carriers across human brain endothelium and subsequently to enter astrocytes. The transfer rate of glucose-coated gold nanoparticles across primary human brain endothelium was at least three times faster than across non-brain endothelia. Movement of these nanoparticles occurred across the apical and basal plasma membranes via the cytosol with relatively little vesicular or paracellular migration; antibiotics that interfere with vesicular transport did not block migration. The transfer rate was also dependent on the surface coating of the nanoparticle and incubation temperature. Using a novel 3-dimensional co-culture system, which includes primary human astrocytes and a brain endothelial cell line hCMEC/D3, we demonstrated that the glucose-coated nanoparticles traverse the endothelium, move through the extracellular matrix and localize in astrocytes. The movement of the nanoparticles through the matrix was >10 µm/hour and they appeared in the nuclei of the astrocytes in considerable numbers. These nanoparticles have the correct properties for efficient and selective carriers of therapeutic agents across the blood-brain barrier.

  9. Dexmedetomidine Attenuates Lipopolysaccharide Induced MCP-1 Expression in Primary Astrocyte

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    Liu, Huan; Faez Abdelgawad, Amro

    2017-01-01

    Background. Neuroinflammation which presents as a possible mechanism of delirium is associated with MCP-1, an important proinflammatory factor which is expressed on astrocytes. It is known that dexmedetomidine (DEX) possesses potent anti-inflammatory properties. This study aimed to investigate the potential effects of DEX on the production of MCP-1 in lipopolysaccharide-stimulated astrocytes. Materials and Methods. Astrocytes were treated with LPS (10 ng/ml, 50 ng/ml, 100 ng/ml, and 1000 ng/ml), DEX (500 ng/mL), LPS (100 ng/ml), and DEX (10, 100, and 500 ng/mL) for a duration of three hours; expression levels of MCP-1 were measured by real-time PCR. The double immunofluorescence staining protocol was utilized to determine the expression of α2-adrenoceptors (α2AR) and glial fibrillary acidic protein (GFAP) on astrocytes. Results. Expressions of MCP-1 mRNA in astrocytes were induced dose-dependently by LPS. Administration of DEX significantly inhibited the expression of MCP-1 mRNA (P < 0.001). Double immunofluorescence assay showed that α2AR colocalize with GFAP, which indicates the expression of α2-adrenoceptors in astrocytes. Conclusions. DEX is a potent suppressor of MCP-1 in astrocytes induced with lipopolysaccharide through α2A-adrenergic receptors, which potentially explains its beneficial effects in the treatment of delirium by attenuating neuroinflammation. PMID:28286770

  10. Antidepressant imipramine induces human astrocytes to differentiate into cells with neuronal phenotype.

    Science.gov (United States)

    Cabras, Stefano; Saba, Francesca; Reali, Camilla; Scorciapino, Maria Laura; Sirigu, Annarita; Talani, Giuseppe; Biggio, Giovanni; Sogos, Valeria

    2010-06-01

    Several recent studies have expanded our conception of the role of astrocytes in neurogenesis, proposing that these cells may contribute to this phenomenon not only as a source of trophic substances, but also as stem cells themselves. We recently observed in vitro that human mature astrocytes can be induced to differentiate into cells with a neuronal phenotype. Antidepressant drugs have been shown to increase neurogenesis in the adult rodent hippocampus. In order to better understand the role of astroglia in antidepressant-induced neurogenesis, primary astrocyte cultures were treated with the antidepressant imipramine. Cell morphology was rapidly modified by treatment. In fact, whereas untreated astrocytes showed large, flat morphology, after a few hours of treatment cells exhibited a round-shaped cell body with long, thin processes. The expression of neuronal markers was analysed by immunocytochemistry, Western Blot and RT-PCR at different treatment times. Results showed an increase in neuronal markers such as neurofilament and neuron-specific enolase (NSE), whereas glial fibrillary acidic protein (GFAP) and nestin expression were not significantly modified by treatment. Similar results were obtained with fluoxetine and venlafaxine. Hes1 mRNA significantly increased after 2 h of treatment, suggesting involvement of this transcription factor in this process. These results confirm the role of astrocytes in neurogenesis and suggest that these cells may represent one of the targets of antidepressants.

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

    Energy Technology Data Exchange (ETDEWEB)

    Pizzurro, Daniella M.; Dao, Khoi [Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States); Costa, Lucio G. [Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States); Department of Neuroscience, University of Parma, Parma (Italy)

    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

  12. Astrocytes and the evolution of the human brain.

    Science.gov (United States)

    Robertson, James M

    2014-02-01

    Cells within the astroglial lineage are proposed as the origin of human brain evolution. It is now widely accepted that they direct mammalian fetal neurogenesis, gliogenesis, laminar cytoarchitectonics, synaptic connectivity and neuronal network formation. Furthermore, genetic, anatomical and functional studies have recently identified multiple astrocyte exaptations that strongly suggest a direct relation to the increased size and complexity of the human brain. Copyright © 2013 The Author. Published by Elsevier Ltd.. All rights reserved.

  13. Purification and Characterization of Progenitor and Mature Human Astrocytes Reveals Transcriptional and Functional Differences with Mouse.

    Science.gov (United States)

    Zhang, Ye; Sloan, Steven A; Clarke, Laura E; Caneda, Christine; Plaza, Colton A; Blumenthal, Paul D; Vogel, Hannes; Steinberg, Gary K; Edwards, Michael S B; Li, Gordon; Duncan, John A; Cheshier, Samuel H; Shuer, Lawrence M; Chang, Edward F; Grant, Gerald A; Gephart, Melanie G Hayden; Barres, Ben A

    2016-01-01

    The functional and molecular similarities and distinctions between human and murine astrocytes are poorly understood. Here, we report the development of an immunopanning method to acutely purify astrocytes from fetal, juvenile, and adult human brains and to maintain these cells in serum-free cultures. We found that human astrocytes have abilities similar to those of murine astrocytes in promoting neuronal survival, inducing functional synapse formation, and engulfing synaptosomes. In contrast to existing observations in mice, we found that mature human astrocytes respond robustly to glutamate. Next, we performed RNA sequencing of healthy human astrocytes along with astrocytes from epileptic and tumor foci and compared these to human neurons, oligodendrocytes, microglia, and endothelial cells (available at http://www.brainrnaseq.org). With these profiles, we identified novel human-specific astrocyte genes and discovered a transcriptome-wide transformation between astrocyte precursor cells and mature post-mitotic astrocytes. These data represent some of the first cell-type-specific molecular profiles of the healthy and diseased human brain.

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

    Directory of Open Access Journals (Sweden)

    Stephen J A Davies

    Full Text Available 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

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

    Science.gov (United States)

    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

  16. Evaluation of PFOS-mediated neurotoxicity in rat primary neurons and astrocytes cultured separately or in co-culture.

    Science.gov (United States)

    Li, Zhenwei; Liu, Qi; Liu, Chang; Li, Chunna; Li, Yachen; Li, Shuangyue; Liu, Xiaohui; Shao, Jing

    2017-02-01

    Perfluorooctane sulfonate (PFOS) is a potential neurotoxicant reported by epidemiological investigations and experimental studies, while the underlying mechanisms are still unclear. Astrocytes not only support for the construction of neurons, but also conduct neuronal functions through glutamate-glutamine cycle in astrocyte-neuron crosstalk. In the present study, the effect of PFOS exposure on rat primary hippocampal neurons or cortex astrocytes was evaluated. Then the role of the astrocytes in PFOS-induced toxic effect on neurons was explored with astrocyte-neuron co-culture system. Exposure of rat primary hippocampal neurons to PFOS has led to oxidation-antioxidation imbalance, increased apoptosis and abnormal autophagy. The adverse effect of PFOS on rat primary cortex astrocytes manifested in the form of altered extracellular glutamate and glutamine concentrations, decreased glutamine synthase activity, as well as decreased gene expression of glutamine synthase, glutamate transporters and glutamine transporters in the glutamate-glutamine cycle. Especially, the alleviation of PFOS-inhibited neurite outgrowth in neurons could be observed in astrocyte-neuron co-culture system, though the ability of astrocytes in fostering neurite outgrowth was affected by PFOS. These results indicated that both astrocytes and neurons might be the targets of PFOS-induced neurotoxicity, and astrocytes could protect against PFOS-inhibited neurite outgrowth in primary cultured neurons. Our research might render some information in explaining the mechanisms of PFOS-induced neurotoxicity. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Overexpression of the human major vault protein in astrocytic brain tumor cells.

    Science.gov (United States)

    Berger, W; Spiegl-Kreinecker, S; Buchroithner, J; Elbling, L; Pirker, C; Fischer, J; Micksche, M

    2001-11-01

    Evidence has shown that the major human vault protein (MVP), which is identical to lung resistance-related protein (LRP), may be causally involved in a special type of multidrug resistance (MDR). The purpose of this study was to investigate the expression and cellular localization of MVP in cells derived from brain tumors and other tumors of neuroectodermal origin. Using both established cell lines (n = 22) and primary explants (n = 30), we show that a distinct overexpression of the MVP gene at the mRNA (RT-PCR) and protein (Western blot) levels is a characteristic feature of cells derived from astrocytic brain tumors. Primary cultures obtained from meningioma specimens also expressed high MVP levels, in contrast to neuroblastoma and medulloblastoma cells, which rarely contained detectable amounts of MVP. Normal human astrocytes cultured in vitro expressed MVP, although at low amounts compared with most malignant cell types. Basal MVP expression correlated with resistance against diverse antineoplastic drugs including anthracyclins, cisplatin and etoposide. By Western blot, MVP was also detected in all tumor samples taken from 7 glioma and 3 meningioma patients. Taken together, these data suggest overexpression of MVP as one explanation for the low efficacy of chemotherapeutic treatment of astrocytic brain tumors. Copyright 2001 Wiley-Liss, Inc.

  18. In vitro differentiation of cultured human CD34+ cells into astrocytes

    Directory of Open Access Journals (Sweden)

    Katari Venkatesh

    2013-01-01

    Full Text Available Background: Astrocytes are abundantly present as glial cells in the brain and play an important role in the regenerative processes. The possible role of stem cell derived astrocytes in the spinal cord injuries is possible related to their influence at the synaptic junctions. Aim: The present study is focused on in vitro differentiation of cultured human CD34+ cells into astrocytes. Materials and Methods: Granulocyte-colony stimulating factor mobilized human CD34+ cells were isolated from peripheral blood using apheresis method from a donor. These cells were further purified by fluorescence-activated cell sorting and cultured in Dulbecco′s modified eagle′s medium. Thus, cultured cells were induced with astrocyte defined medium (ADM and in the differentiated astrocytes serine/threonine protein kinases (STPK and glutamine synthetase (GLUL activities were estimated. The expression of glial fibrillary acidic protein (GFAP and GLUL were confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR. Results: The cultured human CD34+ cells differentiated into astrocytes after 11 h of incubation in ADM. The RT-PCR experiment showed the expression of GLUL (1.5 kb and GFAP (2.9 kb in differentiated astrocytes. The high enzyme activities of GLUL and STPK in differentiated astrocytes compared with cultured human CD34+ cells confirmed astrocyte formation. Conclusion: In the present study, in vitro differentiation of stem cells with retinoic acid induction may result in the formation of astrocytes.

  19. Human Stem Cell-Derived Spinal Cord Astrocytes with Defined Mature or Reactive Phenotypes

    Directory of Open Access Journals (Sweden)

    Laurent Roybon

    2013-09-01

    Full Text Available Differentiation of astrocytes from human stem cells has significant potential for analysis of their role in normal brain function and disease, but existing protocols generate only immature astrocytes. Using early neuralization, we generated spinal cord astrocytes from mouse or human embryonic or induced pluripotent stem cells with high efficiency. Remarkably, short exposure to fibroblast growth factor 1 (FGF1 or FGF2 was sufficient to direct these astrocytes selectively toward a mature quiescent phenotype, as judged by both marker expression and functional analysis. In contrast, tumor necrosis factor alpha and interleukin-1β, but not FGFs, induced multiple elements of a reactive inflammatory phenotype but did not affect maturation. These phenotypically defined, scalable populations of spinal cord astrocytes will be important both for studying normal astrocyte function and for modeling human pathological processes in vitro.

  20. [A protocol for primary dissociated astrocyte and neuron co-culture].

    Science.gov (United States)

    Shi, Ying; Zhou, Mi; Jiang, Min

    2013-02-25

    Cultured primary hippocampal neurons are ideal tool for investigating the subcellular localization and trafficking of neuronal proteins. The aim of the present study was to establish a method to co-culture hippocampal neurons and cortical astrocytes, which would guarantee well conditions of neurons. Newborn Sprague-Dawley (SD) rats were sacrificed by decapitation. The cortex of cerebrum was cut into pieces, and the cortical tissue was digested with trypsin. The liquid suspension of single cells was planted onto a 25 cm² culture flask. On the fourth day of culture, the tissue cells except astrocytes were removed by intensive agitation of culture flask. Purified astrocytes were allowed to grow continuously until they reached most area of flask. At this time point, we replaced the culture media with neuronal cell media containing cytarabine, and planted the primary culture of rat hippocampal neurons onto the feed layer of cortical astrocytes. The microscopic observation results showed that, the astrocytes evenly grew without obvious boundaries between each other, and exhibited good purity. The co-cultured hippocampal neurons were in good condition, developed intertwined network of axons and dendrites, lived for a long time, and could tolerate gene transfection. Above all, this method is relatively simple from a technical point of view, yet provides healthy and reliable neuronal culture.

  1. Role of carnitine palmitoyltransferase I in the control of ketogenesis in primary cultures of rat astrocytes.

    Science.gov (United States)

    Blázquez, C; Sánchez, C; Velasco, G; Guzmán, M

    1998-10-01

    The role of carnitine palmitoyltransferase I (CPT-I) in the control of ketogenesis was studied in primary cultures of rat astrocytes. Ketone bodies were the major product of [14C]palmitate oxidation by cultured astrocytes, whereas CO2 made a minor contribution to the total oxidation products. Using tetradecylglycidate as a specific, cell-permeable inhibitor of CPT-I, a flux control coefficient of 0.77 +/- 0.07 was calculated for CPT-I over the flux of [14C]palmitate to ketone bodies. CPT-I from astrocytes was sensitive to malonyl-CoA (IC50 = 3.4 +/- 0.8 microM) and cross-reacted on western blots with an antibody raised against liver CPT-I. On the other hand, astrocytes expressed significant acetyl-CoA carboxylase (ACC) activity, and consequently they contained considerable amounts of malonyl-CoA. Western blot analysis of ACC isoforms showed that ACC in astrocytes--like in neurons, liver, and white adipose tissue--mostly comprised the 265-kDa isoform, whereas the 280-kDa isoform--which was highly expressed in skeletal muscle--showed much lower abundance. Forskolin was used as a tool to study the modulation of the ketogenic pathway in astrocytes. Thus, forskolin decreased in parallel ACC activity and intracellular malonyl-CoA levels, whereas it stimulated CPT-I activity and [14C]palmitate oxidation to both ketone bodies and CO2. Results show that in cultured astrocytes (a) CPT-I exerts a very high degree of control over ketogenesis from palmitate, (b) the ACC/malonyl-CoA/CPT-I system is similar to that of liver, and (c) the ACC/malonyl-CoA/CPT-I system is subject to regulation by cyclic AMP.

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

    Science.gov (United States)

    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.

  3. A Cellular Star Atlas: Using Astrocytes from Human Pluripotent Stem Cells for Disease Studies

    Directory of Open Access Journals (Sweden)

    Robert eKrencik

    2013-03-01

    Full Text Available What roles do astrocytes play in human disease? This question remains unanswered for nearly every human neurological disorder. Yet, because of their abundance and complexity astrocytes can impact neurological function in many ways. The differentiation of human pluripotent stem cells (hPSCs into neuronal and glial subtypes, including astrocytes, is becoming routine, thus their use as tools for modeling neurodevelopment and disease will provide one important approach to answer this question. When designing experiments, careful consideration must be given to choosing paradigms for differentiation, maturation, and functional analysis of these temporally asynchronous cellular populations in culture. In the case of astrocytes, they display heterogeneous characteristics depending upon species of origin, brain region, developmental stage, environmental factors, and disease states, all of which may render experimental results highly variable. In this review, challenges and future directions are discussed for using hPSC-derived astroglial progenitors and mature astrocytes for neurodevelopmental studies with a focus on exploring human astrocyte effects upon neuronal function. As new technologies emerge to measure the functions of astrocytes in vitro and in vivo, there is also a need for a standardized source of human astrocytes that are most relevant to the diseases of interest.

  4. DJ-1 immunoreactivity in human brain astrocytes is dependent on infarct presence and infarct age.

    Science.gov (United States)

    Mullett, Steven J; Hamilton, Ronald L; Hinkle, David A

    2009-04-01

    DJ-1 is a protein with anti-oxidative stress and anti-apoptotic properties that is abundantly expressed in reactive CNS astrocytes in chronic neurodegenerative disorders such as Parkinson's disease (PD), Alzheimer's disease (AD), and Pick's disease. Genetic mutations which eliminate DJ-1 expression in humans are sufficient to produce an early-onset form of familial PD, PARK7, suggesting that DJ-1 is a critical component of the neuroprotective arsenal of the brain. Previous studies in parkinsonism/dementia brain tissues have revealed that reactive astrocytes within and surrounding incidentally identified infarcts were often robustly immunoreactive for DJ-1, especially if the infarcts showed histological features consistent with older age. Given this, we sought to evaluate astrocytic DJ-1 expression in human stroke more extensively, and with a particular emphasis on determining whether immunohistochemical DJ-1 expression in astrocytes correlates with histological infarct age. The studies presented here show that DJ-1 is abundantly expressed in reactive infarct region astrocytes in both gray and white matter, that subacute and chronic infarct region astrocytes are much more robustly DJ-1+ than are acute infarct and non-infarct region astrocytes, and that DJ-1 staining intensity in astrocytes generally correlates with that of the reactive astrocyte marker GFAP. Confocal imaging of DJ-1 and GFAP dual-labelled human brain sections were used to confirm the localization to and expression of DJ-1 in astrocytes. Neuronal DJ-1 staining was minimal under all infarct and non-infarct conditions. Our data support the conclusion that the major cellular DJ-1 response to stroke in the human brain is astrocytic, and that there is a temporal correlation between DJ-1 expression in these cells and advanced infarct age.

  5. A novel gliotic P2 receptor mediating cyclooxygenase-2 induction in rat and human astrocytes.

    Science.gov (United States)

    Brambilla, R; Ceruti, S; Malorni, W; Cattabeni, F; Abbracchio, M P

    2000-07-01

    In astrocytic cultures maintained in vitro, a brief challenge with the ATP analog alpha,beta methyleneATP (alpha,betameATP) results, 3 days later, in marked elongation of astrocytic processes, an event that resembles the astrocytic hypertrophy known to occur in vivo during reactive astrogliosis. alpha,beta meATP-induced effects were observed in primary astrocytes obtained from both rat striatum and cortex (a brain area highly involved in chronic neurodegenerative pathologies), as well as in human astrocytoma cells (ADF cells). Purine-induced gliosis could be reversed by the non-selective P2X/P2Y receptor antagonist pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS), but not by oxidized ATP (an antagonist of the P2X(7) receptor), in line with previous studies of our laboratory suggesting the involvement of a P2Y receptor subtype. Induction of reactive gliosis was preceded by increased expression of cyclooxygenase-2 (COX-2), an enzyme whose excessive activation has been implicated in both acute and chronic neurodegenerative diseases. The selective COX-2 inhibitor NS-398 prevented both purine-induced astrogliosis and the associated COX-2 induction, suggesting that inhibition of the transcription of the COX-2 gene may also contribute to the anti-inflammatory properties of this agent. Significant blockade of both alpha,beta meATP-mediated reactive gliosis and COX-2 induction was also observed with PPADS. These data suggest that COX-2 mediates P2Y receptor-induced reactive astrogliosis, and that antagonists selective for this receptor subtype may represent a novel class of anti-inflammatory agents of potential interest in acute and chronic neurological disorders characterized by an inflammatory component and reactive gliosis.

  6. The histone deacetylase inhibitor suberoylanilide hydroxamic acid attenuates human astrocyte neurotoxicity induced by interferon-γ

    Directory of Open Access Journals (Sweden)

    Hashioka Sadayuki

    2012-05-01

    Full Text Available Abstract Backgrounds Increasing evidence shows that the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA possesses potent anti-inflammatory and immunomodulatory properties. It is tempting to evaluate the potential of SAHA as a therapeutic agent in various neuroinflammatory and neurodegenerative disorders. Methods We examined the effects of SAHA on interferon (IFN-γ-induced neurotoxicity of human astrocytes and on IFN-γ-induced phosphorylation of signal transducer and activator of transcription (STAT 3 in human astrocytes. We also studied the effects of SAHA on the astrocytic production of two representative IFN-γ-inducible inflammatory molecules, namely IFN-γ-inducible T cell α chemoattractant (I-TAC and intercellular adhesion molecule-1 (ICAM-1. Results SAHA significantly attenuated the toxicity of astrocytes activated by IFN-γ towards SH-SY5Y human neuronal cells. In the IFN-γ-activated astrocytes, SAHA reduced the STAT3 phosphorylation. SAHA also inhibited the IFN-γ-induced astrocytic production of I-TAC, but not ICAM-1. These results indicate that SAHA suppresses IFN-γ-induced neurotoxicity of human astrocytes through inhibition of the STAT3 signaling pathway. Conclusion Due to its anti-neurotoxic and anti-inflammatory properties, SAHA appears to have the therapeutic or preventive potential for a wide range of neuroinflammatory disorders associated with activated astrocytes.

  7. Human optic nerve head astrocytes culture in vitro:Ⅰ. the primary culture and passage%人眼视神经乳头星形胶质细胞体外培养:Ⅰ.原代培养和传代试验

    Institute of Scientific and Technical Information of China (English)

    戴惟葭; Shelley Culp-Stewwart; Anna Cheng; John Flanagan; C Ross Ethier

    2008-01-01

    AIM: To culture astrocytes from human donor eyes in order to understand the function of astrocytes in remodelling events in the glaucomatous optic nerve head (ONH).METHODS: Primary cultures were prepared by explantation of human ONH tissue in order to get astrocytes. Laminar criborsa (LC) cells were prepared concurrently for comparison. Astrocyte cultures could be separated from LC cells by selecting medium.Similar procedures were used for LC.RESULTS: Primary ceils grew from human optic nerve head explants 4-8 weeks after explantation. Astrocytes had different morphologies and growth characteristics from LC cells. Type 1B astrocyte cells could grow in medium without FBS. Purified cultures were obtained by second passage and could be harvested by third to fifth passage, which were prepared to use for further study, including being characterized by positive glial fibrillary acidic protein (GFAP) and neural cell adhesion molecule (NCAM) staining.CONCLUSION: Precise dissection of fragment is the most important step to get clear explants for primary culture. Economic and rapid method could be useful to select cells by different mediums, which will help us to get more purified cells for further study.%目的:探索人眼视神经乳头星形胶质细胞体外培养的方法,为进一步研究星形胶质细胞在青光眼性视神经病变中的作用打下基础.方法:取材新鲜人眼视神经乳头组织和筛板组织,进行星形胶质细胞和筛板细胞的体外培养与传代试验.结果:组织块培养4~8wk后,原代细胞开始生长,星形胶质细胞在形态学和生长特性上与筛板细胞明显不同, β1型星形胶质细胞可以在无血清的培养液中生长良好,通过无血清培养液选择性培养可以在第二代传代过程中分离出纯化的星形胶质细胞,并可在第三至第四代收获大量细胞以备后续的研究.结论:精细准确的组织解剖分离对于获得纯化细胞至关重要,采用无血清培养液选

  8. Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M. (Cleveland Clinic Foundation, OH (USA))

    1990-08-01

    The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures.

  9. Astrocytes derived from trisomic human embryonic stem cells express markers of astrocytic cancer cells and premalignant stem-like progenitors

    Directory of Open Access Journals (Sweden)

    Iverson Linda E

    2010-04-01

    Full Text Available Abstract Background Trisomic variants of human embryonic stem cells (hESCs arise spontaneously in culture. Although trisomic hESCs share many properties with diploid hESCs, they also exhibit features of cancer stem cells. Since most hESC-based therapies will utilize differentiated derivatives, it is imperative to investigate the potential of trisomic hESCs to undergo malignant transformation during differentiation prior to their use in the clinical setting. Methods Diploid and trisomic hESCs were differentiated into astrocytic progenitors cells (APCs, RNA extracted and hybridized to human exon-specific microarrays. Global gene expression profiles of diploid and trisomic APCs were compared to that of an astrocytoma cell line and glioblastoma samples, analyzed by others, using the same microarray platform. Results Bioinformatic analysis of microarray data indicates that differentiated trisomic APCs exhibit global expression profiles with similarities to the malignant astrocytoma cell line. An analogous trend is observed in comparison to glioblastoma samples indicating that trisomic APCs express markers of astrocytic cancer cells. The analysis also allowed identification of transcripts predicted to be differentially expressed in brain tumor stem cells. These data indicate that in vitro differentiation of trisomic hESCs along astrocytic pathways give rise to cells exhibiting properties of premalignant astrocytic stem/progenitor cells. Conclusions Given their occult nature, opportunities to study premalignant stem/progenitor cells in human have been few. The ability to propagate and direct the differentiation of aneuploid hESCs provides a powerful in vitro system for investigating biological properties of human cells exhibiting features of premalignant stem cells. This in vitro culture system can be used to elucidate changes in gene expression occurring enroute to malignant transformation and to identify molecular markers of cancer stem

  10. 3-bromopyruvate inhibits glycolysis, depletes cellular glutathione, and compromises the viability of cultured primary rat astrocytes.

    Science.gov (United States)

    Ehrke, Eric; Arend, Christian; Dringen, Ralf

    2015-07-01

    The pyruvate analogue 3-bromopyruvate (3-BP) is an electrophilic alkylator that is considered a promising anticancer drug because it has been shown to kill cancer cells efficiently while having little toxic effect on nontumor cells. To test for potential adverse effects of 3-BP on brain cells, we exposed cultured primary rat astrocytes to 3-BP and investigated the effects of this compound on cell viability, glucose metabolism, and glutathione (GSH) content. The presence of 3-BP severely compromised cell viability and slowed cellular glucose consumption and lactate production in a time- and concentration-dependent manner, with half-maximal effects observed at about 100 µM 3-BP after 4 hr of incubation. The cellular hexokinase activity was not affected in 3-BP-treated astrocytes, whereas within 30 min after application of 3-BP the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was inhibited, and cellular GSH content was depleted in a concentration-dependent manner, with half-maximal effects observed at about 30 µM 3-BP. The depletion of cellular GSH after exposure to 100 µM 3-BP was not prevented by the presence of 10 mM of the monocarboxylates lactate or pyruvate, suggesting that 3-BP is not taken up into astrocytes predominantly by monocarboxylate transporters. The data suggest that inhibition of glycolysis by inactivation of GAPDH and GSH depletion contributes to the toxicity that was observed for 3-BP-treated cultured astrocytes.

  11. Direct Generation of Human Neuronal Cells from Adult Astrocytes by Small Molecules

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    Longfei Gao

    2017-03-01

    Full Text Available Astrocytes, due to the proximity to neuronal lineage and capability to proliferate, are ideal starting cells to regenerate neurons. Human fetal astrocytes have been successfully converted into neuronal cells by small molecules, which offered a broader range of further applications than transcription factor-mediated neuronal reprogramming. Here we report that human adult astrocytes could also be converted into neuronal cells by a different set of small molecules. These induced cells exhibited typical neuronal morphologies, expressed neuronal markers, and displayed neuronal electrophysiological properties. Genome-wide RNA-sequencing analysis showed that the global gene expression profile of induced neuronal cells resembled that of human embryonic stem cell-differentiated neurons. When transplanted into post-natal mouse brains, these induced neuronal cells could survive and become electrophysiologically mature. Altogether, our study provides a strategy to directly generate transgene-free neuronal cells from human adult astrocytes by small molecules.

  12. Generation of primary cultures of bovine brain endothelial cells and setup of cocultures with rat astrocytes

    DEFF Research Database (Denmark)

    Helms, Hans C; Brodin, Birger

    2014-01-01

    In vitro models of the blood-brain barrier are useful tools to study blood-brain barrier function as well as drug permeation from the systemic circulation to the brain parenchyma. However, a large number of the available in vitro models fail to reflect the tightness of the in vivo blood-brain...... barrier. The present protocol describes the setup of an in vitro coculture model based on primary cultures of endothelial cells from bovine brain microvessels and primary cultures of rat astrocytes. The model displays a high electrical tightness and expresses blood-brain barrier marker proteins....

  13. Astrocyte differentiation of human pluripotent stem cells: new tools for neurological disorder research

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    Abinaya Chandrasekaran

    2016-09-01

    Full Text Available Astrocytes have a central role in brain development and function, and so have gained increasing attention over the past two decades. Consequently, our knowledge about their origin, differentiation and function has increased significantly, with new research showing that astrocytes cultured alone or co-cultured with neurons have the potential to improve our understanding of various central nervous system (CNS diseases, such as Amyotrophic lateral sclerosis, Alzheimer’s disease or Alexander disease. The generation of astrocytes derived from pluripotent stem cells (PSCs opens up a new area for studying neurologic diseases in vitro; these models could be exploited to identify and validate potential drugs by detecting adverse effects in the early stages of drug development. However, as it is now known that a range of astrocyte populations exist in the brain, it will be important in vitro to develop standardized protocols for the in vitro generation of astrocyte subsets with defined maturity status and phenotypic properties. This will then open new possibilities for co-cultures with neurons and the generation of neural organoids for research purposes. The aim of this review article is to compare and summarize the currently available protocols and their strategies to generate human astrocytes from PSCs. Furthermore, we discuss the potential role of human-induced PSCs derived astrocytes in disease modeling.

  14. Staphylococcus epidermidis polysaccharide intercellular adhesin induces IL-8 expression in human astrocytes via a mechanism involving TLR2.

    LENUS (Irish Health Repository)

    Stevens, Niall T

    2009-03-01

    Staphylococcus epidermidis is an opportunistic biofilm-forming pathogen associated with neurosurgical device-related meningitis. Expression of the polysaccharide intercellular adhesin (PIA) on its surface promotes S. epidermidis biofilm formation. Here we investigated the pro-inflammatory properties of PIA against primary and transformed human astrocytes. PIA induced IL-8 expression in a dose- and\\/or time-dependent manner from U373 MG cells and primary normal human astrocytes. This effect was inhibited by depletion of N-acetyl-beta-d-glucosamine polymer from the PIA preparation with Lycopersicon esculentum lectin or sodium meta-periodate. Expression of dominant-negative versions of the TLR2 and TLR4 adaptor proteins MyD88 and Mal in U373 MG cells inhibited PIA-induced IL-8 production. Blocking IL-1 had no effect. PIA failed to induce IL-8 production from HEK293 cells stably expressing TLR4. However, in U373 MG cells which express TLR2, neutralization of TLR2 impaired PIA-induced IL-8 production. In addition to IL-8, PIA also induced expression of other cytokines from U373 MG cells including IL-6 and MCP-1. These data implicate PIA as an important immunogenic component of the S. epidermidis biofilm that can regulate pro-inflammatory cytokine production from human astrocytes, in part, via TLR2.

  15. Human glial chimeric mice reveal astrocytic dependence of JC virus infection

    DEFF Research Database (Denmark)

    Kondo, Yoichi; Windrem, Martha S; Zou, Lisa;

    2014-01-01

    with humanized white matter by engrafting human glial progenitor cells (GPCs) into neonatal immunodeficient and myelin-deficient mice. Intracerebral delivery of JCV resulted in infection and subsequent demyelination of these chimeric mice. Human GPCs and astrocytes were infected more readily than...... oligodendrocytes, and viral replication was noted primarily in human astrocytes and GPCs rather than oligodendrocytes, which instead expressed early viral T antigens and exhibited apoptotic death. Engraftment of human GPCs in normally myelinated and immunodeficient mice resulted in humanized white matter...... that was chimeric for human astrocytes and GPCs. JCV effectively propagated in these mice, which indicates that astroglial infection is sufficient for JCV spread. Sequencing revealed progressive mutation of the JCV capsid protein VP1 after infection, suggesting that PML may evolve with active infection...

  16. WIN 55,212-2, agonist of cannabinoid receptors, prevents amyloid β1-42 effects on astrocytes in primary culture.

    Science.gov (United States)

    Aguirre-Rueda, Diana; Guerra-Ojeda, Sol; Aldasoro, Martin; Iradi, Antonio; Obrador, Elena; Mauricio, Maria D; Vila, Jose M; 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.

  17. WIN 55,212-2, agonist of cannabinoid receptors, prevents amyloid β1-42 effects on astrocytes in primary culture.

    Directory of Open Access Journals (Sweden)

    Diana Aguirre-Rueda

    Full Text Available 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.

  18. Exposure to cell phone radiation up-regulates apoptosis genes in primary cultures of neurons and astrocytes.

    Science.gov (United States)

    Zhao, Tian-Yong; Zou, Shi-Ping; Knapp, Pamela E

    2007-01-22

    The health effects of cell phone radiation exposure are a growing public concern. This study investigated whether expression of genes related to cell death pathways are dysregulated in primary cultured neurons and astrocytes by exposure to a working Global System for Mobile Communication (GSM) cell phone rated at a frequency of 1900MHz. Primary cultures were exposed to cell phone emissions for 2h. We used array analysis and real-time RT-PCR to show up-regulation of caspase-2, caspase-6 and Asc (apoptosis associated speck-like protein containing a card) gene expression in neurons and astrocytes. Up-regulation occurred in both "on" and "stand-by" modes in neurons, but only in "on" mode in astrocytes. Additionally, astrocytes showed up-regulation of the Bax gene. The effects are specific since up-regulation was not seen for other genes associated with apoptosis, such as caspase-9 in either neurons or astrocytes, or Bax in neurons. The results show that even relatively short-term exposure to cell phone radiofrequency emissions can up-regulate elements of apoptotic pathways in cells derived from the brain, and that neurons appear to be more sensitive to this effect than astrocytes.

  19. Nanosecond UV lasers stimulate transient Ca2+ elevations in human hNT astrocytes

    Science.gov (United States)

    Raos, B. J.; Graham, E. S.; Unsworth, C. P.

    2017-06-01

    Objective. Astrocytes respond to various stimuli resulting in intracellular Ca2+ signals that can propagate through organized functional networks. Recent literature calls for the development of techniques that can stimulate astrocytes in a fast and highly localized manner to emulate more closely the characteristics of astrocytic Ca2+ signals in vivo. Approach. In this article we demonstrate, for the first time, how nanosecond UV lasers are capable of reproducibly stimulating Ca2+ transients in human hNT astrocytes. Main results. We report that laser pulses with a beam energy of 4-29 µJ generate transient increases in cytosolic Ca2+. These Ca2+ transients then propagate to adjacent astrocytes as intercellular Ca2+ waves. Significance. We propose that nanosecond laser stimulation provides a valuable tool for enabling the study of Ca2+ dynamics in human astrocytes at both a single cell and network level. Compared to previously developed techniques nanosecond laser stimulation has the advantage of not requiring loading of photo-caged or -sensitising agents, is non-contact, enables stimulation with a high spatiotemporal resolution and is comparatively cost effective.

  20. 6-Hydroxydopamine-induced glutathione alteration occurs via glutathione enzyme system in primary cultured astrocytes

    Institute of Scientific and Technical Information of China (English)

    Ji ZHANG; Jun HU; Jian-hua DING; Hong-hong YAO; Gang HU

    2005-01-01

    Aim: To define the role of enzymes involved in glutathione metabolism in 6-hydroxydopamine (6-OHDA)-induced glutathione alteration in primary cultured astrocytes.Methods: Total glutathione (GSx) levels were determined using the modified enzymatic microtiter plate assay.The mRNA levels ofγ-glutamylcysteine synthetase (γGCS), γ-glutamyltransferase (γGT), glutathione peroxidase (GPx), GR (glutathione reductase), and glutathione transferases (GST) were determined using RT-PCR.γGT activity was determined using γGT assay kits.Results: In primary cultured astrocytes, 6-OHDA induced a significant elevation of cellular GSx levels after treatment for 24 h.However, the GSx levels decreased after 24 h and the values were even lower than the value in the control group without 6-OHDA at 48 h.RT-PCR data showed that the mRNA levels of γGCS, the ratelimiting enzyme of γ-L-glutamyl-L-cysteinylglycine (GSH) synthesis, were increased by 6-OHDA after treatment for 24 h and 48 h; the mRNA levels of GPx, GR, and GST did not alter in 6-OHDA-treated astrocytes after treatment for 24 h and 48 h; and 6-OHDA increased the mRNA levels and the activity of γGT after treatment for 48 h,which induced a decrease in GSx levels, despite the up-regulation of γGCS after exposure to 6-OHDA for 48 h.Conclusion: The change in γGCS correlated with the increase in GSH levels induced by 6-OHDA after treatment for 24 h.GSx levels decreased because of increased γGT mRNA levels and γGT activity induced by 6-OHDA after treatment for 48 h.

  1. An isogenic blood-brain barrier model comprising brain endothelial cells, astrocytes, and neurons derived from human induced pluripotent stem cells.

    Science.gov (United States)

    Canfield, Scott G; Stebbins, Matthew J; Morales, Bethsymarie Soto; Asai, Shusaku W; Vatine, Gad D; Svendsen, Clive N; Palecek, Sean P; Shusta, Eric V

    2017-03-01

    The blood-brain barrier (BBB) is critical in maintaining a physical and metabolic barrier between the blood and the brain. The BBB consists of brain microvascular endothelial cells (BMECs) that line the brain vasculature and combine with astrocytes, neurons and pericytes to form the neurovascular unit. We hypothesized that astrocytes and neurons generated from human-induced pluripotent stem cells (iPSCs) could induce BBB phenotypes in iPSC-derived BMECs, creating a robust multicellular human BBB model. To this end, iPSCs were used to form neural progenitor-like EZ-spheres, which were in turn differentiated to neurons and astrocytes, enabling facile neural cell generation. The iPSC-derived astrocytes and neurons induced barrier tightening in primary rat BMECs indicating their BBB inductive capacity. When co-cultured with human iPSC-derived BMECs, the iPSC-derived neurons and astrocytes significantly elevated trans-endothelial electrical resistance, reduced passive permeability, and improved tight junction continuity in the BMEC cell population, while p-glycoprotein efflux transporter activity was unchanged. A physiologically relevant neural cell mixture of one neuron: three astrocytes yielded optimal BMEC induction properties. Finally, an isogenic multicellular BBB model was successfully demonstrated employing BMECs, astrocytes, and neurons from the same donor iPSC source. It is anticipated that such an isogenic facsimile of the human BBB could have applications in furthering understanding the cellular interplay of the neurovascular unit in both healthy and diseased humans. Read the Editorial Highlight for this article on page 843. © 2016 International Society for Neurochemistry.

  2. Network analysis of human glaucomatous optic nerve head astrocytes

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    Bhattacharya Sanjoy K

    2009-05-01

    Full Text Available Abstract Background Astrocyte activation is a characteristic response to injury in the central nervous system, and can be either neurotoxic or neuroprotective, while the regulation of both roles remains elusive. Methods To decipher the regulatory elements controlling astrocyte-mediated neurotoxicity in glaucoma, we conducted a systems-level functional analysis of gene expression, proteomic and genetic data associated with reactive optic nerve head astrocytes (ONHAs. Results Our reconstruction of the molecular interactions affected by glaucoma revealed multi-domain biological networks controlling activation of ONHAs at the level of intercellular stimuli, intracellular signaling and core effectors. The analysis revealed that synergistic action of the transcription factors AP-1, vitamin D receptor and Nuclear Factor-kappaB in cross-activation of multiple pathways, including inflammatory cytokines, complement, clusterin, ephrins, and multiple metabolic pathways. We found that the products of over two thirds of genes linked to glaucoma by genetic analysis can be functionally interconnected into one epistatic network via experimentally-validated interactions. Finally, we built and analyzed an integrative disease pathology network from a combined set of genes revealed in genetic studies, genes differentially expressed in glaucoma and closely connected genes/proteins in the interactome. Conclusion Our results suggest several key biological network modules that are involved in regulating neurotoxicity of reactive astrocytes in glaucoma, and comprise potential targets for cell-based therapy.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  5. Hypoxia Epigenetically Confers Astrocytic Differentiation Potential on Human Pluripotent Cell-Derived Neural Precursor Cells

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    Tetsuro Yasui

    2017-06-01

    Full Text Available Human neural precursor cells (hNPCs derived from pluripotent stem cells display a high propensity for neuronal differentiation, but they require long-term culturing to differentiate efficiently into astrocytes. The mechanisms underlying this biased fate specification of hNPCs remain elusive. Here, we show that hypoxia confers astrocytic differentiation potential on hNPCs through epigenetic gene regulation, and that this was achieved by cooperation between hypoxia-inducible factor 1α and Notch signaling, accompanied by a reduction of DNA methylation level in the promoter region of a typical astrocyte-specific gene, Glial fibrillary acidic protein. Furthermore, we found that this hypoxic culture condition could be applied to rapid generation of astrocytes from Rett syndrome patient-derived hNPCs, and that these astrocytes impaired neuronal development. Thus, our findings shed further light on the molecular mechanisms regulating hNPC differentiation and provide attractive tools for the development of therapeutic strategies for treating astrocyte-mediated neurological disorders.

  6. Changes in the Transcriptome of Human Astrocytes Accompanying Oxidative Stress-Induced Senescence

    Science.gov (United States)

    Crowe, Elizabeth P.; Tuzer, Ferit; Gregory, Brian D.; Donahue, Greg; Gosai, Sager J.; Cohen, Justin; Leung, Yuk Y.; Yetkin, Emre; Nativio, Raffaella; Wang, Li-San; Sell, Christian; Bonini, Nancy M.; Berger, Shelley L.; Johnson, F. Brad; Torres, Claudio

    2016-01-01

    Aging is a major risk factor for many neurodegenerative disorders. A key feature of aging biology that may underlie these diseases is cellular senescence. Senescent cells accumulate in tissues with age, undergo widespread changes in gene expression, and typically demonstrate altered, pro-inflammatory profiles. Astrocyte senescence has been implicated in neurodegenerative disease, and to better understand senescence-associated changes in astrocytes, we investigated changes in their transcriptome using RNA sequencing. Senescence was induced in human fetal astrocytes by transient oxidative stress. Brain-expressed genes, including those involved in neuronal development and differentiation, were downregulated in senescent astrocytes. Remarkably, several genes indicative of astrocytic responses to injury were also downregulated, including glial fibrillary acidic protein and genes involved in the processing and presentation of antigens by major histocompatibility complex class II proteins, while pro-inflammatory genes were upregulated. Overall, our findings suggest that senescence-related changes in the function of astrocytes may impact the pathogenesis of age-related brain disorders. PMID:27630559

  7. Changes in the Transcriptome of Human Astrocytes Accompanying Oxidative Stress-induced Senescence

    Directory of Open Access Journals (Sweden)

    Elizabeth P. Crowe

    2016-08-01

    Full Text Available Aging is a major risk factor for many neurodegenerative disorders. A key feature of aging biology that may underlie these diseases is cellular senescence. Senescent cells accumulate in tissues with age, undergo widespread changes in gene expression, and typically demonstrate altered, pro-inflammatory profiles. Astrocyte senescence has been implicated in neurodegenerative disease, and to better understand senescence-associated changes in astrocytes, we investigated changes in their transcriptome using RNA sequencing. Senescence was induced in human fetal astrocytes by transient oxidative stress. Brain-expressed genes, including those involved in neuronal development and differentiation, were downregulated in senescent astrocytes. Remarkably, several genes indicative of astrocytic responses to injury were also downregulated, including GFAP and genes involved in the processing and presentation of antigens by major histocompatibility complex class II proteins, while pro-inflammatory genes were upregulated. Overall, our findings suggest that senescence-related changes in the function of astrocytes may impact the pathogenesis of age-related brain disorders.

  8. In vitro evaluation of the effects of electrospun PCL nanofiber mats containing the microalgae Spirulina (Arthrospira) extract on primary astrocytes.

    Science.gov (United States)

    Kim, Sung Hoon; Shin, Chungwhan; Min, Seul Ki; Jung, Sang-Myung; Shin, Hwa Sung

    2012-02-01

    The blue-green microalgae, Spirulina, a harmless food and pharmaceutical additive, has several bioactive compounds that have therapeutic functions. Polycaprolactone (PCL) is a biocompatible and biodegradable polymer that has widely been used for tissue engineering. The electrospun PCL nanofiber containing Spirulina (PCL-Spirulina) was fabricated and tested as a potential extracellular matrix material for a culture of primary astrocytes, which play important roles in CNS injured systems. Spirulina extract was observed to increase growth and metabolic activity of rat primary astrocytes without any harm once added to the culture media. However, PCL-Spirulina nanofiber was proven to alleviate astrocyte activity. Through this research and to the best of our knowledge, we first suggest a novel composite nanomaterial, an electrospun PCL-Spirulina nanofiber that could be used to treat CNS injured systems. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Osteopontin is induced by TGF-β2 and regulates metabolic cell activity in cultured human optic nerve head astrocytes.

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    Carolin Neumann

    Full Text Available The aqueous humor (AH component transforming growth factor (TGF-β2 is strongly correlated to primary open-angle glaucoma (POAG, and was shown to up-regulate glaucoma-associated extracellular matrix (ECM components, members of the ECM degradation system and heat shock proteins (HSP in primary ocular cells. Here we present osteopontin (OPN as a new TGF-β2 responsive factor in cultured human optic nerve head (ONH astrocytes. Activation was initially demonstrated by Oligo GEArray microarray and confirmed by semiquantitative (sq RT-PCR, realtime RT-PCR and western blot. Expressions of most prevalent OPN receptors CD44 and integrin receptor subunits αV, α4, α 5, α6, α9, β1, β3 and β5 by ONH astrocytes were shown by sqRT-PCR and immunofluorescence labeling. TGF-β2 treatment did not affect their expression levels. OPN did not regulate gene expression of described TGF-β2 targets shown by sqRT-PCR. In MTS-assays, OPN had a time- and dose-dependent stimulating effect on the metabolic activity of ONH astrocytes, whereas TGF-β2 significantly reduced metabolism. OPN signaling via CD44 mediated a repressive outcome on metabolic activity, whereas signaling via integrin receptors resulted in a pro-metabolic effect. In summary, our findings characterize OPN as a TGF-β2 responsive factor that is not involved in TGF-β2 mediated ECM and HSP modulation, but affects the metabolic activity of astrocytes. A potential involvement in a protective response to TGF-β2 triggered damage is indicated, but requires further investigation.

  10. Characterization of the transcriptional profile in primary astrocytes after oxidative stress induced by Paraquat

    DEFF Research Database (Denmark)

    Olesen, Birgitte S. M. Thuesen; Clausen, Jørgen; Vang, Ole

    2008-01-01

    antioxidative defense systems. The purpose of the present study was to investigate the antioxidative response at the transcriptional level following exposure of primary astrocytes to a pro-oxidant, Paraquat (PQ). This was done by investigating the time-dependent expression of selected genes encoding...... the antioxidative enzymes Mn- and CuZn superoxide dismutase (SOD) and catalase as well as the transcription factor component AP-1. Paraquat induced the expression of Mn- and CuZn SOD, catalase and decreases the expression of c-jun (a part of AP-1). Furthermore, the gene expression profiles were investigated after......, and Bcl-2-associated death promoter. Thus, we could demonstrate a PQ-inducible effect of the mRNA of antioxidative enzymes, as well as the mRNAs of possible enzymes involved in the protection against oxidative stress....

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

    Science.gov (United States)

    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

  12. Gene expression profiling of human neural progenitor cells following the serum-induced astrocyte differentiation.

    Science.gov (United States)

    Obayashi, Shinya; Tabunoki, Hiroko; Kim, Seung U; Satoh, Jun-ichi

    2009-05-01

    Neural stem cells (NSC) with self-renewal and multipotent properties could provide an ideal cell source for transplantation to treat spinal cord injury, stroke, and neurodegenerative diseases. However, the majority of transplanted NSC and neural progenitor cells (NPC) differentiate into astrocytes in vivo under pathological environments in the central nervous system, which potentially cause reactive gliosis. Because the serum is a potent inducer of astrocyte differentiation of rodent NPC in culture, we studied the effect of the serum on gene expression profile of cultured human NPC to identify the gene signature of astrocyte differentiation of human NPC. Human NPC spheres maintained in the serum-free culture medium were exposed to 10% fetal bovine serum (FBS) for 72 h, and processed for analyzing on a Whole Human Genome Microarray of 41,000 genes, and the microarray data were validated by real-time RT-PCR. The serum elevated the levels of expression of 45 genes, including ID1, ID2, ID3, CTGF, TGFA, METRN, GFAP, CRYAB and CSPG3, whereas it reduced the expression of 23 genes, such as DLL1, DLL3, PDGFRA, SOX4, CSPG4, GAS1 and HES5. Thus, the serum-induced astrocyte differentiation of human NPC is characterized by a counteraction of ID family genes on Delta family genes. Coimmunoprecipitation analysis identified ID1 as a direct binding partner of a proneural basic helix-loop-helix (bHLH) transcription factor MASH1. Luciferase assay indicated that activation of the DLL1 promoter by MASH1 was counteracted by ID1. Bone morphogenetic protein 4 (BMP4) elevated the levels of ID1 and GFAP expression in NPC under the serum-free culture conditions. Because the serum contains BMP4, these results suggest that the serum factor(s), most probably BMP4, induces astrocyte differentiation by upregulating the expression of ID family genes that repress the proneural bHLH protein-mediated Delta expression in human NPC.

  13. Cultured human astrocytes secrete large cholesteryl ester- andtriglyceride-rich lipoproteins along with endothelial lipase

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Lin; Liu, Yanzhu; Forte, Trudy M.; Chisholm, Jeffrey W.; Parks, John S.; Shachter, Neil S.

    2003-12-01

    We cultured normal human astrocytes and characterized their secreted lipoproteins. Human astrocytes secreted lipoproteins in the size range of plasma VLDL (Peak 1), LDL (Peak 2), HDL (Peak 3) and a smaller peak (Peak 4), as determined by gel filtration chromatography, nondenaturing gradient gel electrophoresis and transmission electron microscopy. Cholesterol enrichment of astrocytes led to a particular increase in Peak 1. Almost all Peak 2, 3 and 4 cholesterol and most Peak 1 cholesterol was esterified (unlike mouse astrocyte lipoproteins, which exhibited similar peaks but where cholesterol was predominantly non-esterified). Triglycerides were present at about 2/3 the level of cholesterol. LCAT was detected along with two of its activators, apolipoprotein (apo) A-IV and apoC-I. ApoA-I and apoA-II mRNA and protein were absent. ApoJ was present equally in all peaks but apoE was present predominantly in peaks 3 and 4. ApoB was not detected. The electron microscopic appearance of Peak 1 lipoproteins suggested partial lipolysis leading to the detection of a heparin-releasable triglyceride lipase consistent with endothelial lipase. The increased neuronal delivery of lipids from large lipoprotein particles, for which apoE4 has greater affinity than does apoE3, may be a mechanism whereby the apoE {var_epsilon}4 allele contributes to neurodegenerative risk.

  14. MicroRNA Expression Patterns in Human Astrocytes in Relation to Anatomical Location and Age.

    Science.gov (United States)

    Rao, Vijayaraghava T S; Ludwin, Samuel K; Fuh, Shih-Chieh; Sawaya, Robin; Moore, Craig S; Ho, Ming-Kai; Bedell, Barry J; Sarnat, Harvey B; Bar-Or, Amit; Antel, Jack P

    2016-02-01

    Anatomic distribution and age are variables linked to functions of astrocytes under physiologic and pathologic conditions. We measured the relative expression of a panel of microRNAs (miRNAs) in astrocytes captured by laser micro-dissection from normal human adult white and grey matter, human fetal white matter and germinal matrix samples. Although expression of most miRNAs was comparable between adult and fetal samples, regional differences were observed. In the adult cerebral cortex, expression of miRNAs in morphologically distinct inter-laminar astrocytes underlying the glial limitans differed from those in deeper cortical layers, suggesting functional specialization possibly related to structural stability and defense from potentially harmful factors in the cerebrospinal fluid. Differences between adult white and grey matter miRNA expression included higher expression of pro-inflammatory miRNAs in the former, potentially contributing to differences in inflammation between grey and white matter plaques in multiple sclerosis. Lower expression of miRNAs in fetal versus adult white matter astrocytes likely reflects the immaturity of these migrating cells. Highly expressed miRNAs in the fetal germinal matrix are probably relevant in development and also recapitulate some responses to injury. Future studies can address regional alterations of miRNA expression in pathological conditions.

  15. Characterization of primary and secondary cultures of astrocytes prepared from mouse cerebral cortex

    DEFF Research Database (Denmark)

    Skytt, Dorte Marie; Madsen, Karsten Kirkegaard; Pajecka, Kamilla;

    2010-01-01

    Astrocyte cultures were prepared from cerebral cortex of new-born and 7-day-old mice and additionally, the cultures from new-born animals were passaged as secondary cultures. The cultures were characterized by immunostaining for the astrocyte markers glutamine synthetase (GS), glial fibrillary ac...

  16. Growth enhancement effect of BzATP on primary cultured astrocytes from rat brain

    Institute of Scientific and Technical Information of China (English)

    Hua-Zheng LIANG; Ying LIU; Zhu-Rong YE

    2006-01-01

    Objective To explore whether BzATP could promote the growth of primary cultured astrocytes (AS) of rat and its possible mechanism, and whether TGF-β1 was involved in the event. Methods The primary cultured AS were derived from new born Sprague-Dawley rats.Glial fibrillary acidic protein (GFAP) immunofluorescent stain was used to check the purity of cultured AS. Morphometry was used to detect the changes of AS. The proliferation index of AS was detected by BrdU incorporation assay. Western blot was used to detect the changes of GFAP under different conditions. Changes of TGF-β1 gene transcription were detected by RT-PCR. ELISA was utilized to detect the variation of TGF-β1 protein in the supernate. Results The purity of primary cultured AS reached to 99%. BzATP promoted the hypertrophy of AS including the elongation of AS processes and the enlargement of cell bodies, BzATP also promoted the expression of GFAP in existence of Ca2+, but had no effect on cell proliferation. BzATP increased the transcription of TGF-β1 mRNA and the release of TGF-β1 protein in existence of Ca2+. TGF-β1 neutralizing antibody partially inhibited the expression of GFAP induced by BzATP, but had no effect on AS proliferation and cell morphology. Conclusion BzATP enhanced the hypertrophy of primary cultured AS, increased the expression of GFAP partially through TGF-β1. Mechanisms of the enhancement of AS growth induced by BzATP other than TGF-51 pathway remains to be elucidated.

  17. High efficiency adenovirus-mediated expression of truncated N-terminal huntingtin fragment (htt552) in primary rat astrocytes

    Institute of Scientific and Technical Information of China (English)

    Linhui Wang; Fang Lin; Junchao Wu; Zhenghong Qin

    2009-01-01

    Huntington's disease (HD) is caused by an expansion of polyglutamine tract in N-terminus of huntingtin (htt).The mutation of htt leads to dysfunction and premature death of striatal and cortical neurons. However, the effects of htt mutation on glia remain largely unknown.This study aimed to establish a glia HD model using an adenoviral vector to express wild-type and mutant N-terminal huntingtin fragment 1-552 amino acids (htt552) in rat primary cortical astrocytes. We have eval-uated optimal conditions for the infection of astrocytes with adenovirai vectors, and the kinetics of the expression of htt552 in astrocytes. The majority of astroeytes expressed the transgene after infection. At 24 h post-infection, the highest rate of infection was 89 + 3% for the wild-type (htt552-18Q) with a multiplicity of infection (m.o.i.) of 80, and the highest rate of infection was 91 +4% for the mutant type (htt552-100Q) with the same viral dose. The duration of expression of htt552 lasted for about 7 days with a relatively high level from 1 to 4 days post-infection. Mutant huntingtin (htt552-100Q) pro-duced the characteristic HD pathology after 3 days by the appearance of cytoplasmic aggregates and intranue-lear inclusions. The result of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliu mbromide)assay showed that the inhibition of viability by virus on astrocytes was also dose-dependent. To obtain high infection rate and low toxicity, the viral dose with an m.o.i, of 40 was optimal to our cell model. The present study demonstrates that adenovirai-mediated expression of mutant htt provides an advantageous system for his-tological and biochemical analysis of HD pathogenesis in primary cortical astrocyte cultures.

  18. Human astrocytes derived from glial restricted progenitors support regeneration of the injured spinal cord.

    Science.gov (United States)

    Haas, Christopher; Fischer, Itzhak

    2013-06-15

    Cellular transplantation using neural stem cells and progenitors is a promising therapeutic strategy that has the potential to replace lost cells, modulate the injury environment, and create a permissive environment for the regeneration of injured host axons. Our research has focused on the use of human glial restricted progenitors (hGRP) and derived astrocytes. In the current study, we examined the morphological and phenotypic properties of hGRP prepared from the fetal central nervous system by clinically-approved protocols, compared with astrocytes derived from hGRP prepared by treatment with ciliary neurotrophic factor or bone morphogenetic protein 4. These differentiation protocols generated astrocytes that showed morphological differences and could be classified along an immature to mature spectrum, respectively. Despite these differences, the cells retained morphological and phenotypic plasticity upon a challenge with an alternate differentiation protocol. Importantly, when hGRP and derived astrocytes were transplanted acutely into a cervical dorsal column lesion, they survived and promoted regeneration of long ascending host sensory axons into the graft/lesion site, with no differences among the groups. Further, hGRP taken directly from frozen stocks behaved similarly and also supported regeneration of host axons into the lesion. Our results underscore the dynamic and permissive properties of human fetal astrocytes to promote axonal regeneration. They also suggest that a time-consuming process of pre-differentiation may not be necessary for therapeutic efficacy, and that the banking of large quantities of readily available hGRP can be an appropriate source of permissive cells for transplantation.

  19. Insights into Human Astrocyte Response to H5N1 Infection by Microarray Analysis

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    Xian Lin

    2015-05-01

    Full Text Available Influenza virus infects not only the respiratory system but also the central nervous system (CNS, leading to influenza-associated encephalopathy and encephalitis. Astrocytes are essential for brain homeostasis and neuronal function. These cells can also be infected by influenza virus. However, genome-wide changes in response to influenza viral infection in astrocytes have not been defined. In this study, we performed gene profiling of human astrocytes in response to H5N1. Innate immune and pro-inflammatory responses were strongly activated at 24 h post-infection (hpi. Antiviral genes, as well as several cytokines and chemokines, including CXCL9, CXCL10, and CXCL11, were robustly induced. Phosphorylation of p65 and p38 can be activated by viral infection, suggesting their potential critical roles in H5N1-induced pro-inflammatory response. Moreover, H5N1 infection significantly upregulated the gene expressions related to the neuroactive ligand-receptor interaction pathway at 24 hpi, such as MC2R, CHRNG, P2RY13, GABRA1, and HRH2, which participant in synaptic transmission and may take part in CNS disorders induced by H5N1 infection. Targeting key components of innate immune response and the neuroactive ligand-receptor interaction pathway may provide a strategy to control H5N1-induced encephalopathy and encephalitis. This research can contribute to the understanding of H5N1 pathogenesis in astrocytes.

  20. Human immunodeficiency virus type 1 efficiently binds to human fetal astrocytes and induces neuroinflammatory responses independent of infection

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    Potash Mary

    2007-05-01

    Full Text Available Abstract Background HIV-1 infects human astrocytes in vitro and in vivo but the frequency of infected cells is low and its biological significance is unknown. In studies in vitro, recombinant gp120 alone can induce profound effects on astrocyte biology, suggesting that HIV-1 interaction with astrocytes and its functional consequences extend beyond the limited levels of infection in these cells. Here we determined the relative efficiencies of HIV-1 binding and infection in human fetal astrocytes (HFA, mainly at the single cell level, using HIV-1 tagged with green fluorescence protein (GFP-Vpr fusion proteins, termed HIV-GFP, to detect virus binding and HIV-1 expressing Rev and NefGFP fusion proteins to detect productive infection. Results Essentially all HFA in a population bound HIV-GFP specifically and independently of CCR5 and CXCR4. The dynamics of this binding at 37°C resembled binding of an HIV fusion mutant to CD4-positive cells, indicating that most of HIV-GFP arrested infection of HFA at the stage of virus-cell fusion. Despite extensive binding, only about 1% of HFA were detectably infected by HIV-RevGFP or HIV-NefGFP, but this proportion increased to the majority of HFA when the viruses were pseudotyped with vesicular stomatitis virus envelope glycoprotein G, confirming that HFA impose a restriction upon HIV-1 entry. Exposure of HFA to HIV-1 through its native proteins rapidly induced synthesis of interleukin-6 and interleukin-8 with increased mRNA detected within 3 h and increased protein detected within 18 h of exposure. Conclusion Our results indicate that HIV-1 binding to human astrocytes, although extensive, is not generally followed by virus entry and replication. Astrocytes respond to HIV-1 binding by rapidly increased cytokine production suggesting a role of this virus-brain cell interaction in HIV-1 neuropathogenesis.

  1. Sex differences in the inflammatory response of primary astrocytes to lipopolysaccharide

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    Santos-Galindo María

    2011-07-01

    Full Text Available Abstract Background Numerous neurological and psychiatric disorders show sex differences in incidence, age of onset, symptomatology or outcome. Astrocytes, one of the glial cell types of the brain, show sex differences in number, differentiation and function. Since astrocytes are involved in the response of neural tissue to injury and inflammation, these cells may participate in the generation of sex differences in the response of the brain to pathological insults. To explore this hypothesis, we have examined whether male and female astrocytes show a different response to an inflammatory challenge and whether perinatal testosterone influences this response. Methods Cortical astrocyte cultures were prepared from postnatal day 1 (one day after birth male or female CD1 mice pups. In addition, cortical astrocyte cultures were also prepared from female pups that were injected at birth with 100 μg of testosterone propionate or vehicle. Cultures were treated for 5 hours with medium containing lipopolysaccharide (LPS or with control medium. The mRNA levels of IL6, interferon-inducible protein 10 (IP10, TNFα, IL1β, Toll-like receptor 4 (TLR4, steroidogenic acute regulatory protein and translocator protein were assessed by quantitative real-time polymerase chain reaction. Statistical significance was assessed by unpaired t-test or by one-way analysis of variance followed by the Tukey post hoc test. Results The mRNA levels of IL6, TNFα and IL1β after LPS treatment were significantly higher in astrocytes derived from male or androgenized females compared to astrocytes derived from control or vehicle-injected females. In contrast, IP10 mRNA levels after LPS treatment were higher in astrocytes derived from control or vehicle-injected females than in those obtained from males or androgenized females. The different response of male and female astrocytes to LPS was due neither to differences in the basal expression of the inflammatory molecules nor to

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

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    Xu L

    2015-09-01

    Full Text Available Liming Xu,1,2,* Mo Dan,1,* Anliang Shao,1 Xiang Cheng,1,3 Cuiping Zhang,4 Robert A Yokel,5 Taro Takemura,6 Nobutaka Hanagata,6 Masami Niwa,7,8 Daisuke Watanabe7,81National Institutes for Food and Drug Control, No 2, Temple of Heaven, Beijing, 2School of Information and Engineering, Wenzhou Medical University, Wenzhou, 3School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu, 4Beijing Neurosurgical Institute, Capital Medical University, Beijing, People’s Republic of China; 5College of Pharmacy, University of Kentucky, Lexington, KY, USA; 6Nanotechnology Innovation Station for Nanoscale Science and Technology, National Institute for Materials Science, Tsukuba, Ibaraki, 7Department of Pharmacology, Nagasaki University, 8BBB Laboratory, PharmaCo-Cell Company, Ltd., Nagasaki, Japan*These authors contributed equally to this workBackground: 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

  3. Human iPS cell-derived astrocyte transplants preserve respiratory function after spinal cord injury.

    Science.gov (United States)

    Li, Ke; Javed, Elham; Scura, Daniel; Hala, Tamara J; Seetharam, Suneil; Falnikar, Aditi; Richard, Jean-Philippe; Chorath, Ashley; Maragakis, Nicholas J; Wright, Megan C; Lepore, Angelo C

    2015-09-01

    Transplantation-based replacement of lost and/or dysfunctional astrocytes is a promising therapy for spinal cord injury (SCI) that has not been extensively explored, despite the integral roles played by astrocytes in the central nervous system (CNS). Induced pluripotent stem (iPS) cells are a clinically-relevant source of pluripotent cells that both avoid ethical issues of embryonic stem cells and allow for homogeneous derivation of mature cell types in large quantities, potentially in an autologous fashion. Despite their promise, the iPS cell field is in its infancy with respect to evaluating in vivo graft integration and therapeutic efficacy in SCI models. Astrocytes express the major glutamate transporter, GLT1, which is responsible for the vast majority of glutamate uptake in spinal cord. Following SCI, compromised GLT1 expression/function can increase susceptibility to excitotoxicity. We therefore evaluated intraspinal transplantation of human iPS cell-derived astrocytes (hIPSAs) following cervical contusion SCI as a novel strategy for reconstituting GLT1 expression and for protecting diaphragmatic respiratory neural circuitry. Transplant-derived cells showed robust long-term survival post-injection and efficiently differentiated into astrocytes in injured spinal cord of both immunesuppressed mice and rats. However, the majority of transplant-derived astrocytes did not express high levels of GLT1, particularly at early times post-injection. To enhance their ability to modulate extracellular glutamate levels, we engineered hIPSAs with lentivirus to constitutively express GLT1. Overexpression significantly increased GLT1 protein and functional GLT1-mediated glutamate uptake levels in hIPSAs both in vitro and in vivo post-transplantation. Compared to human fibroblast control and unmodified hIPSA transplantation, GLT1-overexpressing hIPSAs reduced (1) lesion size within the injured cervical spinal cord, (2) morphological denervation by respiratory phrenic motor

  4. Generation of GFAP::GFP astrocyte reporter lines from human adult fibroblast-derived iPS cells using zinc-finger nuclease technology.

    Science.gov (United States)

    Zhang, Ping-Wu; Haidet-Phillips, Amanda M; Pham, Jacqueline T; Lee, Youngjin; Huo, Yuqing; Tienari, Pentti J; Maragakis, Nicholas J; Sattler, Rita; Rothstein, Jeffrey D

    2016-01-01

    Astrocytes are instrumental to major brain functions, including metabolic support, extracellular ion regulation, the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental, psychiatric and neurodegenerative disorders. The generation of adult human fibroblast-derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human-derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes), we generated homogenous populations of iPS astrocytes using zinc-finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno-associated virus integration site 1 (AAVS1) locus in disease and control-derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP, S100β, NFIA and ALDH1L1. In addition, mature pure GFP-iPS astrocytes exhibited a well-described functional astrocytic activity in vitro characterized by neuron-dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP-iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion, the generation of GFAP::GFP-iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte-driven disease pathogenesis and therapy.

  5. Exposure to Cell Phone Radiation Up-Regulates Apoptosis Genes in Primary Cultures of Neurons and Astrocytes

    OpenAIRE

    Zhao, Tian-Yong; Zou, Shi-Ping; Pamela E Knapp

    2006-01-01

    The health effects of cell phone radiation exposure are a growing public concern. This study investigated whether expression of genes related to cell death pathways are dysregulated in primary cultured neurons and astrocytes by exposure to a working GSM (Global System for Mobile Communication) cell phone rated at a frequency of 1900 MHz. Primary cultures were exposed to cell phone emissions for 2 hrs. We used array analysis and real-time RT-PCR to show up-regulation of caspase-2, caspase-6 an...

  6. QKI-7 regulates expression of interferon-related genes in human astrocyte glioma cells.

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    Lin Jiang

    Full Text Available BACKGROUND: The human QKI gene, called quaking homolog, KH domain RNA binding (mouse, is a candidate gene for schizophrenia encoding an RNA-binding protein. This gene was shown to be essential for myelination in oligodendrocytes. QKI is also highly expressed in astrocytes, but its function in these cells is not known. METHODS/PRINCIPAL FINDINGS: We studied the effect of small interference RNA (siRNA-mediated QKI depletion on global gene expression in human astrocyte glioma cells. Microarray measurements were confirmed with real-time quantitative polymerase chain reaction (qPCR. The presence of QKI binding sites (QRE was assessed by a bioinformatic approach. Viability and cell morphology were also studied. The most significant alteration after QKI silencing was the decreased expression of genes involved in interferon (IFN induction (P = 6.3E-10, including IFIT1, IFIT2, MX1, MX2, G1P2, G1P3, GBP1 and IFIH1. All eight genes were down-regulated after silencing of the splice variant QKI-7, but were not affected by QKI-5 silencing. Interestingly, four of them were up-regulated after treatment with the antipsychotic agent haloperidol that also resulted in increased QKI-7 mRNA levels. CONCLUSIONS/SIGNIFICANCE: The coordinated expression of QKI-7 splice variant and IFN-related genes supports the idea that this particular splice variant has specific functions in astrocytes. Furthermore, a role of QKI-7 as a regulator of an inflammatory gene pathway in astrocytes is suggested. This hypothesis is well in line with growing experimental evidence on the role of inflammatory components in schizophrenia.

  7. Kinetics and autoradiography of high affinity uptake of serotonin by primary astrocyte cultures

    Energy Technology Data Exchange (ETDEWEB)

    Katz, D.M.; Kimelberg, H.K.

    1985-07-01

    Primary astrocyte cultures prepared from the cerebral cortices of neonatal rats showed significant accumulation of serotonin (5-hydroxytryptamine; (/sup 3/H)-5-HT). At concentrations in the range of 0.01 to 0.7 microM (/sup 3/H)-5-HT, this uptake was 50 to 85% Na+ dependent and gave a Km of 0.40 +/- 0.11 microM (/sup 3/H)-5-HT and a Vmax of 6.42 +/- 0.85 (+/- SEM) pmol of (/sup 3/H)-5-HT/mg of protein/4 min for the Na+-dependent component. In the absence of Na+ the uptake was nonsaturable. Omission of the monoamine oxidase inhibitor pargyline markedly reduced the Na+-dependent component of (/sup 3/H)-5-HT uptake but had a negligible effect on the Na+-independent component. This suggest significant oxidative deamination of serotonin after it has been taken up by the high affinity system, followed by release of its metabolite. The authors estimated that this system enabled the cells to concentrate (/sup 3/H)-5-HT up to 44-fold at an external (/sup 3/H)-5-HT concentration of 10(-7) M. Inhibition of (/sup 3/H)-5-HT uptake by a number of clinically effective antidepressants was also consistent with a specific high affinity uptake mechanism for 5-HT, the order of effectiveness of inhibition being chlorimipramine greater than fluoxetine greater than imipramine = amitriptyline greater than desmethylimipramine greater than iprindole greater than mianserin. Uptake of (/sup 3/H)-5-HT was dependent on the presence of Cl- as well as Na+ in the medium, and the effect of omission of both ions was nonadditive. Varying the concentration of K+ in the media from 1 to 50 mM had a limited effect on (/sup 3/H)-5-HT uptake.

  8. PPARgamma agonist curcumin reduces the amyloid-beta-stimulated inflammatory responses in primary astrocytes.

    Science.gov (United States)

    Wang, Hong-Mei; Zhao, Yan-Xin; Zhang, Shi; Liu, Gui-Dong; Kang, Wen-Yan; Tang, Hui-Dong; Ding, Jian-Qing; Chen, Sheng-Di

    2010-01-01

    Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder. Accumulating data indicate that astrocytes play an important role in the neuroinflammation related to the pathogenesis of AD. It has been shown that microglia and astrocytes are activated in AD brain and amyloid-beta (Abeta) can increase the expression of cyclooxygenase 2 (COX-2), interleukin-1, and interleukin-6. Suppressing the inflammatory response caused by activated astrocytes may help to inhibit the development of AD. Curcumin is a major constituent of the yellow curry spice turmeric and proved to be a potential anti-inflammatory drug in arthritis and colitis. There is a low age-adjusted prevalence of AD in India, a country where turmeric powder is commonly used as a culinary compound. Curcumin has been shown to suppress activated astroglia in amyloid-beta protein precursor transgenic mice. The real mechanism by which curcumin inhibits activated astroglia is poorly understood. Here we report that the expression of COX-2 and glial fibrillary acidic protein were enhanced and that of peroxisome proliferator-activated receptor gamma (PPARgamma) was decreased in Abeta(25-35)-treated astrocytes. In line with these results, nuclear factor-kappaB translocation was increased in the presence of Abeta. All these can be reversed by the pretreatment of curcumin. Furthermore, GW9662, a PPARgamma antagonist, can abolish the anti-inflammatory effect of curcumin. These results show that curcumin might act as a PPARgamma agonist to inhibit the inflammation in Abeta-treated astrocytes.

  9. Human induced pluripotent stem cell (hiPSC)-derived neurons respond to convulsant drugs when co-cultured with hiPSC-derived astrocytes.

    Science.gov (United States)

    Ishii, Misawa Niki; Yamamoto, Koji; Shoji, Masanobu; Asami, Asano; Kawamata, Yuji

    2017-08-15

    Accurate risk assessment for drug-induced seizure is expected to be performed before entering clinical studies because of its severity and fatal damage to drug development. Induced pluripotent stem cell (iPSC) technology has allowed the use of human neurons and glial cells in toxicology studies. Recently, several studies showed the advantage of co-culture system of human iPSC (hiPSC)-derived neurons with rodent/human primary astrocytes regarding neuronal functions. However, the application of hiPSC-derived neurons for seizure risk assessment has not yet been fully addressed, and not at all when co-cultured with hiPSC-derived astrocytes. Here, we characterized hiPSC-derived neurons co-cultured with hiPSC-derived astrocytes to discuss how hiPSC-derived neurons are useful to assess seizure risk of drugs. First, we detected the frequency of spikes and synchronized bursts hiPSC-derived neurons when co-cultured with hiPSC-derived astrocytes for 8 weeks. This synchronized burst was suppressed by the treatment with 6-cyano-7-nitroquinoxaline-2,3-dione, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist, and D-(-)-2-amino-5-phosphonopentanoic acid, an N-Methyl-d-aspartate (NMDA) receptor antagonist. These data suggested that co-cultured hiPSC-derived neurons formed synaptic connections mediated by AMPA and NMDA receptors. We also demonstrated that co-cultured hiPSC-derived neurons showed epileptiform activity upon treatment with gabazine or kaliotoxin. Finally, we performed single-cell transcriptome analysis in hiPSC-derived neurons and found that hiPSC-derived astrocytes activated the pathways involved in the activities of AMPA and NMDA receptor functions, neuronal polarity, and axon guidance in hiPSC-derived neurons. These data suggested that hiPSC-derived astrocytes promoted the development of action potential, synaptic functions, and neuronal networks in hiPSC-derived neurons, and then these functional alterations result in the epileptiform

  10. Influence of drugs on gap junctions in glioma cell lines and primary astrocytes in vitro

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    Zahra eMoinfar

    2014-05-01

    Full Text Available Gap junctions (GJs are hemichannels on cell membrane. Once they are intercellulary connected to the neighboring cells, they build a functional syncytium which allows rapid transfer of ions and molecules between cells. This characteristic makes GJs a potential modulator in proliferation, migration and development of the cells. So far, several types of GJs are recognized on different brain cells as well as in glioma. Astrocytes, as one of the major cells that maintain neuronal homeostasis, express different types of GJs that let them communicate with neurons, oligodendrocytes and endothelial cells of the blood brain barrier; however, the main GJ in astrocytes is connexin 43. There are different cerebral diseases in which astrocyte GJs might play a role. Several drugs have been reported to modulate gap junctional communication in the brain which can consequently have beneficial or detrimental effects on the course of treatment in certain diseases. However, the exact cellular mechanism behind those pharmaceutical efficacies on GJs is not well-understood. Accordingly, how specific drugs would affect GJs and what some consequent specific brain diseases would be are the interests of the authors of this chapter. We would focus on pharmaceutical effects on GJs on astrocytes in specific diseases where GJs could possibly play a role including: 1 migraine and a novel therapy for migraine with aura, 2 neuroautoimmune diseases and immunomodulatory drugs in the treatment of demyelinating diseases of the central nervous system such as multiple sclerosis, 3 glioma and antineoplastic and anti-inflammatory agents that are used in treating brain tumors and 4 epilepsy and anticonvulsants that are widely used for seizures therapy. All of the above-mentioned therapeutic categories can possibly affect GJs expression of astrocytes and the role is discussed in the upcoming chapter.

  11. Ceramide in primary astrocytes from cerebellum: metabolism and role in cell proliferation.

    Science.gov (United States)

    Riboni, Laura; Tettamanti, Guido; Viani, Paola

    2002-04-01

    Cerebellar astrocytes are equipped with an efficient molecular machinery able to control the levels, and possibly the subcellular location, of ceramide. The major metabolic routes that contribute to the maintenance and variation of the cellular ceramide include ceramide biosynthesis, by de novo pathway or sphingosine recycling, ceramide formation from complex sphingolipids degradation and ceramide catabolism. In cerebellar astrocytes from rat cerebellum a peculiar metabolism of sphingomyelin occurs. This includes the preponderance of acidic sphingomyelinase, paralleled by a deficiency of the neutral Mg2+-dependent enzyme, as well as the presence of an extra-Golgi form of sphingomyelin synthase, which shares many characteristics with PC-PLC. Moreover these cells are characterized by a high efficiency in converting sphingosine to ceramide, possibly functional to the role played by astrocytes in the prevention of neuronal damage by high sphingosine concentration. Recent evidence demonstrates that a change of ceramide level is one of the key steps in the chain of reactions elicited by mitogenic stimuli. In fact, low cellular levels of ceramide characterize, and appear to be required for, the proliferation of cerebellar astrocytes. In particular mitogenic stimuli, such as basic fibroblast growth factor (bFGF), rapidly down regulate the cellular levels of ceramide by stimulating sphingomyelin synthase. Ceramide acts as an intracellular physiological inhibitor of cell growth, being able to counteract the effect of bFGF by inhibiting the MAP kinase pathway. Although many questions remain in this field, the present knowledge strongly supports that ceramide represents a crucial member within lipid mediators, involved in the signaling pathways underlying cell proliferation in cerebellar astrocytes.

  12. The tricarboxylic acid cycle activity in cultured primary astrocytes is strongly accelerated by the protein tyrosine kinase inhibitor tyrphostin 23

    DEFF Research Database (Denmark)

    Hohnholt, Michaela C; Blumrich, Eva-Maria; Waagepetersen, Helle S

    2017-01-01

    production. In addition, T23-treatment strongly increased the molecular carbon labeling of the TCA cycle intermediates citrate, succinate, fumarate and malate, and significantly increased the incorporation of (13)C-labelling into the amino acids glutamate, glutamine and aspartate. These results clearly......Tyrphostin 23 (T23) is a well-known inhibitor of protein tyrosine kinases and has been considered as potential anti-cancer drug. T23 was recently reported to acutely stimulate the glycolytic flux in primary cultured astrocytes. To investigate whether T23 also affects the tricarboxylic acid (TCA...

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

    OpenAIRE

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

    2014-01-01

    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 ex...

  14. Astrocyte pathology in a human neural stem cell model of frontotemporal dementia caused by mutant TAU protein

    Science.gov (United States)

    Hallmann, Anna-Lena; Araúzo-Bravo, Marcos J.; Mavrommatis, Lampros; Ehrlich, Marc; Röpke, Albrecht; Brockhaus, Johannes; Missler, Markus; Sterneckert, Jared; Schöler, Hans R.; Kuhlmann, Tanja; Zaehres, Holm; Hargus, Gunnar

    2017-01-01

    Astroglial pathology is seen in various neurodegenerative diseases including frontotemporal dementia (FTD), which can be caused by mutations in the gene encoding the microtubule-associated protein TAU (MAPT). Here, we applied a stem cell model of FTD to examine if FTD astrocytes carry an intrinsic propensity to degeneration and to determine if they can induce non-cell-autonomous effects in neighboring neurons. We utilized CRISPR/Cas9 genome editing in human induced pluripotent stem (iPS) cell-derived neural progenitor cells (NPCs) to repair the FTD-associated N279K MAPT mutation. While astrocytic differentiation was not impaired in FTD NPCs derived from one patient carrying the N279K MAPT mutation, FTD astrocytes appeared larger, expressed increased levels of 4R-TAU isoforms, demonstrated increased vulnerability to oxidative stress and elevated protein ubiquitination and exhibited disease-associated changes in transcriptome profiles when compared to astrocytes derived from one control individual and to the isogenic control. Interestingly, co-culture experiments with FTD astrocytes revealed increased oxidative stress and robust changes in whole genome expression in previously healthy neurons. Our study highlights the utility of iPS cell-derived NPCs to elucidate the role of astrocytes in the pathogenesis of FTD. PMID:28256506

  15. Infection with Theiler's murine encephalomyelitis virus directly induces proinflammatory cytokines in primary astrocytes via NF-kappaB activation: potential role for the initiation of demyelinating disease.

    Science.gov (United States)

    Palma, JoAnn P; Kwon, Daeho; Clipstone, Neil A; Kim, Byung S

    2003-06-01

    Theiler's virus infection in the central nervous system (CNS) induces a demyelinating disease very similar to human multiple sclerosis. We have assessed cytokine gene activation upon Theiler's murine encephalomyelitis virus (TMEV) infection and potential mechanisms in order to delineate the early events in viral infection that lead to immune-mediated demyelinating disease. Infection of SJL/J primary astrocyte cultures induces selective proinflammatory cytokine genes (interleukin-12p40 [IL-12p40], IL-1, IL-6, tumor necrosis factor alpha, and beta interferon [IFN-beta]) important in the innate immune response to infection. We find that TMEV-induced cytokine gene expression is mediated by the NF-kappaB pathway based on the early nuclear NF-kappaB translocation and suppression of cytokine activation in the presence of specific inhibitors of the NF-kappaB pathway. Further studies show this to be partly independent of dsRNA-dependent protein kinase (PKR) and IFN-alpha/beta pathways. Altogether, these results demonstrate that infection of astrocytes and other CNS-resident cells by TMEV provides the early NF-kappaB-mediated signals that directly activate various proinflammatory cytokine genes involved in the initiation and amplification of inflammatory responses in the CNS known to be critical for the development of immune-mediated demyelination.

  16. Hydrogel scaffolds promote neural gene expression and structural reorganization in human astrocyte cultures

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    V. Bleu Knight

    2017-01-01

    Full Text Available Biomaterial scaffolds have the potential to enhance neuronal development and regeneration. Understanding the genetic responses of astrocytes and neurons to biomaterials could facilitate the development of synthetic environments that enable the specification of neural tissue organization with engineered scaffolds. In this study, we used high throughput transcriptomic and imaging methods to determine the impact of a hydrogel, PuraMatrix™, on human glial cells in vitro. Parallel studies were undertaken with cells grown in a monolayer environment on tissue culture polystyrene. When the Normal Human Astrocyte (NHA cell line is grown in a hydrogel matrix environment, the glial cells adopt a structural organization that resembles that of neuronal-glial cocultures, where neurons form clusters that are distinct from the surrounding glia. Statistical analysis of next generation RNA sequencing data uncovered a set of genes that are differentially expressed in the monolayer and matrix hydrogel environments. Functional analysis demonstrated that hydrogel-upregulated genes can be grouped into three broad categories: neuronal differentiation and/or neural plasticity, response to neural insult, and sensory perception. Our results demonstrate that hydrogel biomaterials have the potential to transform human glial cell identity, and may have applications in the repair of damaged brain tissue.

  17. Hydrogel scaffolds promote neural gene expression and structural reorganization in human astrocyte cultures

    Science.gov (United States)

    Knight, V. Bleu

    2017-01-01

    Biomaterial scaffolds have the potential to enhance neuronal development and regeneration. Understanding the genetic responses of astrocytes and neurons to biomaterials could facilitate the development of synthetic environments that enable the specification of neural tissue organization with engineered scaffolds. In this study, we used high throughput transcriptomic and imaging methods to determine the impact of a hydrogel, PuraMatrix™, on human glial cells in vitro. Parallel studies were undertaken with cells grown in a monolayer environment on tissue culture polystyrene. When the Normal Human Astrocyte (NHA) cell line is grown in a hydrogel matrix environment, the glial cells adopt a structural organization that resembles that of neuronal-glial cocultures, where neurons form clusters that are distinct from the surrounding glia. Statistical analysis of next generation RNA sequencing data uncovered a set of genes that are differentially expressed in the monolayer and matrix hydrogel environments. Functional analysis demonstrated that hydrogel-upregulated genes can be grouped into three broad categories: neuronal differentiation and/or neural plasticity, response to neural insult, and sensory perception. Our results demonstrate that hydrogel biomaterials have the potential to transform human glial cell identity, and may have applications in the repair of damaged brain tissue.

  18. Multiple oxygen tension environments reveal diverse patterns of transcriptional regulation in primary astrocytes.

    Directory of Open Access Journals (Sweden)

    Wayne Chadwick

    Full Text Available The central nervous system normally functions at O(2 levels which would be regarded as hypoxic by most other tissues. However, most in vitro studies of neurons and astrocytes are conducted under hyperoxic conditions without consideration of O(2-dependent cellular adaptation. We analyzed the reactivity of astrocytes to 1, 4 and 9% O(2 tensions compared to the cell culture standard of 20% O(2, to investigate their ability to sense and translate this O(2 information to transcriptional activity. Variance of ambient O(2 tension for rat astrocytes resulted in profound changes in ribosomal activity, cytoskeletal and energy-regulatory mechanisms and cytokine-related signaling. Clustering of transcriptional regulation patterns revealed four distinct response pattern groups that directionally pivoted around the 4% O(2 tension, or demonstrated coherent ascending/decreasing gene expression patterns in response to diverse oxygen tensions. Immune response and cell cycle/cancer-related signaling pathway transcriptomic subsets were significantly activated with increasing hypoxia, whilst hemostatic and cardiovascular signaling mechanisms were attenuated with increasing hypoxia. Our data indicate that variant O(2 tensions induce specific and physiologically-focused transcript regulation patterns that may underpin important physiological mechanisms that connect higher neurological activity to astrocytic function and ambient oxygen environments. These strongly defined patterns demonstrate a strong bias for physiological transcript programs to pivot around the 4% O(2 tension, while uni-modal programs that do not, appear more related to pathological actions. The functional interaction of these transcriptional 'programs' may serve to regulate the dynamic vascular responsivity of the central nervous system during periods of stress or heightened activity.

  19. Group B Streptococcus interactions with human meningeal cells and astrocytes in vitro.

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    Khalil Alkuwaity

    Full Text Available BACKGROUND: Streptococcus agalactiae (Group B Streptococcus, GBS is a leading cause of life-threatening neonatal meningitis and survivors often suffer permanent neurological damage. How this organism interacts with the meninges and subsequently with astrocytes that constitute the underlying cortical glia limitans superficialis is not known. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we demonstrate dose-dependent adherence of GBS over time to human meningioma cells and fetal astrocytes in vitro, which was not influenced by expression of either β-haemolysin/cytolysin (β-h/c toxin, different capsule serotypes or by absence of capsule (p>0.05. Internalization of GBS by both cell types was, however, a slow and an infrequent event (only 0.02-0.4% of associated bacteria were internalised by 9 h. Expression of β-h/c toxin did not play a role in invasion (p>0.05, whereas capsule expression lead to a reduction (p<0.05 in the numbers of intracellular bacteria recovered. GBS strains induced cytotoxicity as demonstrated by the measurement of lactate dehydrogenase (LDH enzyme release by 9 h and by viable staining. Increasing levels of meningioma cell death correlated with bacterial growth and the phenotype of β-h/c toxin production, i.e. from weakly, to normo- to hyper-haemolytic. However, cytotoxicity was significantly greater (p<0.05 towards astrocytes, and infection with initial MOI≥0.003 induced 70-100% LDH release. By comparing wild-type (β-h/c(+ and mutant (ΔcylE β-h/c(- strains and β-h/c toxin extracts and by using the surfactant dipalmitoylphosphatidylcholine in cytotoxicity inhibition experiments, β-h/c toxin was demonstrated as principally responsible for cell death. CONCLUSIONS/SIGNIFICANCE: This study has described key events in the interactions of GBS with meningeal cells and astrocytes in vitro and a major virulence role for β-h/c toxin. Understanding the mechanisms involved will help to identify potential therapies for improving

  20. TSPO PIGA Ligands Promote Neurosteroidogenesis and Human Astrocyte Well-Being

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    Eleonora Da Pozzo

    2016-06-01

    Full Text Available The steroidogenic 18 kDa translocator protein (TSPO is an emerging, attractive therapeutic tool for several pathological conditions of the nervous system. Here, 13 high affinity TSPO ligands belonging to our previously described N,N-dialkyl-2-phenylindol-3-ylglyoxylamide (PIGA class were evaluated for their potential ability to affect the cellular Oxidative Metabolism Activity/Proliferation index, which is used as a measure of astrocyte well-being. The most active PIGA ligands were also assessed for steroidogenic activity in terms of pregnenolone production, and the values were related to the metabolic index in rat and human models. The results showed a positive correlation between the increase in the Oxidative Metabolism Activity/Proliferation index and the pharmacologically induced stimulation of steroidogenesis. The specific involvement of steroid molecules in mediating the metabolic effects of the PIGA ligands was demonstrated using aminoglutethimide, a specific inhibitor of the first step of steroid biosynthesis. The most promising steroidogenic PIGA ligands were the 2-naphthyl derivatives that showed a long residence time to the target, in agreement with our previous data. In conclusion, TSPO ligand-induced neurosteroidogenesis was involved in astrocyte well-being.

  1. RNAseq profiling of primary microglia and astrocyte cultures in near-term ovine fetus: A glial in vivo-in vitro multi-hit paradigm in large mammalian brain.

    Science.gov (United States)

    Cortes, M; Cao, M; Liu, H L; Burns, P; Moore, C; Fecteau, G; Desrochers, A; Barreiro, L B; Antel, J P; Frasch, M G

    2017-01-30

    The chronically instrumented fetal sheep is a widely used animal model to study fetal brain development in health and disease, but no methods exist yet to interrogate dedicated brain cell populations to identify their molecular and genomic phenotype. For example, the molecular mechanisms whereby microglia or astrocytes contribute to inflammation in the brain remain incompletely understood. Here we present a protocol to derive primary pure microglial or astrocyte cultures from near-term fetal sheep brain, after the animals have been chronically instrumented and studied in vivo. Next, we present the implementation of whole transcriptome sequencing (RNAseq) pipeline to deeper elucidate the phenotype of such primary sheep brain glial cultures. We validate the new primary cultures method for cell purity and test the function of the glial cells on protein (IL-1β) and transcriptome (RNAseq) levels in response to a lipopolysaccharide (LPS) challenge in vitro. This method represents the first implementation of pure microglial or astrocytes cultures in fetal sheep brain. The presented approach opens new possibilities for testing not only supernatant protein levels in response to an in vitro challenge, but also to evaluate changes in the transcriptome of glial cells derived from a large mammalian brain bearing high resemblance to the human brain. Moreover, the presented approach lends itself to modeling the complex multi-hit paradigms of antenatal and perinatal cerebral insults in vivo and in vitro. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Changes in mitochondrial morphology induced by calcium or rotenone in primary astrocytes occur predominantly through ros-mediated remodeling.

    Science.gov (United States)

    Deheshi, Samineh; Dabiri, Bahram; Fan, Susu; Tsang, Michelle; Rintoul, Gordon L

    2015-06-01

    Morphological changes in mitochondria have been primarily attributed to fission and fusion, while the more pliable transformations of mitochondria (remodeling, rounding, or stretching) have been largely overlooked. In this study, we quantify the contributions of fission and remodeling to changes in mitochondrial morphology induced by the Ca(2+) ionophore 4Br-A23187 and the metabolic toxin rotenone. We also examine the role of reactive oxygen species (ROS) in the regulation of mitochondrial remodeling. In agreement with our previous studies, mitochondrial remodeling, not fission, is the primary contributor to Ca(2+) -mediated changes in mitochondrial morphology induced by 4Br-A23187 in rat cortical astrocytes. Treatment with rotenone produced similar results. In both paradigms, remodeling was selectively blocked by antioxidants whereas fission was not, suggesting a ROS-mediated mechanism for mitochondrial remodeling. In support of this hypothesis, inhibition of endogenous ROS by overnight incubation in antioxidants resulted in elongated reticular networks of mitochondria. Examination of inner and outer mitochondrial membranes revealed that they largely acted in concert during the remodeling process. While mitochondrial morphology is traditionally ascribed to a net output of fission and fusion processes, in this study we provide evidence that the acute pliability of mitochondria can be a dominant factor in determining their morphology. More importantly, our results suggest that the remodeling process is independently regulated through a ROS-signaling mechanism. Mitochondrial morphology is traditionally ascribed to a balance of fission and fusion processes. We have shown that mitochondria can undergo more pliable transformations; remodeling, rounding, or stretching. We demonstrate that remodeling, not fission, is the primary contributor to calcium mediated changes in mitochondrial morphology in primary astrocytes. Others have shown fission is mediated by calcineurin

  3. Neurosphere Based Differentiation of Human iPSC Improves Astrocyte Differentiation

    DEFF Research Database (Denmark)

    Zhou, Shuling; Szczesna, Karolina; Ochalek, Anna;

    2016-01-01

    of brain tissue. In this study, we determined that culturing iPSC-derived NPCs as three-dimensional (3D) floating neurospheres resulted in increased expression of the neural progenitor cell (NPC) markers, PAX6 and NESTIN. Expansion of NPCs in 3D culture methods also resulted in a more homogenous PAX6...... expression when compared to 2D culture methods. Furthermore, the 3D propagation method for NPCs resulted in a significant higher expression of the astrocyte markers  GFAP and aquaporin 4 (AQP4) in the differentiated cells. Thus, our 3D propagation method could constitute a useful tool to promote NPC......Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) are traditionally maintained and proliferated utilizing two-dimensional (2D) adherent monolayer culture systems. However, NPCs cultured using this system hardly reflect the intrinsic spatial development...

  4. Rare mutations of the DMBT1 gene in human astrocytic gliomas

    DEFF Research Database (Denmark)

    Mueller, Wolf; Mollenhauer, Jan; Stockhammer, Florian

    2002-01-01

    The Deleted in Malignant Brain Tumors 1 gene (DMBT1) has been proposed as a tumor suppressor gene candidate in human brain tumors, based on the observation of homozygous deletions affecting the DMBT1 region or part of the gene. In order to support this hypothesis, we performed a mutational analysis...... of the entire coding region of DMBT1, employing SSCP analysis and direct DNA sequencing in a series of 79 astrocytic gliomas. Five somatic mutations were detected. Two mutations, one of which resulted in an amino acid exchange, occurred in glioblastomas. One pilocytic astrocytoma carried two missense mutations...... and another pilocytic astrocytoma contained a somatic mutation, not affecting the presumed protein. In addition, 21 of the 27 single nucleotide polymorphisms identified in this study have not been recognized previously. The data indicate, that small mutations are not a frequent finding in gliomas....

  5. Influence of basement membrane proteins and endothelial cell-derived factors on the morphology of human fetal-derived astrocytes in 2D.

    Directory of Open Access Journals (Sweden)

    Amanda F Levy

    Full Text Available Astrocytes are the most prevalent type of glial cell in the brain, participating in a variety of diverse functions from regulating cerebral blood flow to controlling synapse formation. Astrocytes and astrocyte-conditioned media are widely used in models of the blood-brain barrier (BBB, however, very little is known about astrocyte culture in 2D. To test the hypothesis that surface coating and soluble factors influence astrocyte morphology in 2D, we quantitatively analyzed the morphology of human fetal derived astrocytes on glass, matrigel, fibronectin, collagen IV, and collagen I, and after the addition soluble factors including platelet-derived growth factor (PDGF, laminin, basic fibroblast growth factor (bFGF, and leukemia inhibitory factor (LIF. Matrigel surface coatings, as well as addition of leukemia inhibitory factor (LIF to the media, were found to have the strongest effects on 2D astrocyte morphology, and may be important in improving existing BBB models. In addition, the novel set of quantitative parameters proposed in this paper provide a test for determining the influence of compounds on astrocyte morphology, both to screen for new endothelial cell-secreted factors that influence astrocytes, and to determine in a high-throughput way which factors are important for translation to more complex, 3D BBB models.

  6. Distinct Contributions of Astrocytes and Pericytes to Neuroinflammation Identified in a 3D Human Blood-Brain Barrier on a Chip.

    Science.gov (United States)

    Herland, Anna; van der Meer, Andries D; FitzGerald, Edward A; Park, Tae-Eun; Sleeboom, Jelle J F; Ingber, Donald E

    2016-01-01

    Neurovascular inflammation is a major contributor to many neurological disorders, but modeling these processes in vitro has proven to be difficult. Here, we microengineered a three-dimensional (3D) model of the human blood-brain barrier (BBB) within a microfluidic chip by creating a cylindrical collagen gel containing a central hollow lumen inside a microchannel, culturing primary human brain microvascular endothelial cells on the gel's inner surface, and flowing medium through the lumen. Studies were carried out with the engineered microvessel containing endothelium in the presence or absence of either primary human brain pericytes beneath the endothelium or primary human brain astrocytes within the surrounding collagen gel to explore the ability of this simplified model to identify distinct contributions of these supporting cells to the neuroinflammatory response. This human 3D BBB-on-a-chip exhibited barrier permeability similar to that observed in other in vitro BBB models created with non-human cells, and when stimulated with the inflammatory trigger, tumor necrosis factor-alpha (TNF-α), different secretion profiles for granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6) were observed depending on the presence of astrocytes or pericytes. Importantly, the levels of these responses detected in the 3D BBB chip were significantly greater than when the same cells were co-cultured in static Transwell plates. Thus, as G-CSF and IL-6 have been reported to play important roles in neuroprotection and neuroactivation in vivo, this 3D BBB chip potentially offers a new method to study human neurovascular function and inflammation in vitro, and to identify physiological contributions of individual cell types.

  7. Distinct Contributions of Astrocytes and Pericytes to Neuroinflammation Identified in a 3D Human Blood-Brain Barrier on a Chip

    Science.gov (United States)

    FitzGerald, Edward A.; Park, Tae-Eun; Sleeboom, Jelle J. F.; Ingber, Donald E.

    2016-01-01

    Neurovascular inflammation is a major contributor to many neurological disorders, but modeling these processes in vitro has proven to be difficult. Here, we microengineered a three-dimensional (3D) model of the human blood-brain barrier (BBB) within a microfluidic chip by creating a cylindrical collagen gel containing a central hollow lumen inside a microchannel, culturing primary human brain microvascular endothelial cells on the gel’s inner surface, and flowing medium through the lumen. Studies were carried out with the engineered microvessel containing endothelium in the presence or absence of either primary human brain pericytes beneath the endothelium or primary human brain astrocytes within the surrounding collagen gel to explore the ability of this simplified model to identify distinct contributions of these supporting cells to the neuroinflammatory response. This human 3D BBB-on-a-chip exhibited barrier permeability similar to that observed in other in vitro BBB models created with non-human cells, and when stimulated with the inflammatory trigger, tumor necrosis factor-alpha (TNF-α), different secretion profiles for granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6) were observed depending on the presence of astrocytes or pericytes. Importantly, the levels of these responses detected in the 3D BBB chip were significantly greater than when the same cells were co-cultured in static Transwell plates. Thus, as G-CSF and IL-6 have been reported to play important roles in neuroprotection and neuroactivation in vivo, this 3D BBB chip potentially offers a new method to study human neurovascular function and inflammation in vitro, and to identify physiological contributions of individual cell types. PMID:26930059

  8. The Epigenetic Reader BRD2 as a Specific Modulator of PAI-1 Expression in Lipopolysaccharide-Stimulated Mouse Primary Astrocytes.

    Science.gov (United States)

    Choi, Chang Soon; Hong, Seong Hwi; Sim, Seobo; Cho, Kyu Suk; Kim, Ji-Woon; Yang, Sung Min; Jeon, Se Jin; You, Jueng Soo; Shin, Chan Young

    2015-11-01

    The post translational modification of lysine acetylation is a key mechanism that regulates chromatin structure. Epigenetic readers, such as the BET domains, are responsible for reading histone lysine acetylation which is a hallmark of open chromatin structure, further providing a scaffold that can be accessed by RNA polymerases as well as transcription factors. Recently, several reports have assessed and highlighted the roles of epigenetic readers in various cellular contexts. However, little is known about their role in the regulation of inflammatory genes, which is critical in exquisitely tuning inflammatory responses to a variety of immune stimuli. In this study, we investigated the role of epigenetic readers BRD2 and BRD4 in the lipopolysaccharide (LPS)-induced immune responses in mouse primary astrocytes. Inflammatory stimulation by LPS showed that the levels of Brd2 mRNA and protein were increased, while Brd4 mRNA levels did not change. Knocking down of Brd2 mRNA using specific small interfering RNA (siRNA) in cultured mouse primary astrocytes inhibited LPS-induced mRNA expression and secretion of plasminogen activator inhibitor-1 (PAI-1). However, no other pro-inflammatory cytokines, such as Il-6, Il-1β and Tnf-α, were affected. Indeed, treatment with bromodomain-containing protein inhibitor, JQ1, blocked Pai-1 mRNA expression through the inhibition of direct BRD2 protein-binding and active histone modification on Pai-1 promoter. Taken together, our data suggest that BRD2 is involved in the modulation of neuroinflammatory responses through PAI-1 and via the regulation of epigenetic reader BET protein, further providing a potential novel therapeutic strategy in neuroinflammatory diseases.

  9. Generation of human pluripotent stem cell reporter lines for the isolation of and reporting on astrocytes generated from ventral midbrain and ventral spinal cord neural progenitors

    Directory of Open Access Journals (Sweden)

    Staffan Holmqvist

    2015-07-01

    Full Text Available Astrocytes play a critical role during the development and the maintenance of the CNS in health and disease. Yet, their lack of accessibility from fetuses and from the brain of diseased patients has hindered our understanding of their full implication in developmental and pathogenic processes. Human pluripotent stem cells (PSCs are an alternative source to obtain large quantities of astrocytes in vitro, for mechanistic studies of development and disease. However, these studies often require highly pure populations of astrocytes, which are not always achieved, depending on the PSC lines and protocols used. Here, we describe the generation and characterization of human PSC reporter lines expressing TagRFP driven by the ABC1D region of the human GFAP promoter, as new cellular model for generating homogenous population of astrocytes generated from CNS regionally defined PSC-derived neural progenitors. GFAABC1D::TagRFP-expressing astrocytes can be purified by fluorescent-activated cell sorting and maintain a bright expression for several additional weeks. These express canonical astrocyte markers NF1A, S100β, CX43, GLAST, GS and CD44. These new cellular models, from which highly pure populations of fluorescence-expressing astrocytes can be obtained, provide a new platform for studies where pure or fluorescently labeled astrocyte populations are necessary, for example to assess pro-inflammatory cytokine and chemokine release in response to specific treatment, and uptake and degradation of fluorescently labeled pathogenic proteins, as reported in this study.

  10. High-efficient generation of induced pluripotent stem cells from human astrocytes.

    Science.gov (United States)

    Ruiz, Sergio; Brennand, Kristen; Panopoulos, Athanasia D; Herrerías, Aída; Gage, Fred H; Izpisua-Belmonte, Juan Carlos

    2010-12-09

    The reprogramming of human somatic cells to induced pluripotent stem (hiPS) cells enables the possibility of generating patient-specific autologous cells for regenerative medicine. A number of human somatic cell types have been reported to generate hiPS cells, including fibroblasts, keratinocytes and peripheral blood cells, with variable reprogramming efficiencies and kinetics. Here, we show that human astrocytes can also be reprogrammed into hiPS (ASThiPS) cells, with similar efficiencies to keratinocytes, which are currently reported to have one of the highest somatic reprogramming efficiencies. ASThiPS lines were indistinguishable from human embryonic stem (ES) cells based on the expression of pluripotent markers and the ability to differentiate into the three embryonic germ layers in vitro by embryoid body generation and in vivo by teratoma formation after injection into immunodeficient mice. Our data demonstrates that a human differentiated neural cell type can be reprogrammed to pluripotency and is consistent with the universality of the somatic reprogramming procedure.

  11. High-efficient generation of induced pluripotent stem cells from human astrocytes.

    Directory of Open Access Journals (Sweden)

    Sergio Ruiz

    Full Text Available The reprogramming of human somatic cells to induced pluripotent stem (hiPS cells enables the possibility of generating patient-specific autologous cells for regenerative medicine. A number of human somatic cell types have been reported to generate hiPS cells, including fibroblasts, keratinocytes and peripheral blood cells, with variable reprogramming efficiencies and kinetics. Here, we show that human astrocytes can also be reprogrammed into hiPS (ASThiPS cells, with similar efficiencies to keratinocytes, which are currently reported to have one of the highest somatic reprogramming efficiencies. ASThiPS lines were indistinguishable from human embryonic stem (ES cells based on the expression of pluripotent markers and the ability to differentiate into the three embryonic germ layers in vitro by embryoid body generation and in vivo by teratoma formation after injection into immunodeficient mice. Our data demonstrates that a human differentiated neural cell type can be reprogrammed to pluripotency and is consistent with the universality of the somatic reprogramming procedure.

  12. RNA Localization in Astrocytes

    DEFF Research Database (Denmark)

    Thomsen, Rune

    2012-01-01

    Messenger RNA (mRNA) localization is a mechanism by which polarized cells can regulate protein synthesis to specific subcellular compartments in a spatial and temporal manner, and plays a pivotal role in multiple physiological processes from embryonic development to cell differentiation......, regulation of the blood brain barrier and glial scar tissue formation. Despite the involvement in various CNS functions only a limited number of studies have addressed mRNA localization in astrocytes. This PhD project was initially focused on developing and implementing methods that could be used to asses mRNA...... localization in astrocyte protrusions, and following look into the subcellular localization pattern of specific mRNA species of both primary astrocytes isolated from cortical hemispheres of newborn mice, and the mouse astrocyte cell line, C8S. The Boyden chamber cell fractionation assay was optimized, in a way...

  13. Stages of restricted HIV-1 infection in astrocyte cultures derived from human fetal brain tissue.

    Science.gov (United States)

    Messam, C A; Major, E O

    2000-05-01

    The predominant cell types infected by HIV-1 in AIDS associated encephalopathy are cells of the macrophage/microglial lineage. There has been consistent evidence, however, that astrocytes also become infected although not at the same frequency or level of multiplication as microglial cells. HIV-1 antigens and/or nucleic acid have been identified in astrocytes in brain autopsy tissue from both adult and pediatric AIDS cases. In cell cultures, HIV-1 infection of astrocytes results in an initial productive but non-cytopathogenic infection that diminishes to a viral persistence or latent state. Understanding the nature of HIV-1 infection of astrocytes, which represents the largest population of cells in the brain, will contribute to the understanding of AIDS encephalopathy and the dementia that occurs in nearly one-quarter of all AIDS patients.

  14. Comparative effects on rat primary astrocytes and C6 rat glioma cells cultures after 24-h exposure to silver nanoparticles (AgNPs)

    Energy Technology Data Exchange (ETDEWEB)

    Salazar-García, Samuel; Silva-Ramírez, Ana Sonia; Ramirez-Lee, Manuel A.; Rosas-Hernandez, Hector [Universidad Autonoma de San Luis Potosi, Facultad de Ciencias Quimicas (Mexico); Rangel-López, Edgar [Instituto Nacional de Neurologia y Neurocirugia Manuel Velasco Suárez, Laboratorio de Aminoacidos Excitadores (Mexico); Castillo, Claudia G. [Facultad de Medicina, Universidad Autonoma de San Luis Potosi (Mexico); Santamaría, Abel [Instituto Nacional de Neurologia y Neurocirugia Manuel Velasco Suárez, Laboratorio de Aminoacidos Excitadores (Mexico); Martinez-Castañon, Gabriel A. [Universidad Autonoma de San Luis Potosi, Facultad de Estomatologia (Mexico); Gonzalez, Carmen, E-mail: cgonzalez.uaslp@gmail.com, E-mail: gonzalez.castillocarmen@fcq.uaslp.mx [Universidad Autonoma de San Luis Potosi, Facultad de Ciencias Quimicas (Mexico)

    2015-11-15

    The aim of this work was to compare the effects of 24-h exposure of rat primary astrocytes and C6 rat glioma cells to 7.8 nm AgNPs. Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor and current treatments lead to diverse side-effects; for this reason, it is imperative to investigate new approaches, including those alternatives provided by nanotechnology, like nanomaterials (NMs) such as silver nanoparticles. Herein, we found that C6 rat glioma cells, but no primary astrocytes, decreased cell viability after AgNPs treatment; however, both cell types diminished their proliferation. The decrease of glioma C6 cells proliferation was related with necrosis, while in primary astrocytes, the decreased proliferation was associated with the induction of apoptosis. The ionic control (AgNO{sub 3}) exerted a different profile than AgNPs; the bulk form did not modify the basal effect in each determination, whereas cisplatin, a well-known antitumoral drug used as a comparative control, promoted cytotoxicity in both cell types at specific concentrations. Our findings prompt the need to determine the fine molecular and cellular mechanisms involved in the differential biological responses to AgNPs in order to develop new tools or alternatives based on nanotechnology that may contribute to the understanding, impact and use of NMs in specific targets, like glioblastoma cells.

  15. Acrylamide-induced neurotoxicity in primary astrocytes and microglia: Roles of the Nrf2-ARE and NF-κB pathways.

    Science.gov (United States)

    Zhao, Mengyao; Lewis Wang, Fu Sheng; Hu, Xiaosong; Chen, Fang; Chan, Hing Man

    2017-08-01

    Acrylamide (AA) is a common food contaminant formed during food heat processing that has neurotoxic effects. We hypothesize that AA induces oxidative stress in astrocytes and microglia, leading to neurotoxicity. Oxidative status, translocation of Nrf2 and NF-κB, and related down-stream targets were measured in primary astrocytes and microglia obtained from BALB/c mice. The results showed that AA increased reactive oxygen species (ROS) formation and reduced glutathione levels, causing successive events associated with oxidative stress, including 4-hydroxynonenal and 8-hydroxy-2-deoxyguanosine adduct formation, in both cell types. Both Nrf2 and NF-κB pathways were activated, but Nrf2 and its downstream antioxidative genes acted at earlier stages in both cell types before NF-κB activation. After NF-κB activation, related cytokines, including IL-6, TNF-α, G-CSF, and IL-1β, were released and cell viability decreased. Greater ROS generation, faster glutathione reduction, and increased oxidative adduct formation were observed in microglia compared with astrocytes. Moreover, Nrf2/NF-κB and its downstream genes were up-regulated much faster and to greater degrees in microglia than astrocytes. These results clarify the roles of the Nrf2 and NF-κB pathways in AA-induced neurotoxicity. These cellular responses may provide new insights for the development of adverse outcome pathway approaches for risk assessments of AA. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. 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.

    Science.gov (United States)

    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.

  17. 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.

    Science.gov (United States)

    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.

  18. Epigallocatechin-3-gallate induces oxidative phosphorylation by activating cytochrome c oxidase in human cultured neurons and astrocytes.

    Science.gov (United States)

    Castellano-González, Gloria; Pichaud, Nicolas; Ballard, J William O; Bessede, Alban; Marcal, Helder; Guillemin, Gilles J

    2016-02-16

    Mitochondrial dysfunction and resulting energy impairment have been identified as features of many neurodegenerative diseases. Whether this energy impairment is the cause of the disease or the consequence of preceding impairment(s) is still under discussion, however a recovery of cellular bioenergetics would plausibly prevent or improve the pathology. In this study, we screened different natural molecules for their ability to increase intracellular adenine triphosphate purine (ATP). Among them, epigallocatechin-3-gallate (EGCG), a polyphenol from green tea, presented the most striking results. We found that it increases ATP production in both human cultured astrocytes and neurons with different kinetic parameters and without toxicity. Specifically, we showed that oxidative phosphorylation in human cultured astrocytes and neurons increased at the level of the routine respiration on the cells pre-treated with the natural molecule. Furthermore, EGCG-induced ATP production was only blocked by sodium azide (NaN3) and oligomycin, inhibitors of cytochrome c oxidase (CcO; complex IV) and ATP synthase (complex V) respectively. These findings suggest that the EGCG modulates CcO activity, as confirmed by its enzymatic activity. CcO is known to be regulated differently in neurons and astrocytes. Accordingly, EGCG treatment is acting differently on the kinetic parameters of the two cell types. To our knowledge, this is the first study showing that EGCG promotes CcO activity in human cultured neurons and astrocytes. Considering that CcO dysfunction has been reported in patients having neurodegenerative diseases such as Alzheimer's disease (AD), we therefore suggest that EGCG could restore mitochondrial function and prevent subsequent loss of synaptic function.

  19. Porosome in astrocytes.

    Science.gov (United States)

    Lee, Jin-Sook; Cho, Won Jin; Jeftinija, Ksenija; Jeftinija, Srdija; Jena, Bhanu P

    2009-02-01

    Secretion is a universal cellular process occurring in bakers yeast, to the complex multicellular organisms, to humans beings. Neurotransmission, digestion, immune response or the release of hormones occur as a result of cell secretion. Secretory defects result in numerous diseases and hence a molecular understanding of the process is critical. Cell secretion involves the transport of vesicular products from within cells to the outside. Porosomes are permanent cup-shaped supramolecular structures at the cell plasma membrane, where secretory vesicles transiently dock and transiently fuse to release intravesicular contents to the outside. In the past decade, porosomes have been determined to be the universal secretory machinery in cells, present in the exocrine pancreas, endocrine and neuroendocrine cells, and in neurons. In this study, we report for the first time the presence of porosomes in rat brain astrocytes. Using atomic force microscopy on live astrocytes, cup-shaped porosomes measuring 10-15 nm are observed at the cell plasma membrane. Further studies using electron microscopy confirm the presence of porosomes in astrocytes. Analogous to neuronal porosomes, there is a central plug in the astrocyte porosome complex. Immunoisolation and reconstitution of the astrocyte porosome in lipid membrane, demonstrates a structure similar to what is observed in live cells. These studies demonstrate that in astrocytes, the secretory apparatus at the cell plasma membrane is similar to what is found in neurons.

  20. A predictive in vitro model of the impact of drugs with anticholinergic properties on human neuronal and astrocytic systems.

    Directory of Open Access Journals (Sweden)

    Elizabeth K Woehrling

    Full Text Available The link between off-target anticholinergic effects of medications and acute cognitive impairment in older adults requires urgent investigation. We aimed to determine whether a relevant in vitro model may aid the identification of anticholinergic responses to drugs and the prediction of anticholinergic risk during polypharmacy. In this preliminary study we employed a co-culture of human-derived neurons and astrocytes (NT2.N/A derived from the NT2 cell line. NT2.N/A cells possess much of the functionality of mature neurons and astrocytes, key cholinergic phenotypic markers and muscarinic acetylcholine receptors (mAChRs. The cholinergic response of NT2 astrocytes to the mAChR agonist oxotremorine was examined using the fluorescent dye fluo-4 to quantitate increases in intracellular calcium [Ca2+]i. Inhibition of this response by drugs classified as severe (dicycloverine, amitriptyline, moderate (cyclobenzaprine and possible (cimetidine on the Anticholinergic Cognitive Burden (ACB scale, was examined after exposure to individual and pairs of compounds. Individually, dicycloverine had the most significant effect regarding inhibition of the astrocytic cholinergic response to oxotremorine, followed by amitriptyline then cyclobenzaprine and cimetidine, in agreement with the ACB scale. In combination, dicycloverine with cyclobenzaprine had the most significant effect, followed by dicycloverine with amitriptyline. The order of potency of the drugs in combination frequently disagreed with predicted ACB scores derived from summation of the individual drug scores, suggesting current scales may underestimate the effect of polypharmacy. Overall, this NT2.N/A model may be appropriate for further investigation of adverse anticholinergic effects of multiple medications, in order to inform clinical choices of suitable drug use in the elderly.

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

    Science.gov (United States)

    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.

  2. Maxi-anion channel as a candidate pathway for osmosensitive ATP release from mouse astrocytes in primary culture

    Institute of Scientific and Technical Information of China (English)

    Hong-Tao Liu; Abduqodir H Toychiev; Nobuyuki Takahashi; Ravshan Z Sabirov; Yasunobu Okada

    2008-01-01

    In the present study,we aimed to evaluate the pathways contributing to ATP release from mouse astrocytes during hypoosmotic stress.We first examined the expression of mRNAs for proteins constituting possible ATPreleasing pathways that have been suggested over the past several years.In RT-PCR analysis using both control and osmotically swollen astrocytes,amplification of cDNA fragments of expected size was seen for connexins (Cx32,Cx37,Cx43),pannexin 1 (Pxl),the P2X7 receptor,MRP1 and MDRI,but not CFTR.Inhibitors of exocytotie vesicular release,gap junction hemi-channels,CFTR,MRPI,MDR1,the P2X7 receptor,and volume-sensitive outwardly rectifying chloride channels had no significant effects on the massive ATP release from astrocytes.In contrast,the hypotonicity-induced ATP release from astrocytes was most effectively inhibited by gadolinium release from several other cell types.Thus,we propose that the maxi-anion channel constitutes a major pathway for swelling-induced ATP release from cultured mouse astrocytes as well.

  3. Gene expression Analysis of Neurons and Astrocytes Isolated by Laser Capture Microdissection from Frozen Human Brain Tissues.

    Directory of Open Access Journals (Sweden)

    Lidia Tagliafierro

    2016-08-01

    Full Text Available Different cell types and multiple cellular connections characterize the human brain. Gene expression analysis using a specific population of cells is more accurate than conducting analysis of the whole tissue homogenate, particularly in the context of neurodegenerative diseases, where a specific subset of cells is affected by the different pathology. Due to the difficulty to obtain homogenous cell populations, gene expression in specific cell-types (neurons, astrocytes, etc. has been understudied. To leverage the use of archive resources of frozen human brains in studies of neurodegenerative diseases, we developed and calibrated a method to quantify cell-type specific – neuronal, astrocytes – expression profiles of genes implicated in neurodegenerative diseases, including Parkinson’s and Alzheimer’s diseases. Archive human frozen brain tissues were used to prepare slides for rapid immunostaining using cell-specific antibodies. The immunoreactive-cells were isolated by Laser Capture Microdissection (LCM. The enrichment for a particular cell-type of interest was validated in post-analysis stage by the expression of cell-specific markers. We optimized the technique to preserve the RNA integrity, so that the RNA was suitable for downstream expression analyses. Following RNA extraction, the expression levels were determined digitally using nCounter Single Cell Gene Expression assay (NanoString Technologies®. The results demonstrated that using our optimized technique we successfully isolated single neurons and astrocytes from human frozen brain tissues and obtained RNA of a good quality that was suitable for mRNA expression analysis. We present here new advancements compared to previous reported methods, which improve the method’s feasibility and its applicability for a variety of downstream molecular analyses. Our new developed method can be implemented in genetic and functional genomic research of neurodegenerative diseases and has the

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

    Science.gov (United States)

    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.

  5. Chlamydophila (Chlamydia) pneumoniae infection of human astrocytes and microglia in culture displays an active, rather than a persistent, phenotype.

    Science.gov (United States)

    Dreses-Werringloer, Ute; Gérard, Hervé C; Whittum-Hudson, Judith A; Hudson, Alan P

    2006-10-01

    The intracellular pathogen Chlamydia pneumoniae can cause persistent infections during which its morphologic, molecular, and pathogenic characteristics differ importantly from those of active infection. This bacterium was identified within astrocytes and microglia in the brain of late-onset Alzheimer disease patients. We investigated whether infection of these two host cell types displays an active or persistent growth phenotype. The human astrocytoma and microglioma cell lines U-87 MG and CHME-5 (respectively) and the human epithelial cell line HEp-2 were infected by the standard method with C pneumoniae strain AR-39. Cultures were harvested at 24, 48, and 72 hours postinfection and subjected to analysis of inclusion morphology. DNA and RNA were prepared from portions of each infected culture sample and analyzed for relative chromosome accumulation and presence or absence of several specific bacterial mRNAs. Astrocytes and microglial cells infected in vitro with C pneumoniae displayed inclusions that were indistinguishable from those characteristic of active infection of the standard HEp-2 host cell line. Real time polymerase chain reaction (PCR) showed that the relative accumulation of chlamydial chromosome over time during infection of these two cell lines also was virtually identical to that in actively infected HEp-2 cells. Reverse transcriptase PCR (RT-PCR) analyses showed that mRNA from ftsK, pyk, and other chlamydial genes whose expression is abrogated during persistent infection were easily identifiable in infected CHME-5 and U-87 MG cells. In cultured human astrocytes and microglia, C pneumoniae displays an active, not a persistent, growth phenotype. This indicates normal passage through the developmental cycle with its probable concomitant destruction by lysis of some portion of host cells at the termination of that cycle.

  6. Astrocytes in Migration.

    Science.gov (United States)

    Zhan, Jiang Shan; Gao, Kai; Chai, Rui Chao; Jia, Xi Hua; Luo, Dao Peng; Ge, Guo; Jiang, Yu Wu; Fung, Yin-Wan Wendy; Li, Lina; Yu, Albert Cheung Hoi

    2017-01-01

    Cell migration is a fundamental phenomenon that underlies tissue morphogenesis, wound healing, immune response, and cancer metastasis. Great progresses have been made in research methodologies, with cell migration identified as a highly orchestrated process. Brain is considered the most complex organ in the human body, containing many types of neural cells with astrocytes playing crucial roles in monitoring normal functions of the central nervous system. Astrocytes are mostly quiescent under normal physiological conditions in the adult brain but become migratory after injury. Under most known pathological conditions in the brain, spinal cord and retina, astrocytes are activated and become hypertrophic, hyperplastic, and up-regulating GFAP based on the grades of severity. These three observations are the hallmark in glia scar formation-astrogliosis. The reactivation process is initiated with structural changes involving cell process migration and ended with cell migration. Detailed mechanisms in astrocyte migration have not been studied extensively and remain largely unknown. Here, we therefore attempt to review the mechanisms in migration of astrocytes.

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

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jin-Sun; Kim, Hee-Sun, E-mail: hskimp@ewha.ac.kr

    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.

  8. 2-Chloroethanol Induced Upregulation of Matrix Metalloproteinase-2 in Primary Cultured Rat Astrocytes Via MAPK Signal Pathways

    Science.gov (United States)

    Sun, Qi; Liao, Yingjun; Wang, Tong; Tang, Hongge; Wang, Gaoyang; Zhao, Fenghong; Jin, Yaping

    2017-01-01

    This study was to explore the mechanisms underlying 1,2-dichloroethane (1,2-DCE) induced brain edema by focusing on alteration of matrix metalloproteinase-2 (MMP-2) in rat astrocytes induced by 2-chloroethanol (2-CE), an intermediate metabolite of 1,2-DCE in vivo. Protein and mRNA levels of MMP-2, and the phosphorylated protein levels of p38 MAPK (p-p38), extracellular signal regulated protein kinase (p-ERK1/2) and c-Jun N-terminal kinase (p-JNK1/2) in astrocytes were examined by immunostaining, western blot or real-time RT-PCR analysis. Findings from this study disclosed that protein levels of MMP-2 were upregulated by 2-CE in astrocytes. Meanwhile, protein levels of p-p38, p-ERK1/2 and p-JNK1/2 were also increased apparently in the cells treated with 2-CE. Moreover, pretreatment of astrocytes with SB202190 (inhibitor of p38 MAPK), U0126 (inhibitor of ERK1/2) or SP600125 (inhibitor of JNK1/2) could suppress the upregulated expression of p-p38, p-ERK1/2, and p-JNK1/2. In response to suppressed protein levels of p-p38 and p-JNK1/2, the protein levels of MMP-2 also decreased significantly, indicating that activation of MAPK signal pathways were involved in the mechanisms underlying 2-CE-induced upregulation of MMP-2 expression. PMID:28101000

  9. Expression and activation by Epstein Barr virus of human endogenous retroviruses-W in blood cells and astrocytes: inference for multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Giuseppe Mameli

    Full Text Available BACKGROUND: Proposed co-factors triggering the pathogenesis of multiple sclerosis (MS are the Epstein Barr virus (EBV, and the potentially neuropathogenic MSRV (MS-associated retrovirus and syncytin-1, of the W family of human endogenous retroviruses. METHODOLOGY/PRINCIPAL FINDINGS: In search of links, the expression of HERV-W/MSRV/syncytin-1, with/without exposure to EBV or to EBV glycoprotein350 (EBVgp350, was studied on peripheral blood mononuclear cells (PBMC from healthy volunteers and MS patients, and on astrocytes, by discriminatory env-specific RT-PCR assays, and by flow cytometry. Basal expression of HERV-W/MSRV/syncytin-1 occurs in astrocytes and in monocytes, NK, and B, but not in T cells. This uneven expression is amplified in untreated MS patients, and dramatically reduced during therapy. In astrocytes, EBVgp350 stimulates the expression of HERV-W/MSRV/syncytin-1, with requirement of the NF-κB pathway. In EBVgp350-treated PBMC, MSRVenv and syncytin-1 transcription is activated in B cells and monocytes, but not in T cells, nor in the highly expressing NK cells. The latter cells, but not the T cells, are activated by proinflammatory cytokines. CONCLUSIONS/SIGNIFICANCE: In vitro EBV activates the potentially immunopathogenic and neuropathogenic HERV-W/MSRV/syncytin-1, in cells deriving from blood and brain. In vivo, pathogenic outcomes would depend on abnormal situations, as in late EBV primary infection, that is often symptomatic, or/and in the presence of particular host genetic backgrounds. In the blood, HERV-Wenv activation might induce immunopathogenic phenomena linked to its superantigenic properties. In the brain, toxic mechanisms against oligodendrocytes could be established, inducing inflammation, demyelination and axonal damage. Local stimulation by proinflammatory cytokines and other factors might activate further HERV-Ws, contributing to the neuropathogenity. In MS pathogenesis, a possible model could include EBV as

  10. Expression and functional activity of the ABC-transporter proteins P-glycoprotein and multidrug-resistance protein 1 in human brain tumor cells and astrocytes.

    Science.gov (United States)

    Spiegl-Kreinecker, Sabine; Buchroithner, Johanna; Elbling, Leonilla; Steiner, Elisabeth; Wurm, Gabriele; Bodenteich, Angelika; Fischer, Johannes; Micksche, Michael; Berger, Walter

    2002-03-01

    The poor prognosis of glioma patients is partly based on the minor success obtained from chemotherapeutic treatments. Resistance mechanisms at the tumor cell level may be, in addition to the blood-brain barrier, involved in the intrinsic chemo-insensitivity of brain tumors. We investigated the expression of the drug-transporter proteins P-glycoprotein (P-gp) and multidrug-resistance protein 1 (MRP1) in cell lines (N = 24) and primary cell cultures (N = 36) from neuroectodermal tumors, as well as in brain tumor extracts (N = 18) and normal human astrocytes (N = 1). We found that a considerable expression of P-gp was relatively rare in glioma cells, in contrast to MRP1, which was constitutively overexpressed in cells derived from astrocytomas as well as glioblastomas. Also, normal astrocytes cultured in vitro expressed high amounts of MRPI but no detectable P-gp. Meningioma cells frequently co-expressed P-gp and MRP1, while, most of the neuroblastoma cell lines express higher P-gp but lower MRP1 levels as compared to the other tumor types. Both, a drug-exporting and a chemoprotective function of P-gp as well as MRP1 could be demonstrated in selected tumor cells by a significant upregulation of cellular 3H-daunomycin accumulation and daunomycin cytotoxicity via administration of transporter antagonists. Summing up, our data suggest that P-gp contributes to cellular resistance merely in a small subgroup of gliomas, but frequently in neuroblastomas and meningiomas. In contrast, MRP1 is demonstrated to play a constitutive role in the intrinsic chemoresistance of gliomas and their normal cell counterpart.

  11. Taurine Biosynthesis by Neurons and Astrocytes*

    Science.gov (United States)

    Vitvitsky, Victor; Garg, Sanjay K.; Banerjee, Ruma

    2011-01-01

    The physiological roles of taurine, a product of cysteine degradation and one of the most abundant amino acids in the body, remain elusive. Taurine deficiency leads to heart dysfunction, brain development abnormalities, retinal degradation, and other pathologies. The taurine synthetic pathway is proposed to be incomplete in astrocytes and neurons, and metabolic cooperation between these cell types is reportedly needed to complete the pathway. In this study, we analyzed taurine synthesis capability as reported by incorporation of radioactivity from [35S]cysteine into taurine, in primary murine astrocytes and neurons, and in several transformed cell lines (human (SH-SY5Y) and murine (N1E-115) neuroblastoma, human astrocytoma (U-87MG and 1321 N1), and rat glioma (C6)). Extensive incorporation of radioactivity from [35S]cysteine into taurine was observed in rat glioma cells as well as in primary mouse astrocytes and neurons, establishing the presence of an intact taurine synthesis pathway in these cells. Interestingly, exposure of cells to cysteine or cysteamine resulted in elevated intracellular hypotaurine without a corresponding increase in taurine levels, suggesting that oxidation of hypotaurine limits taurine synthesis in cells. Consistent with its role as an organic osmolyte, taurine synthesis was stimulated under hypertonic conditions in neurons. PMID:21778230

  12. Taurine biosynthesis by neurons and astrocytes.

    Science.gov (United States)

    Vitvitsky, Victor; Garg, Sanjay K; Banerjee, Ruma

    2011-09-16

    The physiological roles of taurine, a product of cysteine degradation and one of the most abundant amino acids in the body, remain elusive. Taurine deficiency leads to heart dysfunction, brain development abnormalities, retinal degradation, and other pathologies. The taurine synthetic pathway is proposed to be incomplete in astrocytes and neurons, and metabolic cooperation between these cell types is reportedly needed to complete the pathway. In this study, we analyzed taurine synthesis capability as reported by incorporation of radioactivity from [(35)S]cysteine into taurine, in primary murine astrocytes and neurons, and in several transformed cell lines (human (SH-SY5Y) and murine (N1E-115) neuroblastoma, human astrocytoma (U-87 MG and 1321 N1), and rat glioma (C6)). Extensive incorporation of radioactivity from [(35)S]cysteine into taurine was observed in rat glioma cells as well as in primary mouse astrocytes and neurons, establishing the presence of an intact taurine synthesis pathway in these cells. Interestingly, exposure of cells to cysteine or cysteamine resulted in elevated intracellular hypotaurine without a corresponding increase in taurine levels, suggesting that oxidation of hypotaurine limits taurine synthesis in cells. Consistent with its role as an organic osmolyte, taurine synthesis was stimulated under hypertonic conditions in neurons.

  13. Reactive Astrocytes Expressing Intense Estrogen Receptor-alpha Immunoreactivities Have Much Elongated Cytoplasmic Processes: An Autopsy Case of Human Cerebellar Tissue with Multiple Genitourinary and Gastrointestinal Anomalies

    Science.gov (United States)

    Kim, Eo-Jin; Oh, Chang Seok; Kim, Jaehyup; Kim, Wu Ho; Chung, Yoon Hee

    2007-01-01

    We performed an immunohistochemical study on the estrogen receptor alpha (ER-α) distribution in the cerebellum of a human neonate with multiple congenital anomalies, that had been acquired during autopsy. Although the exact pathology in the brain was not clearly elucidated in this study, an unidentified stressful condition might have induced the astrocytes into reactive states. In this immunohistochemical study on the neonatal cerebellum with multiple congenital anomalies, intense ER-α immunoreactivities (IRs) were localized mainly within the white matter even though ER-α IRs were known to be mainly localized in neurons. Double immunohistochemical staining showed that ER-α IR cells were reactive astrocytes, but not neurons. Interestingly, there were differences in the process length among the reactive astrocytes showing ER-α IRs. Our quantitative data confirmed that among the glial fibrillary acidic protein (GFAP)-expressing reactive astrocytes, the cells exhibiting intense ER-α IRs have much longer cytoplasmic processes and relatively weaker GFAP IRs. Taken together, the elongated processes of reactive astrocytes might be due to decreased expression of GFAP, which might be induced by elevated expression of ER-α even though the elucidation of the exact mechanism needs further studies. PMID:17982251

  14. Essential role of mitogen-activated protein kinase pathways in protease activated receptor 2-mediated nitric-oxide production from rat primary astrocytes.

    Science.gov (United States)

    Park, Gyu Hwan; Jeon, Se Jin; Ryu, Jae Ryun; Choi, Min Sik; Han, Seol-Heui; Yang, Sung-Il; Ryu, Jong Hoon; Cheong, Jae Hoon; Shin, Chan Young; Ko, Kwang Ho

    2009-09-01

    Protease-activated receptors (PARs) play important roles in the regulation of brain function such as neuroinflammation by transmitting the signal from proteolytic enzymes such as thrombin and trypsin. We and others have reported that a member of the family, PAR-2 is activated by trypsin, whose involvement in the neurophysiological process is increasingly evident, and is involved in the neuroinflammatory processes including morphological changes of astrocytes. In this study, we investigated the role of PAR-2 in the production of nitric oxide (NO) in rat primary astrocytes. Treatment of PAR-2 agonist trypsin increased NO production in a dose-dependent manner, which was mediated by the induction of inducible nitric-oxide synthase. The trypsin-mediated production of NO was mimicked by PAR-2 agonist peptide and reduced by either pharmacological PAR-2 antagonist peptide or by siRNA-mediated inhibition of PAR-2 expression, which suggests the critical role of PAR-2 in this process. NO production by PAR-2 was mimicked by PMA, a PKC activator, and was attenuated by Go6976, a protein kinase C (PKC) inhibitor. PAR-2 stimulation activated three subtypes of mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. NO production by PAR-2 was blocked by inhibition of ERK, p38, and JNK pathways. PAR-2 stimulation also activated nuclear factor-kappaB (NF-kappaB) DNA binding and transcriptional activity as well as IkappaBalpha phosphorylation. Inhibitors of NF-kappaB pathway inhibited PAR-2-mediated NO production. In addition, inhibitors of MAPK pathways prevented transcriptional activation of NF-kappaB reporter constructs. These results suggest that PAR-2 activation-mediated NO production in astrocytes is transduced by the activation of MAPKs followed by NF-kappaB pathways.

  15. Expression of the human isoform of glutamate dehydrogenase, hGDH2, augments TCA cycle capacity and oxidative metabolism of glutamate during glucose deprivation in astrocytes.

    Science.gov (United States)

    Nissen, Jakob D; Lykke, Kasper; Bryk, Jaroslaw; Stridh, Malin H; Zaganas, Ioannis; Skytt, Dorte M; Schousboe, Arne; Bak, Lasse K; Enard, Wolfgang; Pääbo, Svante; Waagepetersen, Helle S

    2017-03-01

    A key enzyme in brain glutamate homeostasis is glutamate dehydrogenase (GDH) which links carbohydrate and amino acid metabolism mediating glutamate degradation to CO2 and expanding tricarboxylic acid (TCA) cycle capacity with intermediates, i.e. anaplerosis. Humans express two GDH isoforms, GDH1 and 2, whereas most other mammals express only GDH1. hGDH1 is widely expressed in human brain while hGDH2 is confined to astrocytes. The two isoforms display different enzymatic properties and the nature of these supports that hGDH2 expression in astrocytes potentially increases glutamate oxidation and supports the TCA cycle during energy-demanding processes such as high intensity glutamatergic signaling. However, little is known about how expression of hGDH2 affects the handling of glutamate and TCA cycle metabolism in astrocytes. Therefore, we cultured astrocytes from cerebral cortical tissue of hGDH2-expressing transgenic mice. We measured glutamate uptake and metabolism using [(3) H]glutamate, while the effect on metabolic pathways of glutamate and glucose was evaluated by use of (13) C and (14) C substrates and analysis by mass spectrometry and determination of radioactively labeled metabolites including CO2 , respectively. We conclude that hGDH2 expression increases capacity for uptake and oxidative metabolism of glutamate, particularly during increased workload and aglycemia. Additionally, hGDH2 expression increased utilization of branched-chain amino acids (BCAA) during aglycemia and caused a general decrease in oxidative glucose metabolism. We speculate, that expression of hGDH2 allows astrocytes to spare glucose and utilize BCAAs during substrate shortages. These findings support the proposed role of hGDH2 in astrocytes as an important fail-safe during situations of intense glutamatergic activity. GLIA 2017;65:474-488.

  16. Primary ciliogenesis defects are associated with human astrocytoma/glioblastoma cells

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    Rattner Jerome B

    2009-12-01

    Full Text Available Abstract Background Primary cilia are non-motile sensory cytoplasmic organelles that have been implicated in signal transduction, cell to cell communication, left and right pattern embryonic development, sensation of fluid flow, regulation of calcium levels, mechanosensation, growth factor signaling and cell cycle progression. Defects in the formation and/or function of these structures underlie a variety of human diseases such as Alström, Bardet-Biedl, Joubert, Meckel-Gruber and oral-facial-digital type 1 syndromes. The expression and function of primary cilia in cancer cells has now become a focus of attention but has not been studied in astrocytomas/glioblastomas. To begin to address this issue, we compared the structure and expression of primary cilia in a normal human astrocyte cell line with five human astrocytoma/glioblastoma cell lines. Methods Cultured normal human astrocytes and five human astrocytoma/glioblastoma cell lines were examined for primary cilia expression and structure using indirect immunofluorescence and electron microscopy. Monospecific antibodies were used to detect primary cilia and map the relationship between the primary cilia region and sites of endocytosis. Results We show that expression of primary cilia in normal astrocytes is cell cycle related and the primary cilium extends through the cell within a unique structure which we show to be a site of endocytosis. Importantly, we document that in each of the five astrocytoma/glioblastoma cell lines fully formed primary cilia are either expressed at a very low level, are completely absent or have aberrant forms, due to incomplete ciliogenesis. Conclusions The recent discovery of the importance of primary cilia in a variety of cell functions raises the possibility that this structure may have a role in a variety of cancers. Our finding that the formation of the primary cilium is disrupted in cells derived from astrocytoma/glioblastoma tumors provides the first

  17. Identification of soluble CD14 as an endogenous agonist for toll-like receptor 2 on human astrocytes by genome-scale functional screening of glial cell derived proteins

    NARCIS (Netherlands)

    Bsibsi, M.; Bajramovic, J.J.; Duijvenvoorden, E. van; Persoon, C.; Ravid, R.; Noort, J.M. van; Vogt, M.H.J.

    2007-01-01

    Human astrocytes express a limited repertoire of Toll-like receptor (TLR) family members including TLR1-4, which are expressed on the cell surface. Also, TLR3 but not TLR4 activation on astrocytes induces expression of several factors involved in neuroprotection and down-regulation of inflammation r

  18. Identification of soluble CD14 as an endogenous agonist for toll-like receptor 2 on human astrocytes by genome-scale functional screening of glial cell derived proteins

    NARCIS (Netherlands)

    Bsibsi, M.; Bajramovic, J.J.; Duijvenvoorden, E. van; Persoon, C.; Ravid, R.; Noort, J.M. van; Vogt, M.H.J.

    2007-01-01

    Human astrocytes express a limited repertoire of Toll-like receptor (TLR) family members including TLR1-4, which are expressed on the cell surface. Also, TLR3 but not TLR4 activation on astrocytes induces expression of several factors involved in neuroprotection and down-regulation of inflammation r

  19. Therapeutic potential of mesenchymal stromal cells and MSC conditioned medium in Amyotrophic Lateral Sclerosis (ALS--in vitro evidence from primary motor neuron cultures, NSC-34 cells, astrocytes and microglia.

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    Hui Sun

    Full Text Available Administration of mesenchymal stromal cells (MSC improves functional outcome in the SOD1G93A mouse model of the degenerative motor neuron disorder amyotrophic lateral sclerosis (ALS as well as in models of other neurological disorders. We have now investigated the effect of the interaction between MSC and motor neurons (derived from both non-transgenic and mutant SOD1G93A transgenic mice, NSC-34 cells and glial cells (astrocytes, microglia (derived again from both non-transgenic and mutant SOD1G93A ALS transgenic mice in vitro. In primary motor neurons, NSC-34 cells and astrocytes, MSC conditioned medium (MSC CM attenuated staurosporine (STS - induced apoptosis in a concentration-dependent manner. Studying MSC CM-induced expression of neurotrophic factors in astrocytes and NSC-34 cells, we found that glial cell line-derived neurotrophic factor (GDNF and ciliary neurotrophic factor (CNTF gene expression in astrocytes were significantly enhanced by MSC CM, with differential responses of non-transgenic and mutant astrocytes. Expression of Vascular Endothelial Growth Factor (VEGF in NSC-34 cells was significantly upregulated upon MSC CM-treatment. MSC CM significantly reduced the expression of the cytokines TNFα and IL-6 and iNOS both in transgenic and non-transgenic astrocytes. Gene expression of the neuroprotective chemokine Fractalkine (CX3CL1 was also upregulated in mutant SOD1G93A transgenic astrocytes by MSC CM treatment. Correspondingly, MSC CM increased the respective receptor, CX3CR1, in mutant SOD1G93A transgenic microglia. Our data demonstrate that MSC modulate motor neuronal and glial response to apoptosis and inflammation. MSC therefore represent an interesting candidate for further preclinical and clinical evaluation in ALS.

  20. Amitriptyline induces brain-derived neurotrophic factor (BDNF) mRNA expression through ERK-dependent modulation of multiple BDNF mRNA variants in primary cultured rat cortical astrocytes and microglia.

    Science.gov (United States)

    Hisaoka-Nakashima, Kazue; Kajitani, Naoto; Kaneko, Masahiro; Shigetou, Takahiro; Kasai, Miho; Matsumoto, Chie; Yokoe, Toshiki; Azuma, Honami; Takebayashi, Minoru; Morioka, Norimitsu; Nakata, Yoshihiro

    2016-03-01

    A significant role of brain-derived neurotrophic factor (BDNF) has been previously implicated in the therapeutic effect of antidepressants. To ascertain the contribution of specific cell types in the brain that produce BDNF following antidepressant treatment, the effects of the tricyclic antidepressant amitriptyline on rat primary neuronal, astrocytic and microglial cortical cultures were examined. Amitriptyline increased the expression of BDNF mRNA in astrocytic and microglial cultures but not neuronal cultures. Antidepressants with distinct mechanisms of action, such as clomipramine, duloxetine and fluvoxamine, also increased BDNF mRNA expression in astrocytic and microglial cultures. There are multiple BDNF mRNA variants (exon I, IIA, IV and VI) expressed in astrocytes and microglia and the variant induced by antidepressants has yet to be elaborated. Treatment with antidepressants increased the expression of exon I, IV and VI in astrocyte and microglia. Clomipramine alone significantly upregulated expression of exon IIA. The amitriptyline-induced expression of both total and individual BDNF mRNA variants (exon I, IV and VI) were blocked by MEK inhibitor U0126, indicating MEK/ERK signaling is required in the expression of BDNF. These findings indicate that non-neural cells are a significant target of antidepressants and further support the contention that glial production of BDNF is crucial role in the therapeutic effect of antidepressants. The current data suggest that targeting of glial function could lead to the development of antidepressants with a truly novel mechanism of action.

  1. Evaluating the Role of Viral Proteins in HIV-Mediated Neurotoxicity Using Primary Human Neuronal Cultures.

    Science.gov (United States)

    Rao, Vasudev R; Eugenin, Eliseo A; Prasad, Vinayaka R

    2016-01-01

    Despite the inability of HIV-1 to infect neurons, over half of the HIV-1-infected population in the USA suffers from neurocognitive dysfunction. HIV-infected immune cells in the periphery enter the central nervous system by causing a breach in the blood-brain barrier. The damage to the neurons is mediated by viral and host toxic products released by activated and infected immune and glial cells. To evaluate the toxicity of any viral isolate, viral protein, or host inflammatory protein, we describe a protocol to assess the neuronal apoptosis and synaptic compromise in primary cultures of human neurons and astrocytes.

  2. Dynamic reactive astrocytes after focal ischemia

    Institute of Scientific and Technical Information of China (English)

    Shinghua Ding

    2014-01-01

    Astrocytes are specialized and most numerous glial cell type in the central nervous system and play important roles in physiology. Astrocytes are also critically involved in many neural disor-ders including focal ischemic stroke, a leading cause of brain injury and human death. One of the prominent pathological features of focal ischemic stroke is reactive astrogliosis and glial scar for-mation associated with morphological changes and proliferation. This review paper discusses the recent advances in spatial and temporal dynamics of morphology and proliferation of reactive astrocytes after ischemic stroke based on results from experimental animal studies. As reactive astrocytes exhibit stem cell-like properties, knowledge of dynamics of reactive astrocytes and glial scar formation will provide important insights for astrocyte-based cell therapy in stroke.

  3. Platelet-derived growth factor (PDGF-BB-mediated induction of monocyte chemoattractant protein 1 in human astrocytes: implications for HIV-associated neuroinflammation

    Directory of Open Access Journals (Sweden)

    Bethel-Brown Crystal

    2012-12-01

    Full Text Available Abstract Chemokine (C-C motif ligand 2, also known as monocyte chemoattractant protein 1 (MCP-1 is an important factor for the pathogenesis of HIV-associated neurocognitive disorders (HAND. The mechanisms of MCP-1-mediated neuropathogenesis, in part, revolve around its neuroinflammatory role and the recruitment of monocytes into the central nervous system (CNS via the disrupted blood-brain barrier (BBB. We have previously demonstrated that HIV-1/HIV-1 Tat upregulate platelet-derived growth factor (PDGF-BB, a known cerebrovascular permeant; subsequently, the present study was aimed at exploring the regulation of MCP-1 by PDGF-BB in astrocytes with implications in HAND. Specifically, the data herein demonstrate that exposure of human astrocytes to HIV-1 LAI elevated PDGF-B and MCP-1 levels. Furthermore, treating astrocytes with the human recombinant PDGF-BB protein significantly increased the production and release of MCP-1 at both the RNA and protein levels. MCP-1 induction was regulated by activation of extracellular-signal-regulated kinase (ERK1/2, c-Jun N-terminal kinase (JNK and p38 mitogen-activated protein (MAP kinases and phosphatidylinositol 3-kinase (PI3K/Akt pathways and the downstream transcription factor, nuclear factor κB (NFκB. Chromatin immunoprecipitation (ChIP assays demonstrated increased binding of NFκB to the human MCP-1 promoter following PDGF-BB exposure. Conditioned media from PDGF-BB-treated astrocytes increased monocyte transmigration through human brain microvascular endothelial cells (HBMECs, an effect that was blocked by STI-571, a tyrosine kinase inhibitor (PDGF receptor (PDGF-R blocker. PDGF-BB-mediated release of MCP-1 was critical for increased permeability in an in vitro BBB model as evidenced by blocking antibody assays. Since MCP-1 is linked to disease severity, understanding its modulation by PDGF-BB could aid in understanding the proinflammatory responses in HAND. These results suggest that astrocyte

  4. The NRF2 transcriptional target, OSGIN1, contributes to monomethyl fumarate-mediated cytoprotection in human astrocytes

    Science.gov (United States)

    Brennan, Melanie S.; Matos, Maria F.; Richter, Karl E.; Li, Bing; Scannevin, Robert H.

    2017-01-01

    Dimethyl fumarate (DMF) is indicated for the treatment of relapsing multiple sclerosis and may exert therapeutic effects via activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathway. Following oral DMF administration, central nervous system (CNS) tissue is predominantly exposed to monomethyl fumarate (MMF), the bioactive metabolite of DMF, which can stabilize NRF2 and induce antioxidant gene expression; however, the detailed NRF2-dependent mechanisms modulated by MMF that lead to cytoprotection are unknown. Our data identify a mechanism for MMF-mediated cytoprotection in human astrocytes that functions in an OSGIN1-dependent manner, specifically via upregulation of the OSGIN1-61 kDa isoform. NRF2-dependent OSGIN1 expression induced P53 nuclear translocation following MMF administration, leading to cell-cycle inhibition and cell protection against oxidative challenge. This study provides mechanistic insight into MMF-mediated cytoprotection via NRF2, OSGIN1, and P53 in human CNS-derived cells and contributes to our understanding of how DMF may act clinically to ameliorate pathological processes in neurodegenerative disease. PMID:28181536

  5. Bryostatin activates HIV-1 latent expression in human astrocytes through a PKC and NF-ĸB-dependent mechanism.

    Science.gov (United States)

    Díaz, Laura; Martínez-Bonet, Marta; Sánchez, Javier; Fernández-Pineda, Alejandra; Jiménez, José Luis; Muñoz, Eduardo; Moreno, Santiago; Álvarez, Susana; Muñoz-Fernández, Ma Ángeles

    2015-07-22

    Multiple studies have shown that HIV-1 patients may develop virus reservoirs that impede eradication; these reservoirs include the central nervous system (CNS). Despite an undetectable viral load in patients treated with potent antiretrovirals, current therapy is unable to purge the virus from these latent reservoirs. To broaden the inhibitory range and effectiveness of current antiretrovirals, the potential of bryostatin was investigated as a latent HIV-1 activator. We used primary astrocytes, NHA cells, and astrocytoma cells U-87. Infected cells with HIV-1(NL4.3) were treated with bryostatin alone or in combination with different inhibitors. HIV-1 production was quantified by using ELISA. Transcriptional activity was measured using luciferase reporter gene assays by using lipofectin. We performed cotransfection experiments of the LTR promoter with the active NF-κB member p65/relA. To confirm the NF-κB role, Western blot and confocal microscopy were performed. Bryostatin reactivates latent viral infection in the NHA and U87 cells via activation of protein kinase C (PKC)-alpha and -delta, because the PKC inhibitors rottlerin and GF109203X abrogated the bryostatin effect. No alteration in cell proliferation was found. Moreover, bryostatin strongly stimulated LTR transcription by activating the transcription factor NF-κB. Bryostatin could be a beneficial adjunct to the treatment of HIV-1 brain infection.

  6. Progressive Motor Neuron Pathology and the Role of Astrocytes in a Human Stem Cell Model of VCP-Related ALS

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    Claire E. Hall

    2017-05-01

    Full Text Available Motor neurons (MNs and astrocytes (ACs are implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS, but their interaction and the sequence of molecular events leading to MN death remain unresolved. Here, we optimized directed differentiation of induced pluripotent stem cells (iPSCs into highly enriched (> 85% functional populations of spinal cord MNs and ACs. We identify significantly increased cytoplasmic TDP-43 and ER stress as primary pathogenic events in patient-specific valosin-containing protein (VCP-mutant MNs, with secondary mitochondrial dysfunction and oxidative stress. Cumulatively, these cellular stresses result in synaptic pathology and cell death in VCP-mutant MNs. We additionally identify a cell-autonomous VCP-mutant AC survival phenotype, which is not attributable to the same molecular pathology occurring in VCP-mutant MNs. Finally, through iterative co-culture experiments, we uncover non-cell-autonomous effects of VCP-mutant ACs on both control and mutant MNs. This work elucidates molecular events and cellular interplay that could guide future therapeutic strategies in ALS.

  7. Hemin inhibits NO production by IL-1β-stimulated human astrocytes through induction of heme oxygenase-1 and reduction of p38 MAPK activation

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    Sheng Wen S

    2010-09-01

    Full Text Available Abstract Background Heme oxygenase (HO-1 has been shown to attenuate oxidative injury and reduce apoptosis. HO-1 can be induced by various stimuli released during cellular injury, such as heme. Deleterious free heme is degraded by HO-1 to carbon monoxide, iron and biliverdin, which have potent anti-oxidant and anti-inflammatory properties. In this study, we tested the hypothesis that upregulation of HO-1 would inhibit production of the free radical (NO by interlukin (IL-1β-activated human astrocytes. Methods To measure NO production, inducible NO synthase (iNOS, HO-1 expression and mitogen-activated protein (MAP kinase activation we used hemin as an HO-1 inducer and tin protoporphyrin (SnPP IX as an inhibitor of HO-1 activity in human astrocyte cultures prior to IL-1β exposure. Transfection of astrocyte cultures was performed using a pLEX expression vector carrying the human HO-1 sequence prior to IL-1β treatment. Supernatants of astrocyte cultures pretreated with inhibitors of p38 MAPK or MEK1/2 prior to IL-1β exposure were collected for NO assay. Results IL-1β treatment of astrocytes alone induced undetectable amounts of HO-1 protein by western blot. However, HO-1 mRNA expression was modestly up-regulated in response to IL-1β stimulation. Pretreatment with hemin alone substantially induced both HO-1 mRNA and protein expression, and HO-1 mRNA expression was further enhanced when hemin was combined with IL-1β treatment. In contrast, IL-1β-induced iNOS mRNA expression and NO production were markedly inhibited by hemin treatment. When pretreated with SnPP, the inhibitory effect of hemin on IL-1β-induced NO production and iNOS expression was reversed, suggesting the involvement of HO-1. IL-1β-induced p38 MAPK activation, which is known to be required for NO production, was also down-regulated by hemin. Conclusion These findings support the hypothesis that up-regulation of HO-1 in astrocytes is associated with down-regulation of i

  8. Light-controlled astrocytes promote human mesenchymal stem cells toward neuronal differentiation and improve the neurological deficit in stroke rats.

    Science.gov (United States)

    Tu, Jie; Yang, Fan; Wan, Jun; Liu, Yunhui; Zhang, Jie; Wu, Bifeng; Liu, Yafeng; Zeng, Shaoqun; Wang, Liping

    2014-01-01

    Astrocytes are key components of the central nervous system (CNS) and release factors to support neural stem cell proliferation, differentiation, and migration. Adenosine 5'-triphosphate (ATP) is one of the key factors released upon activation of astrocytes that regulates the neural stem cell's function. However, it is not clear whether ATP derived from the depolarized astrocytes plays a vital role in promoting the neuronal differentiation of mesenchymal stem cells (MSCs) in vitro and in vivo. Herein, for the first time, we co-cultured MSCs with light-stimulated-channelrhodopsin-2 (ChR2)-astrocytes, and observed that the neuronal differentiation of MSCs was enhanced by expressing more neuronal markers, Tuj1 and NeuN. The ChR2-astrocyte-conditioned medium also stimulated MSCs differentiating into neuronal lineage cells by expressing more Tuj1 and Pax6, which was blocked by the P2X receptor antagonist, TNP-ATP. Then we found that light-depolarization of astrocytes significantly increased ATP accumulation in their bathing medium without impairing the cell membrane. We further found that ATP up-regulated the Tuj1, Pax6, FZD8 and β-catenin mRNA levels of MSCs, which could be reversed by application of TNP-ATP. Together these in vitro data provided convergent evidence that ATP from light-depolarized-astrocytes activated the wnt/β-catenin signaling of MSCs through binding to the P2X receptors, and promoted the neuronal differentiation of MSCs. Finally but importantly, our study also demonstrated in stroke rats that light-controlled astrocytes stimulated endogenous ATP release into the ischemic area to influence the transplanted MSCs, resulting in promoting the MSCs towards neuronal differentiation and improvements of neurological deficit. Copyright © 2013 Wiley Periodicals, Inc.

  9. Mutant ubiquitin attenuates interleukin-1β- and tumor necrosis factor-α-induced pro-inflammatory signaling in human astrocytic cells.

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    Kyungsun Choi

    Full Text Available A frameshift mutation of ubiquitin called ubiquitin(+1 (UBB(+1 was found in the aging and Alzheimer's disease brains and thought to be associated with neuronal dysfuction and degeneration. Even though ubiquitylation has been known to regulate vital cellular functions mainly through proteasome-dependent degradation of polyubiquitinated substrates, proteolysis-independent roles of ubiquitylation have emerged as key mechanisms in various signaling cascades. In this study, we have investigated the effect of UBB(+1 on proinflammatory signaling such as interleukin-1β (IL-1β and tumor necrosis factor-α (TNF-α in human astrocytes. Treatment with TNF-α and IL-1β induced expression of CCL2 and CXCL8 by human astrocytic cells; while ectopic expression of UBB(+1 significantly abrogated the proinflammatory cytokine-induced expression of chemokines. Ectopic expression of UBB(+1 suppressed TNF-α- and IL-1β-induced activation of NF-κB and JNK signaling pathway. Furthermore, we have demonstrated that polyubiquitylation of TRAFs and subsequent phosphorylation of TAK1 were significantly inhibited by stable expression of UBB(+1. Collectively, these results suggest that UBB(+1 may affect proinflammatory signaling in the central nervous system via inhibitory mechanisms of ubiquitin-dependent signaling in human astrocytes.

  10. Active sulforhodamine 101 uptake into hippocampal astrocytes.

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    Christian Schnell

    Full Text Available 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.

  11. Specific human astrocyte subtype revealed by affinity purified GFAP antibody; unpurified serum cross-reacts with neurofilament-L in Alzheimer.

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    Jinte Middeldorp

    Full Text Available The human GFAP splice variants GFAPDelta164 and GFAPDeltaexon6 both result in a GFAP protein isoform with a unique out-of-frame carboxy-terminus that can be detected by the GFAP+1 antibody. We previously reported that GFAP+1 was expressed in astrocytes and in degenerating neurons in Alzheimer's disease brains. In this study we aimed at further investigating the neuronal GFAP+1 expression and we started by affinity purifying the GFAP+1 antibody. The purified antibody resulted in a loss of neuronal GFAP+1 signal, although other antibodies directed against the amino- and carboxy-terminus of GFAPalpha still revealed GFAP-immunopositive neurons, as described before. With an in-depth analysis of a western blot, followed by mass spectrometry we discovered that the previously detected neuronal GFAP+1 expression was due to cross-reactivity of the antibody with neurofilament-L (NF-L. This was confirmed by double-label fluorescent immunohistochemistry and western blotting with the unpurified GFAP+1 antibody and an antibody against NF-L. Our data imply that NF-L can accumulate in some tangle-like structures in Alzheimer brains. More importantly, the purified GFAP+1 antibody clearly revealed a specific subtype of astrocytes in the adult human brain. These large astrocytes are present throughout the brain, e.g., along the subventricular zone, in the hippocampus, in the striatum and in the spinal cord of controls, Alzheimer, and Parkinson patients. The presence of a specific GFAP-isoform suggests a specialized function of these astrocytes.

  12. Specific Human Astrocyte Subtype Revealed by Affinity Purified GFAP+1 Antibody; Unpurified Serum Cross-Reacts with Neurofilament-L in Alzheimer

    Science.gov (United States)

    Middeldorp, Jinte; van den Berge, Simone A.; Aronica, Eleonora; Speijer, Dave; Hol, Elly M.

    2009-01-01

    The human GFAP splice variants GFAPΔ164 and GFAPΔexon6 both result in a GFAP protein isoform with a unique out-of-frame carboxy-terminus that can be detected by the GFAP+1 antibody. We previously reported that GFAP+1 was expressed in astrocytes and in degenerating neurons in Alzheimer's disease brains. In this study we aimed at further investigating the neuronal GFAP+1 expression and we started by affinity purifying the GFAP+1 antibody. The purified antibody resulted in a loss of neuronal GFAP+1 signal, although other antibodies directed against the amino- and carboxy-terminus of GFAPα still revealed GFAP-immunopositive neurons, as described before. With an in-depth analysis of a western blot, followed by mass spectrometry we discovered that the previously detected neuronal GFAP+1 expression was due to cross-reactivity of the antibody with neurofilament-L (NF-L). This was confirmed by double-label fluorescent immunohistochemistry and western blotting with the unpurified GFAP+1 antibody and an antibody against NF-L. Our data imply that NF-L can accumulate in some tangle-like structures in Alzheimer brains. More importantly, the purified GFAP+1 antibody clearly revealed a specific subtype of astrocytes in the adult human brain. These large astrocytes are present throughout the brain, e.g., along the subventricular zone, in the hippocampus, in the striatum and in the spinal cord of controls, Alzheimer, and Parkinson patients. The presence of a specific GFAP-isoform suggests a specialized function of these astrocytes. PMID:19888461

  13. A reliable primary human CNS culture protocol for morphological studies of dendritic and synaptic elements.

    Science.gov (United States)

    Hammond, Robert R; Iskander, Sam; Achim, Cristian L; Hearn, Stephen; Nassif, Jane; Wiley, Clayton A

    2002-08-30

    Primary dissociated human fetal forebrain cultures were grown in defined serum-free conditions. At 4 weeks in vitro the cultures contained abundant morphologically well differentiated neurons with complex dendritic arbors. Astrocytic proliferation was negligible without the use of antimitotic agents. Confocal scanning laser microscopy (CSLM) and electron microscopy confirmed the presence of a dense neuropil, numerous cell-cell contacts and synapses. Neurons expressed a variety of proteins including growth associated protein-43 (GAP43), microtubule associated protein-2ab (MAP), class-III beta tubulin (C3BT), neurofilaments (NF), synaptophysin (SYN), parvalbumin (PA) and calbindin (CB). The cultures have proven to be reliable and simple to initiate and maintain for many weeks without passaging. They are useful in investigations of dendritic growth and injury of primary human CNS neurons.

  14. Differences in distribution and regulation of astrocytic aquaporin-4 in human and rat hydrocephalic brain

    DEFF Research Database (Denmark)

    Skjolding, Anders Daehli; Holst, Anders Vedel; Broholm, Helle

    2013-01-01

    in human hydrocephalic cortex relative to controls was quantified by western blotting (n=28). A second biopsy (n=13) was processed for immunohistochemistry (GFAP, CD68, CD34 and aquaporin-4) and double immunofluorescence (aquaporin-4+GFAP and aquaporin-4+CD34). Brain tissue from human controls and kaolin...

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

    Science.gov (United States)

    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

  16. The NRTIs lamivudine, stavudine and zidovudine have reduced HIV-1 inhibitory activity in astrocytes.

    Directory of Open Access Journals (Sweden)

    Lachlan R Gray

    Full Text Available 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.

  17. Neurosphere based differentiation of human iPSC improves astrocyte differentiation

    DEFF Research Database (Denmark)

    Zhou, Shuling; Szczesna, Karolina; Ochalek, Anna

    2016-01-01

    Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) are traditionally maintained and proliferated utilizing two-dimensional (2D) adherent monolayer culture systems. However, NPCs cultured using this system hardly reflect the intrinsic spatial development...

  18. Mature astrocytes in the adult human neocortex express the early neuronal marker doublecortin.

    NARCIS (Netherlands)

    Verwer, R.W.H.; Sluiter, A.A.; Balesar, R.A.; Baayen, J.C.; Noske, D.P.; Dirven, C.M.; Wouda, J.; van Dam, A.M.; Lucassen, P.J.; Swaab, D.F.

    2007-01-01

    Doublecortin (DCX) is a microtubule-associated protein expressed by migrating neuroblasts and is considered to be a reliable marker of neurogenesis. DCX has been used to study the relation between neurogenesis in adult human brain and neurological and neurodegenerative disease processes in the searc

  19. Memory in astrocytes: a hypothesis

    Directory of Open Access Journals (Sweden)

    Caudle Robert M

    2006-01-01

    Full Text Available Abstract Background Recent work has indicated an increasingly complex role for astrocytes in the central nervous system. Astrocytes are now known to exchange information with neurons at synaptic junctions and to alter the information processing capabilities of the neurons. As an extension of this trend a hypothesis was proposed that astrocytes function to store information. To explore this idea the ion channels in biological membranes were compared to models known as cellular automata. These comparisons were made to test the hypothesis that ion channels in the membranes of astrocytes form a dynamic information storage device. Results Two dimensional cellular automata were found to behave similarly to ion channels in a membrane when they function at the boundary between order and chaos. The length of time information is stored in this class of cellular automata is exponentially related to the number of units. Therefore the length of time biological ion channels store information was plotted versus the estimated number of ion channels in the tissue. This analysis indicates that there is an exponential relationship between memory and the number of ion channels. Extrapolation of this relationship to the estimated number of ion channels in the astrocytes of a human brain indicates that memory can be stored in this system for an entire life span. Interestingly, this information is not affixed to any physical structure, but is stored as an organization of the activity of the ion channels. Further analysis of two dimensional cellular automata also demonstrates that these systems have both associative and temporal memory capabilities. Conclusion It is concluded that astrocytes may serve as a dynamic information sink for neurons. The memory in the astrocytes is stored by organizing the activity of ion channels and is not associated with a physical location such as a synapse. In order for this form of memory to be of significant duration it is necessary

  20. CGRP in human models of primary headaches

    DEFF Research Database (Denmark)

    Ashina, Håkan; Schytz, Henrik Winther; Ashina, Messoud

    2017-01-01

    experiments are likely due to assay variation; therefore, proper validation and standardization of an assay is needed. To what extent CGRP is involved in tension-type headache and cluster headache is unknown. CONCLUSION: Human models of primary headaches have elucidated the role of CGRP in headache......OBJECTIVE: To review the role of CGRP in human models of primary headaches and to discuss methodological aspects and future directions. DISCUSSION: Provocation experiments demonstrated a heterogeneous CGRP migraine response in migraine patients. Conflicting CGRP plasma results in the provocation...... pathophysiology and sparked great interest in developing new treatment strategies using CGRP antagonists and antibodies. Future studies applying more refined human experimental models should identify biomarkers of CGRP-induced primary headache and reveal whether CGRP provocation experiments could be used...

  1. Interleukin-1 beta down-regulates the expression of metabotropic glutamate receptor 5 in cultured human astrocytes

    NARCIS (Netherlands)

    Aronica, E.; Gorter, J.A.; Rozemuller, A.J.M.; Yankaya, B.; Troost, D.

    2005-01-01

    Expression of metabotropic glutamate receptor 5 (mGluR5) protein is known to be plastic and to depend critically on the astrocytes' microenvironment. In the present study we investigated whether interleukins, which are involved in the immune response following brain injury, could contribute to the

  2. The Anti-Inflammatory Effects of Lipoxygenase and Cyclo-Oxygenase Inhibitors in Inflammation-Induced Human Fetal Glia Cells and the Aβ Degradation Capacity of Human Fetal Astrocytes in an Ex vivo Assay

    Directory of Open Access Journals (Sweden)

    Rea Pihlaja

    2017-05-01

    Full Text Available Chronic inflammation is a common phenomenon present in the background of multiple neurodegenerative diseases, including Alzheimer's disease (AD. The arachidonic acid pathway overproduces proinflammatory eicosanoids during these states and glial cells in the brain gradually lose their vital functions of protecting and supporting neurons. In this study, the role of different key enzymes of the eicosanoid pathway mediating inflammatory responses was examined in vitro and ex vivo using human fetal glial cells. Astrocytes and microglia were exposed to proinflammatory agents i.e., cytokines interleukin 1-β (IL-1β and tumor necrosis factor (TNF-α. ELISA assays were used to examine the effects of inhibitors of key enzymes in the eicosanoid pathway. Inhibitors for 5-lipoxygenase (5-LOX and cyclo-oxygenase 2 (COX-2 in both cell types and 5-, 12-, and 15-LOX-inhibitor in astrocytes reduced significantly IL-6 secretion, compared to exposed glial cells without inhibitors. The cytokine antibody array showed that especially treatments with 5, -12, and -15 LOX inhibitor in astrocytes, 5-LOX inhibitor in microglia and COX-2 inhibitor in both glial cell types significantly reduced the expression of multiple proinflammatory cytokines. Furthermore, human fetal astrocytes and microglia were cultured on top of AD-affected and control human brain sections for 30 h. According to the immunochemical evaluation of the level of total Aβ, astrocytes were very efficient at degrading Aβ from AD-affected brain sections ex vivo; simultaneously added enzyme inhibitors did not increase their Aβ degradation capabilities. Microglia were not able to reduce the level of total Aβ during the 30 h incubation time.

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

    Science.gov (United States)

    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

  4. [Primary human demodicosis. A disease sui generis].

    Science.gov (United States)

    Hsu, C-K; Zink, A; Wei, K-J; Dzika, E; Plewig, G; Chen, W

    2015-03-01

    Human Demodex mites (Demodex folliculorum and Demodex brevis) are unique in that they are an obligate human ectoparasite that can inhabit the pilosebaceous unit lifelong without causing obvious host immune response in most cases. The mode of symbiosis between humans and human Demodex mites is unclear, while the pathogenicity of human Demodex mites in many inflammatory skin diseases is now better understood. Primary human demodicosis is a skin disease sui generis not associated with local or systemic immunosuppression. Diagnosis is often underestimated and differentiation from folliculitis, papulopustular rosacea and perioral dermatitis is not always straightforward. Dependent on the morphology and degree of inflammation, the clinical manifestations can be classified into spinulate, papulopustular, nodulocystic, crustic and fulminant demodicosis. Therapy success can be achieved only with acaricides/arachidicides. The effective doses, optimal regimen and antimicrobial resistance remain to be determined.

  5. Isolation and characterization of ischemia-derived astrocytes (IDA with ability to transactivate quiescent astrocytes

    Directory of Open Access Journals (Sweden)

    Alejandro eVillarreal

    2016-06-01

    Full Text Available 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 (IDA 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 (OGD. 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.

  6. Astrocyte, the star avatar: redefined

    Indian Academy of Sciences (India)

    Pankaj Seth; Nitin Koul

    2008-09-01

    Until recently, the neuroscience community held the belief that glial cells such as astrocytes and oligodendrocytes functioned solely as “support” cells of the brain. In this role, glial cells simply provide physical support and housekeeping functions for the more important cells of the brain, the neurons. However, this view has changed radically in recent years with the discovery of previously unrecognized and surprising functions for this underappreciated cell type. In the past decade or so, emerging evidence has provided new insights into novel glial cell activities such as control of synapse formation and function, communication, cerebrovascular tone regulation, immune regulation and adult neurogenesis. Such advances in knowledge have effectively elevated the role of the astrocyte to one that is more important than previously realized. This review summarizes the past and present knowledge of glial cell functions that has evolved over the years, and has resulted in a new appreciation of astrocytes and their value in studying the neurobiology of human brain cells and their functions. In this review, we highlight recent advances in the role of glial cells in physiology, pathophysiology and, most importantly, in adult neurogenesis and “stemness”, with special emphasis on astrocytes.

  7. Modulation of polymorphonuclear neutrophil functions by astrocytes

    Directory of Open Access Journals (Sweden)

    Xie Luokun

    2010-09-01

    Full Text Available Abstract Background Neuroinflammation is a complex process involving cells from the immune system and the central nerve system (CNS. Polymorphonuclear neutrophils (PMN are the most abundant class of white blood cells, and typically the first type of leukocyte recruited to sites of inflammation. In the CNS, astrocytes are the most abundant glial cell population and participate in the local innate immune response triggered by a variety of insults. In the present study, we investigated the impacts of astrocytes on PMN function. Methods Primary astrocyte cultures were derived from postnatal C57BL/6 mice and primary neutrophils were isolated from 8 to 12 weeks old C57BL/6 mice. PMNs respiratory burst was analyzed by H2DCFDA assay. For phagocytosis assay, neutrophils were incubated with FITC-labeled E. coli and the phagocytosis of E coli was determined by flow cytometer. PMNs degranulation was determined by myeloperoxidase assay. Cytokine expression was determined by real-time PCR. To determine the involvement of different signaling pathway, protein lysates were prepared and western blots were conducted to assess the activation of Akt, Erk1/2, and p38. Results Using ex vivo neutrophils and primary astrocyte cultures, our study demonstrated that astrocytes differentially regulate neutrophil functions, depending upon whether the interactions between the two cell types are direct or indirect. Upon direct cell-cell contact, astrocytes attenuate neutrophil apoptosis, respiratory bust, and degranulation, while enhancing neutrophil phagocytic capability and pro-inflammatory cytokine expression. Through indirect interaction with neutrophils, astrocytes attenuate apoptosis and enhance necrosis in neutrophils, augment neutrophil phagocytosis and respiratory burst, and inhibit neutrophil degranulation. In addition, astrocytes could augment Akt, Erk1/2, and p38 activation in neutrophils. Conclusions Astrocytes differentially regulate neutrophil functions through

  8. Methylene Blue Protects Astrocytes against Glucose Oxygen Deprivation by Improving Cellular Respiration

    Science.gov (United States)

    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. PMID:25848957

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

    Science.gov (United States)

    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.

  10. BAG3 protein regulates caspase-3 activation in HIV-1-infected human primary microglial cells

    Science.gov (United States)

    Rosati, Alessandra; Khalili, Kamel; Deshmane, Satish L.; Radhakrishnan, Sujatha; Pascale, Maria; Turco, M. Caterina; Marzullo, Liberato

    2015-01-01

    BAG3, a member of the BAG co-chaperones family, is expressed in several cell types subjected to stressful conditions, such as exposure to high temperature, heavy metals, drugs. Furthermore, it is constitutively expressed in some tumors. Among the biological activities of the protein, there is apoptosis downmodulation; this appears to be exerted through BAG3 interaction with the heat shock protein (Hsp) 70, that influences cell apoptosis at several levels. We recently reported that BAG3 protein was detectable in the cytoplasm of reactive astrocytes in HIV-1-associated encephalopathy biopsies. Here we report that downmodulation of BAG3 protein levels allows caspase-3 activation by HIV-1 infection in human primary microglial cells. This is the first reported evidence of a role for BAG3 in the balance of death versus survival during viral infection. PMID:18821563

  11. miR-21 and 221 upregulation and miR-181b downregulation in human grade II-IV astrocytic tumors.

    Science.gov (United States)

    Conti, Alfredo; Aguennouz, M'Hammed; La Torre, Domenico; Tomasello, Chiara; Cardali, Salvatore; Angileri, Filippo F; Maio, Francesca; Cama, Annamaria; Germanò, Antonino; Vita, Giuseppe; Tomasello, Francesco

    2009-07-01

    MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. Recent evidence indicates that miRNAs can function both as tumor suppressors and as oncogenes. It has been demonstrated that in glioblastoma multiforme miR-21 and 221 are upregulated whereas miR-128 and 181 are downregulated. Expression of miR-21, 221, 128a, 128b, 128c, 181a, 181b, 181c was studied using real-time quantitative reverse transcriptase polymerase chain reaction and northern blotting for human astrocytic tumors with different grade of malignancy. miR-21 and 221 were overexpressed in glioma samples, whereas miRNA 181b was downregulated compared with normal brain tissue. miRNA-21 was hyperexpressed in all tumor samples whereas higher levels of miRNA-221 were found in high-grade gliomas. This study is the first analysis of miRNAs in astrocytic tumor at different stages of malignancy. The different expression pattern observed in tumors at different stages of malignancy is probably dependent on the cell-specific repertoire of target genes of tumors sharing different molecular pathways activity and suggests miRNAs may have also a place in diagnosis and staging of brain tumors.

  12. Calcium-sensing receptor antagonist (calcilytic) NPS 2143 specifically blocks the increased secretion of endogenous Aβ42 prompted by exogenous fibrillary or soluble Aβ25-35 in human cortical astrocytes and neurons-therapeutic relevance to Alzheimer's disease.

    Science.gov (United States)

    Armato, Ubaldo; Chiarini, Anna; Chakravarthy, Balu; Chioffi, Franco; Pacchiana, Raffaella; Colarusso, Enzo; Whitfield, James F; Dal Prà, Ilaria

    2013-10-01

    The "amyloid-β (Aβ) hypothesis" posits that accumulating Aβ peptides (Aβs) produced by neurons cause Alzheimer's disease (AD). However, the Aβs contribution by the more numerous astrocytes remains undetermined. Previously we showed that fibrillar (f)Aβ25-35, an Aβ42 proxy, evokes a surplus endogenous Aβ42 production/accumulation in cortical adult human astrocytes. Here, by using immunocytochemistry, immunoblotting, enzymatic assays, and highly sensitive sandwich ELISA kits, we investigated the effects of fAβ25-35 and soluble (s)Aβ25-35 on Aβ42 and Aβ40 accumulation/secretion by human cortical astrocytes and HCN-1A neurons and, since the calcium-sensing receptor (CaSR) binds Aβs, their modulation by NPS 2143, a CaSR allosteric antagonist (calcilytic). The fAβ25-35-exposed astrocytes and surviving neurons produced, accumulated, and secreted increased amounts of Aβ42, while Aβ40 also accrued but its secretion was unchanged. Accordingly, secreted Aβ42/Aβ40 ratio values rose for astrocytes and neurons. While slightly enhancing Aβ40 secretion by fAβ25-35-treated astrocytes, NPS 2143 specifically suppressed the fAβ25-35-elicited surges of endogenous Aβ42 secretion by astrocytes and neurons. Therefore, NPS 2143 addition always kept Aβ42/Aβ40 values to baseline or lower levels. Mechanistically, NPS 2143 decreased total CaSR protein complement, transiently raised proteasomal chymotrypsin activity, and blocked excess NO production without affecting the ongoing increases in BACE1/β-secretase and γ-secretase activity in fAβ25-35-treated astrocytes. Compared to fAβ25-35, sAβ25-35 also stimulated Aβ42 secretion by astrocytes and neurons and NPS 2143 specifically and wholly suppressed this effect. Therefore, since NPS 2143 thwarts any Aβ/CaSR-induced surplus secretion of endogenous Aβ42 and hence further vicious cycles of Aβ self-induction/secretion/spreading, calcilytics might effectively prevent/stop the progression to full-blown AD.

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

    Science.gov (United States)

    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.

  14. Motor neuron death in ALS – programmed by astrocytes?

    Science.gov (United States)

    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

  15. Glucocorticoid regulation of astrocytic fate and function.

    Directory of Open Access Journals (Sweden)

    Shuang Yu

    Full Text Available Glial loss in the hippocampus has been suggested as a factor in the pathogenesis of stress-related brain disorders that are characterized by dysregulated glucocorticoid (GC secretion. However, little is known about the regulation of astrocytic fate by GC. Here, we show that astrocytes derived from the rat hippocampus undergo growth inhibition and display moderate activation of caspase 3 after exposure to GC. Importantly, the latter event, observed both in situ and in primary astrocytic cultures is not followed by either early- or late-stage apoptosis, as monitored by stage I or stage II DNA fragmentation. Thus, unlike hippocampal granule neurons, astrocytes are resistant to GC-induced apoptosis; this resistance is due to lower production of reactive oxygen species (ROS and a greater buffering capacity against the cytotoxic actions of ROS. We also show that GC influence hippocampal cell fate by inducing the expression of astrocyte-derived growth factors implicated in the control of neural precursor cell proliferation. Together, our results suggest that GC instigate a hitherto unknown dialog between astrocytes and neural progenitors, adding a new facet to understanding how GC influence the cytoarchitecture of the hippocampus.

  16. Bile acid formation in primary human hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Curt Einarsson; Ewa Ellis; Anna Abrahamsson; Bo-G6ran Ericzon; Ingemar Bj rkhem; Magnus Axelson

    2000-01-01

    AIM To evaluate a culture system for bile acid formation in primary human hepatocytes in comparison with HepG2 cells. METHODS Hepatocytes were isolated from normal human liver tissue and were cultured in serum-free William's E medium. The medium was collected and renewed every 24 h. Bile acids and their precursors in media were finally analysed by gas chromatography-mass spectrometry. RESULTS Cholic acid ( CA ) andchenodeoxycholic acid (CDCA) conjugated with glycine or taurine accounted for 70% and 25% of total steroids. A third of CDCA was also conjugated with sulphuric acid. Dexamathasone and thyroid hormorm alone or in combination did not significantly effect bile acid formation. The addition of cyclosporin A (10 μmol/L) inhibited the synthesis of CA and CDCA by about 13% and 30%, respectively. CONCLUSION Isolated human hepatocytes in primary culture behave as in the intact liver by converting cholesterol to conjugated CA and CDCA. This is in contrast to cultured HepG2 cells, which release large amounts of bile acid precursors and unconjugated bile acids into the medium.

  17. Human factors and ergonomics for primary care.

    Science.gov (United States)

    Bowie, Paul; Jeffcott, Shelly

    2016-03-01

    In the second paper of this series, we provide a brief overview of the scientific discipline of human factors and ergonomics (HFE). Traditionally the HFE focus in healthcare has been in acute hospital settings which are perceived to exhibit characteristics more similar to other high-risk industries already applying related principles and methods. This paper argues that primary care is an area which could benefit extensively from an HFE approach, specifically in improving the performance and well-being of people and organisations. To this end, we define the purpose of HFE, outline its three specialist sub-domains (physical, cognitive and organisational HFE) and provide examples of guiding HFE principles and practices. Additionally, we describe HFE issues of significance to primary care education, improvement and research and outline early plans for building capacity and capability in this setting.

  18. Astrocytes: a central element in neurological diseases.

    Science.gov (United States)

    Pekny, Milos; Pekna, Marcela; Messing, Albee; Steinhäuser, Christian; Lee, Jin-Moo; Parpura, Vladimir; Hol, Elly M; Sofroniew, Michael V; Verkhratsky, Alexei

    2016-03-01

    The neurone-centred view of the past disregarded or downplayed the role of astroglia as a primary component in the pathogenesis of neurological diseases. As this concept is changing, so is also the perceived role of astrocytes in the healthy and diseased brain and spinal cord. We have started to unravel the different signalling mechanisms that trigger specific molecular, morphological and functional changes in reactive astrocytes that are critical for repairing tissue and maintaining function in CNS pathologies, such as neurotrauma, stroke, or neurodegenerative diseases. An increasing body of evidence shows that the effects of astrogliosis on the neural tissue and its functions are not uniform or stereotypic, but vary in a context-specific manner from astrogliosis being an adaptive beneficial response under some circumstances to a maladaptive and deleterious process in another context. There is a growing support for the concept of astrocytopathies in which the disruption of normal astrocyte functions, astrodegeneration or dysfunctional/maladaptive astrogliosis are the primary cause or the main factor in neurological dysfunction and disease. This review describes the multiple roles of astrocytes in the healthy CNS, discusses the diversity of astroglial responses in neurological disorders and argues that targeting astrocytes may represent an effective therapeutic strategy for Alexander disease, neurotrauma, stroke, epilepsy and Alzheimer's disease as well as other neurodegenerative diseases.

  19. Bile acid formation in primary human hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Curt Einarsson; Ewa Ellis; Anna Abrahamsson; Bo-G ran Ericzon; Ingemar Bj rkhem; Magnus Axelson

    2000-01-01

    AIM To evaluate a system for bile acid formation in human hepatocytes in comparison with HepG2 cells.METHODS Hepatocytes were isolated from normal human liver tissue and were cultured in serum-freeWilliam's E medium. The medium was collected and renewed every 24 h. Bile acids and their precursors inmedia were finally analysed by gas chromatography-mass spectrometry.RESULTS Cholic acid (CA) and chenodeoxycholic acid (CDCA) conjugated with glycine or taurineaccounted for 70% and 25% of total steroids. One third of CDCA was also conjugated with sulphuric acid.Dexamethasone and thyroid hormone alone or in combination did not significantly affect bile acid formation.The addition of cyclosporin A (10 tm) inhibited the synthesis of CA and CDCA by about 13% and 30%,respectively.CONCLUSION Isolated human hepatocytes in primary culture behave as in the intact liver by convertingalmost quantitatively cholesterol to conjugated CA and CDCA. This is in contrast to cultured HepG2 cells,which release large amounts of bile acid precursors and unconjugated bile acids into the medium.

  20. Proliferative Response of Astrocytes to Oxygen-Glucose Deprivation in Primary Rats%原代大鼠星形胶质细胞对氧糖剥夺的增殖性应答反应

    Institute of Scientific and Technical Information of China (English)

    范玉颖; 张俊梅; 王华; 赵晓春

    2012-01-01

    目的 建立良好的模拟缺氧缺血性脑损伤后星形胶质细胞增殖的体外模型的方法.方法 采用CCK-8(Cell Counting Kit-8)法测定星形胶质细胞活力,5-乙炔基-2′脱氧尿嘧啶核苷(EdU)掺入法检测星形胶质细胞DNA合成,免疫组织化学染色法检测星形胶质细胞增殖细胞核抗原(PCNA)表达.比较原代大鼠星形胶质细胞在氧糖剥夺(OGD),10 mL·L-1氧气、无糖含血清培养基)培养不同时间(0h、3h、6h、9h)的增殖情况.结果 CCK-8检测结果显示随OGD培养时间延长,原代大鼠星形胶质细胞活力逐渐增强,6h达高峰,与相应时间点正常培养组比较差异有统计学意义;随后减弱,9h已接近正常培养组水平.OGD培养后,原代大鼠星形胶质细胞EdU和PCNA阳性细胞率逐渐增加,6h达最高水平,与相应时间点的正常培养组比较差异有统计学意义;而后下降,至9h与正常培养组比较差异无统计学意义.结论 OGD培养6h可成功模拟体内缺氧缺血后星形胶质细胞增殖改变,为进行缺氧缺血性脑损伤后星形胶质细胞过度增殖的相关研究提供了稳定可靠的体外实验模型和理论依据.%Objective To explore the method for building a good in vitro model mimicking astrogliosis after hypoxic - ischemic brain injury during the neonatal period. Methods The vitalities of astrocytes of primary rats were measured by adopting cell counting kit -8( CCK -8) method. Ethynyl deoxyuridine ( EdU) incorporation was used to test DNA synthesis of the astrocytes, and immunohistochemistry staining was used to test the proliferating cell nuclear antigen (PCNA) of astrocytes. The proliferations of the astrocytes of primary rats at different time points(0 h,3 h,6 h and 9 h) after oxygen - glucose deprivation( OGD) ,10 mL · L-1 oxygen,dulbecco's minimum essential medium without glucose) were compared. Results With OGD cultivation prolonged, the vigor of astrocytes of primary rats increased and reached to

  1. Adenosine A2A receptor and ecto-5'-nucleotidase/CD73 are upregulated in hippocampal astrocytes of human patients with mesial temporal lobe epilepsy (MTLE).

    Science.gov (United States)

    Barros-Barbosa, Aurora R; Ferreirinha, Fátima; Oliveira, Ângela; Mendes, Marina; Lobo, M Graça; Santos, Agostinho; Rangel, Rui; Pelletier, Julie; Sévigny, Jean; Cordeiro, J Miguel; Correia-de-Sá, Paulo

    2016-12-01

    Refractoriness to existing medications of up to 80 % of the patients with mesial temporal lobe epilepsy (MTLE) prompts for finding new antiepileptic drug targets. The adenosine A2A receptor emerges as an interesting pharmacological target since its excitatory nature partially counteracts the dominant antiepileptic role of endogenous adenosine acting via inhibitory A1 receptors. Gain of function of the excitatory A2A receptor has been implicated in a significant number of brain pathologies commonly characterized by neuronal excitotoxicity. Here, we investigated changes in the expression and cellular localization of the A2A receptor and of the adenosine-generating enzyme, ecto-5'-nucleotidase/CD73, in the hippocampus of control individuals and MTLE human patients. Western blot analysis indicates that the A2A receptor is more abundant in the hippocampus of MTLE patients compared to control individuals. Immunoreactivity against the A2A receptor predominates in astrocytes staining positively for the glial fibrillary acidic protein (GFAP). No co-localization was observed between the A2A receptor and neuronal cell markers, like synaptotagmin 1/2 (nerve terminals) and neurofilament 200 (axon fibers). Hippocampal astrogliosis observed in MTLE patients was accompanied by a proportionate increase in A2A receptor and ecto-5'-nucleotidase/CD73 immunoreactivities. Given our data, we hypothesize that selective blockade of excessive activation of astrocytic A2A receptors and/or inhibition of surplus adenosine formation by membrane-bound ecto-5'-nucleotidase/CD73 may reduce neuronal excitability, thus providing a novel therapeutic target for drug-refractory seizures in MTLE patients.

  2. Sporadic amyotrophic lateral sclerosis: new hypothesis regarding its etiology and pathogenesis suggests that astrocytes might be the primary target hosting a still unknown external agent.

    Science.gov (United States)

    Sica, Roberto E P; Nicola, Alejandro F De; González Deniselle, María C; Rodriguez, Gabriel; Monachelli, Gisella M Gargiulo; Peralta, Liliana Martinez; Bettini, Mariela

    2011-08-01

    This article briefly describes the already known clinical features and pathogenic mechanisms underlying sporadic amyotrophic lateral sclerosis, namely excitoxicity, oxidative stress, protein damage, inflammation, genetic abnormalities and neuronal death. Thereafter, it puts forward the hypothesis that astrocytes may be the cells which serve as targets for the harmful action of a still unknown environmental agent, while neuronal death may be a secondary event following the initial insult to glial cells. The article also suggests that an emergent virus or a misfolded infectious protein might be potential candidates to accomplish this task.

  3. Sporadic amyotrophic lateral sclerosis: new hypothesis regarding its etiology and pathogenesis suggests that astrocytes might be the primary target hosting a still unknown external agent

    Directory of Open Access Journals (Sweden)

    Roberto E.P. Sica

    2011-08-01

    Full Text Available This article briefly describes the already known clinical features and pathogenic mechanisms underlying sporadic amyotrophic lateral sclerosis, namely excitoxicity, oxidative stress, protein damage, inflammation, genetic abnormalities and neuronal death. Thereafter, it puts forward the hypothesis that astrocytes may be the cells which serve as targets for the harmful action of a still unknown environmental agent, while neuronal death may be a secondary event following the initial insult to glial cells. The article also suggests that an emergent virus or a misfolded infectious protein might be potential candidates to accomplish this task.

  4. Astrocytes : a central element in neurological diseases

    NARCIS (Netherlands)

    Pekny, Milos; Pekna, Marcela; Messing, Albee; Steinhäuser, Christian; Lee, Jin Moo; Parpura, Vladimir; Hol, Elly M.|info:eu-repo/dai/nl/F-1891-2013; Sofroniew, Michael V.; Verkhratsky, Alexei

    2016-01-01

    The neurone-centred view of the past disregarded or downplayed the role of astroglia as a primary component in the pathogenesis of neurological diseases. As this concept is changing, so is also the perceived role of astrocytes in the healthy and diseased brain and spinal cord. We have started to unr

  5. Astrocytes : a central element in neurological diseases

    NARCIS (Netherlands)

    Pekny, Milos; Pekna, Marcela; Messing, Albee; Steinhäuser, Christian; Lee, Jin Moo; Parpura, Vladimir; Hol, Elly M.; Sofroniew, Michael V.; Verkhratsky, Alexei

    2016-01-01

    The neurone-centred view of the past disregarded or downplayed the role of astroglia as a primary component in the pathogenesis of neurological diseases. As this concept is changing, so is also the perceived role of astrocytes in the healthy and diseased brain and spinal cord. We have started to unr

  6. Histone acetylation in astrocytes suppresses GFAP and stimulates a reorganization of the intermediate filament network.

    Science.gov (United States)

    Kanski, Regina; Sneeboer, Marjolein A M; van Bodegraven, Emma J; Sluijs, Jacqueline A; Kropff, Wietske; Vermunt, Marit W; Creyghton, Menno P; De Filippis, Lidia; Vescovi, Angelo; Aronica, Eleonora; van Tijn, Paula; van Strien, Miriam E; Hol, Elly M

    2014-10-15

    Glial fibrillary acidic protein (GFAP) is the main intermediate filament in astrocytes and is regulated by epigenetic mechanisms during development. We demonstrate that histone acetylation also controls GFAP expression in mature astrocytes. Inhibition of histone deacetylases (HDACs) with trichostatin A or sodium butyrate reduced GFAP expression in primary human astrocytes and astrocytoma cells. Because splicing occurs co-transcriptionally, we investigated whether histone acetylation changes the ratio between the canonical isoform GFAPα and the alternative GFAPδ splice variant. We observed that decreased transcription of GFAP enhanced alternative isoform expression, as HDAC inhibition increased the GFAPδ∶GFAPα ratio. Expression of GFAPδ was dependent on the presence and binding of splicing factors of the SR protein family. Inhibition of HDAC activity also resulted in aggregation of the GFAP network, reminiscent of our previous findings of a GFAPδ-induced network collapse. Taken together, our data demonstrate that HDAC inhibition results in changes in transcription, splicing and organization of GFAP. These data imply that a tight regulation of histone acetylation in astrocytes is essential, because dysregulation of gene expression causes the aggregation of GFAP, a hallmark of human diseases like Alexander's disease.

  7. The modulation of phosphatase expression impacts the proliferation efficiency of HSV-1 in infected astrocytes.

    Directory of Open Access Journals (Sweden)

    Lei Yue

    Full Text Available Herpes Simplex Virus 1 (HSV-1 is a major pathogen that causes human neurological diseases, including herpes simplex encephalitis (HSE. Previous studies have shown that astrocytes are involved in HSV-1 systemic pathogenesis in the central nervous system (CNS, although the mechanism remains unclear. In this study, a high-throughput RNAi library screening method was used to analyze the effect of host phosphatase gene regulation on HSV-1 replication using Macaca mulatta primary astrocytes in an in vitro culture system. The results showed that the downregulation of five phosphatase genes (PNKP, SNAP23, PTPRU, LOC714621 and PPM1M significantly inhibited HSV-1 infection, suggesting that these phosphatases were needed in HSV-1 replication in rhesus astrocytes. Although statistically significant, the effect of downregulation of these phosphatases on HSV-1 replication in a human astrocytoma cell line appears to be more limited. Our results suggest that the phosphatase genes in astrocytes may regulate the immunological and pathological reactions caused by HSV-1 CNS infection through the regulation of HSV-1 replication or of multiple signal transduction pathways.

  8. Origin and development of human retinal astrocyte%人视网膜星形胶质细胞发育及起源的研究

    Institute of Scientific and Technical Information of China (English)

    孙立梅; 李永平; 张卉颖; 张文忻

    2012-01-01

    Background Retinal astrocytes are the main glial cells of retina,their origin and evolution progress are always the hot and difficult points in domestic and foreign researches. Objective This study was to explore the origin and development of retinal astrocytes in human fetal retina. Methods Thirty-three human embryonic eyes were collected from the abortion with the embryonic ages of 8-12 gestation weeks (20 eyes),15-17 gestation weeks ( 2 eyes),19-23 gestation weeks ( 4 eyes ),25 - 28 gestation weeks ( 4 eyes ),30 - 32 gestation weeks (3 eyes).The section of eyeball wall was prepared to observe the morphology and structure of different embryonic ages of retinas by regular histopathology examination.The origin of human embryonic retinal astrocytes was assessed by evaluating the change of GFAP expression in different embryonic ages of retinas using immunochemistry and immunofluorescence under the light and laser scanning confocal microscope.Theresearch was approved by the the Ethics Committee. Results The optic cup in embryonic 6- 7 weeks was in the retinal layering phase.Some immature short or round spindle-like cells appeared in primitive non-cell layer in the inner layer of the optic cup in embryonic 9 weeks of eyes.There were no positive GFAP-immunoactive cell was detected until embryonic 15 weeks of eyes.Some spindle-like cells migrated from a single layer primitive neuroepithelium next to optic disc expressing GFAP in the eyes with embryonic 19 weeks,and positive immunostaing for GFAP were detected in stellate cells surround blood vessels,and some seem to form the vessel wall in the ganglion cell layer and nerve fiber layer of the fetal central retina from 25 weeks through 26 weeks.Some positive response cells for GFAP presented in inner layer of the retina closed to ora serrata with the connection to nonpigmented epithelium (NPE) of the ciliary body during this period.Human retinal astrocytes showed typical stellate-like in shape,and the cellular processes

  9. Spinal astrocytes produce and secrete dynorphin neuropeptides.

    Science.gov (United States)

    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.

  10. Enterovirus 71 VP1 activates calmodulin-dependent protein kinase II and results in the rearrangement of vimentin in human astrocyte cells.

    Directory of Open Access Journals (Sweden)

    Cong Haolong

    Full Text Available Enterovirus 71 (EV71 is one of the main causative agents of foot, hand and mouth disease. Its infection usually causes severe central nervous system diseases and complications in infected infants and young children. In the present study, we demonstrated that EV71 infection caused the rearrangement of vimentin in human astrocytoma cells. The rearranged vimentin, together with various EV71 components, formed aggresomes-like structures in the perinuclear region. Electron microscopy and viral RNA labeling indicated that the aggresomes were virus replication sites since most of the EV71 particles and the newly synthesized viral RNA were concentrated here. Further analysis revealed that the vimentin in the virus factories was serine-82 phosphorylated. More importantly, EV71 VP1 protein is responsible for the activation of calmodulin-dependent protein kinase II (CaMK-II which phosphorylated the N-terminal domain of vimentin on serine 82. Phosphorylation of vimentin and the formation of aggresomes were required for the replication of EV71 since the latter was decreased markedly after phosphorylation was blocked by KN93, a CaMK-II inhibitor. Thus, as one of the consequences of CaMK-II activation, vimentin phosphorylation and rearrangement may support virus replication by playing a structural role for the formation of the replication factories. Collectively, this study identified the replication centers of EV71 in human astrocyte cells. This may help us understand the replication mechanism and pathogenesis of EV71 in human.

  11. A protocol for primary dissociated astrocyte and neuron co-culture%原代神经元与星形胶质细胞的混合培养方法

    Institute of Scientific and Technical Information of China (English)

    石莹; 周密; 姜民

    2013-01-01

    Cultured primary hippocampal neurons are ideal tool for investigating the subcellular localization and trafficking of neu-ronal proteins. The aim of the present study was to establish a method to co-culture hippocampal neurons and cortical astrocytes, which would guarantee well conditions of neurons. Newborn Sprague-Dawley (SD) rats were sacrificed by decapitation. The cortex of cerebrum was cut into pieces, and the cortical tissue was digested with trypsin. The liquid suspension of single cells was planted onto a 25 cm2 culture flask. On the fourth day of culture, the tissue cells except astrocytes were removed by intensive agitation of culture flask. Purified astrocytes were allowed to grow continuously until they reached most area of flask. At this time point, we replaced the culture media with neuronal cell media containing cytarabine, and planted the primary culture of rat hippocampal neurons onto the feed layer of cortical astrocytes. The microscopic observation results showed that, the astrocytes evenly grew without obvious boundaries between each other, and exhibited good purity. The co-cultured hippocampal neurons were in good condition, developed intertwined network of axons and dendrites, lived for a long time, and could tolerate gene transfection. Above all, this method is relatively simple from a technical point of view, yet provides healthy and reliable neuronal culture.%原代培养的海马神经元是研究神经细胞中蛋白运输与亚细胞空间定位的有效研究工具.本文旨在建立海马神经元与皮层胶质细胞混合培养的方法,以期提供状态良好的神经元供研究用.新生2~3 d Sprague-Dawley (SD)大鼠断头处死,从大脑半球表面片取2~3片皮层组织,切碎后用胰蛋白酶消化成单细胞悬液,种植于25 cm2培养瓶内.种植后第四天,采用剧烈敲击培养瓶的方法去除非星形胶质细胞的杂质细胞,继续培养贴壁细胞直至细胞接近铺满瓶底,改用含阿糖胞苷

  12. Translational regulation mechanisms of aquaporin-4 supramolecular organization in astrocytes.

    Science.gov (United States)

    Pisani, Francesco; Rossi, Andrea; Nicchia, Grazia Paola; Svelto, Maria; Frigeri, Antonio

    2011-12-01

    The two predominant isoforms of Aquaporin-4 (AQP4), AQP4-M23 and AQP4-M1, assemble in the plasma membrane to form supramolecular structures called Orthogonal Array of Particles (OAPs) whose dimension is tightly associated to the M1/M23 ratio. Here, we explore translational regulation contribution to M1/M23 expression in primary cultures of rat astrocytes, and analyze the role of M1 mRNA 5'untranslated region (5'UTR) in this mechanism. Using isoform-specific RNAi we found that in rat astrocytes primary cultures a large proportion of M23 protein derives from M1 mRNA translation. Furthermore, site-specific mutagenesis of the 5'UTR sequence of AQP4-M1 mRNA indicates that a multiple-site leaky scanning mechanism, an out-of-frame upstream ORF (uORF), and a reinitiation mechanism are able to modulate the M1/M23 ratio and consequently, OAPs formation. These mechanisms are likely to be shared by different species, including human, and they can also be assumed to play a role in those pathophysiological situations where the organization of AQP4 in supramolecular structures (OAPs) is involved. Finally, we report that, when transfected in Hela cells, the longer rat AQP4 isoform, called Mz, which is not present in human impairs OAPs formation. Copyright © 2011 Wiley‐Liss, Inc.

  13. Genistein inhibits differentiation of primary human adipocytes.

    Science.gov (United States)

    Park, Hea Jin; Della-Fera, Mary Anne; Hausman, Dorothy B; Rayalam, Srujana; Ambati, Suresh; Baile, Clifton A

    2009-02-01

    Genistein, a major soy isoflavone, has been reported to exhibit antiadipogenic and proapoptotic potential in vivo and in vitro. It is also a phytoestrogen which has high affinity to estrogen receptor beta. In this study, we determined the effect of genistein on adipogenesis and estrogen receptor (ER) alpha and beta expression during differentiation in primary human preadipocytes. Genistein inhibited lipid accumulation in a dose-dependent manner at concentrations of 6.25 microM and higher, with 50 microM genistein inhibiting lipid accumulation almost completely. Low concentrations of genistein (3.25 microM) increased cell viability and higher concentrations (25 and 50 microM) decreased it by 16.48+/-1.35% (P<.0001) and 50.68+/-1.34% (P<.0001). Oil Red O staining was used to confirm the effects on lipid accumulation. The inhibition of lipid accumulation was associated with inhibition of glycerol-3-phosphate dehydrogenase activity and down-regulation of expression of adipocyte-specific genes, including peroxisome proliferator-activated receptor gamma, CCAAT/enhancer binding protein alpha, glycerol-3-phosphate dehydrogenase, adipocyte fatty acid binding protein, fatty acid synthase, sterol regulatory element-binding protein 1, perilipin, leptin, lipoprotein lipase and hormone-sensitive lipase. These effects of genistein during the differentiation period were associated with down-regulation of ERalpha and ERbeta expression. This study adds to the elucidation of the molecular pathways involved in the inhibition of adipogenesis by phytoestrogens.

  14. [Amyotrophic lateral sclerosis: is the astrocyte the cell primarily involved?].

    Science.gov (United States)

    Sica, Roberto E

    2013-01-01

    So far, amyotrophic lateral sclerosis (ALS) is thought as due to a primary insult of the motor neurons. None of its pathogenic processes proved to be the cause of the illness, nor can be blamed environmental agents. Motor neurons die by apoptosis, leaving the possibility that their death might be due to an unfriendly environment, unable to sustain their health, rather than being directly targeted themselves. These reasons justify an examination of the astrocytes, because they have the most important role controlling the neurons' environment. It is known that astrocytes are plastic, enslaving their functions to the requirements of the neurons to which they are related. Each population of astrocytes is unique, and if it were affected the consequences would reach the neurons that it normally sustains. In regard to the motor neurons, this situation would lead to a disturbed production and release of astrocytic neurotransmitters and transporters, impairing nutritional and trophic support as well. For explaining the spreading of muscle symptoms in ALS, correlated with the type of spreading observed at the cortical and spinal motor neurons pools, the present hypotheses suggests that the illness-causing process is spreading among astrocytes, through their gap junctions, depriving the motor neurons of their support. Also it is postulated that a normal astrocytic protein becomes misfolded and infectious, inducing the misfolding of its wild type, travelling from one protoplasmatic astrocyte to another and to the fibrous astrocytes encircling the pyramidal pathway which joints the upper and lower motoneurones.

  15. Global Patterns in Human Consumption of Net Primary Production

    Science.gov (United States)

    Imhoff, Marc L.; Bounoua, Lahouari; Ricketts, Taylor; Loucks, Colby; Harriss, Robert; Lawrence William T.

    2004-01-01

    The human population and its consumption profoundly affect the Earth's ecosystems. A particularly compelling measure of humanity's cumulative impact is the fraction of the planet's net primary production that we appropriate for our Net primary production-the net amount of solar energy converted to plant organic matter through photosynthesis-can be measured in units of elemental carbon and represents the primary food energy source for the world's ecosystems. Human appropriation of net primary production, apart from leaving less for other species to use, alters the composition of the atmosphere, levels of biodiversity, flows within food webs and the provision of important primary production required by humans and compare it to the total amount generated on the landscape. We then derive a spatial ba!mce sheet of net primary production supply and demand for the world. We show that human appropriation of net primary production varies spatially from almost zero to many times the local primary production. These analyses reveal the uneven footprint of human consumption and related environmental impacts, indicate the degree to which human populations depend on net primary production "imports" and suggest policy options for slowing future growth of human appropriation of net primary production.

  16. Global patterns in human consumption of net primary production

    Science.gov (United States)

    Imhoff, Marc L.; Bounoua, Lahouari; Ricketts, Taylor; Loucks, Colby; Harriss, Robert; Lawrence, William T.

    2004-06-01

    The human population and its consumption profoundly affect the Earth's ecosystems. A particularly compelling measure of humanity's cumulative impact is the fraction of the planet's net primary production that we appropriate for our own use. Net primary production-the net amount of solar energy converted to plant organic matter through photosynthesis-can be measured in units of elemental carbon and represents the primary food energy source for the world's ecosystems. Human appropriation of net primary production, apart from leaving less for other species to use, alters the composition of the atmosphere, levels of biodiversity, energy flows within food webs and the provision of important ecosystem services. Here we present a global map showing the amount of net primary production required by humans and compare it to the total amount generated on the landscape. We then derive a spatial balance sheet of net primary production `supply' and `demand' for the world. We show that human appropriation of net primary production varies spatially from almost zero to many times the local primary production. These analyses reveal the uneven footprint of human consumption and related environmental impacts, indicate the degree to which human populations depend on net primary production `imports' and suggest policy options for slowing future growth of human appropriation of net primary production.

  17. Exposure to cadmium chloride influences astrocyte-elevated gene-1 (AEG-1) expression in MDA-MB231 human breast cancer cells.

    Science.gov (United States)

    Luparello, Claudio; Longo, Alessandra; Vetrano, Marco

    2012-01-01

    It is known that cadmium (Cd) is able to regulate gene expression, drastically affecting the pattern of transcriptional activity and intracellular signalization in normal and pathological human cells. We have already shown that Cd exerts a cytotoxic effect on neoplastic MDA-MB231 cells from the human breast, which is characterized by the onset of a "non-classical" apoptotic kind of death, impairment of mitochondrial activity and drastic changes in gene expression pattern. In the present study, employing a combination of conventional and differential display-PCR techniques, immunocytochemical, ELISA and Western analyses, we extended the knowledge on the transcriptional modulation exerted by the metal demonstrating that in MDA-MB231 cells 5 μM CdCl(2) treatment for 96 h selectively down-regulates astrocyte-elevated gene-1 (AEG-1) and reduces the accumulation of its protein product which appears to be associated with the internal cytomembranes and also present in the nucleoplasm. In addition, due to the acknowledged role of AEG-1 in the intranuclear shuttling of NF-κB p65 subunit, we also showed that CdCl(2) treatment determines the decrease of p65 amount in nuclear extracts and the down-regulation of the NF-κB downstream genes c-fos and c-jun, thus providing a new contribution to the comprehension of the intracellular molecular mechanisms implicated in Cd-breast cancer cell interactions.

  18. In Vitro Modeling of Blood-Brain Barrier with Human iPSC-Derived Endothelial Cells, Pericytes, Neurons, and Astrocytes via Notch Signaling

    Directory of Open Access Journals (Sweden)

    Kohei Yamamizu

    2017-03-01

    Full Text Available The blood-brain barrier (BBB is composed of four cell populations, brain endothelial cells (BECs, pericytes, neurons, and astrocytes. Its role is to precisely regulate the microenvironment of the brain through selective substance crossing. Here we generated an in vitro model of the BBB by differentiating human induced pluripotent stem cells (hiPSCs into all four populations. When the four hiPSC-derived populations were co-cultured, endothelial cells (ECs were endowed with features consistent with BECs, including a high expression of nutrient transporters (CAT3, MFSD2A and efflux transporters (ABCA1, BCRP, PGP, MRP5, and strong barrier function based on tight junctions. Neuron-derived Dll1, which activates Notch signaling in ECs, was essential for the BEC specification. We performed in vitro BBB permeability tests and assessed ten clinical drugs by nanoLC-MS/MS, finding a good correlation with the BBB permeability reported in previous cases. This technology should be useful for research on human BBB physiology, pathology, and drug development.

  19. Targeting astrocytes in major depression

    OpenAIRE

    2015-01-01

    Astrocytes represent a highly heterogeneous population of neural cells primarily responsible for the homeostasis of the central nervous system. Astrocytes express multiple receptors for neurotransmitters, including the serotonin 5-HT2B receptors and interact with neurones at the synapse. Astroglia contribute to neurological diseases through homeostatic response, neuroprotection and reactivity. In major depression, astrocytes show signs of degeneration and are decreased in numbe...

  20. Unravelling and Exploiting Astrocyte Dysfunction in Huntington's Disease.

    Science.gov (United States)

    Khakh, Baljit S; Beaumont, Vahri; Cachope, Roger; Munoz-Sanjuan, Ignacio; Goldman, Steven A; Grantyn, Rosemarie

    2017-07-01

    Astrocytes are abundant within mature neural circuits and are involved in brain disorders. Here, we summarize our current understanding of astrocytes and Huntington's disease (HD), with a focus on correlative and causative dysfunctions of ion homeostasis, calcium signaling, and neurotransmitter clearance, as well as on the use of transplanted astrocytes to produce therapeutic benefit in mouse models of HD. Overall, the data suggest that astrocyte dysfunction is an important contributor to the onset and progression of some HD symptoms in mice. Additional exploration of astrocytes in HD mouse models and humans is needed and may provide new therapeutic opportunities to explore in conjunction with neuronal rescue and repair strategies. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Increased in vitro glial fibrillary acidic protein expression, telomerase activity, and telomere length after productive human immunodeficiency virus-1 infection in murine astrocytes.

    Science.gov (United States)

    Ojeda, Diego; López-Costa, Juan José; Sede, Mariano; López, Ester María; Berria, María Isabel; Quarleri, Jorge

    2014-02-01

    Although HIV-associated neurocognitive disorders (HAND) result from injury and loss of neurons, productive infection routinely takes place in cells of macrophage lineage. In such a complex context, astrocytosis induced by local chemokines/cytokines is one of the hallmarks of HIV neuropathology. Whether this sustained astrocyte activation is able to alter telomere-aging process is unknown. We hypothesized that interaction of HIV with astrocytes may impact astrocyte telomerase activity (TA) and telomere length in a scenario of astrocytic activation measured by expression of glial fibrillary acidic protein (GFAP). To test this hypothesis, cultured murine astrocytes were challenged with pseudotyped HIV/vesicular stomatitis virus (HIV/VSV) to circumvent the absence of viral receptors; and GFAP, telomerase activity, and telomere length were quantified. As an early and transient event after HIV infection, both TA activity and telomere length were significantly augmented (P telomere length, that may attenuate cell proliferation and enhance the astrocyte dysregulation, contributing to HIV neuropathogenesis. Understanding the mechanisms involved in HIV-mediated persistence by altering the telomere-related aging processes could aid in the development of therapeutic modalities for neurological complications of HIV infection.

  2. Expressão das proteínas BCL-2 e BAX em tumores astrocíticos humanos Expression of BCL-2 and BAX proteins in human astrocytic tumors

    Directory of Open Access Journals (Sweden)

    Mário Henrique Girão Faria

    2006-08-01

    Full Text Available INTRODUÇÃO: Os astrocitomas constituem os mais freqüentes tumores primários do sistema nervoso central (SNC. Admite-se que parte do crescimento tumoral seja resultante da inibição da morte celular programada: a apoptose. Tal fenômeno é basicamente regulado pelo equilíbrio entre moléculas antiapoptóticas (ex.: B-cell lymphoma protein 2 [BCL-2] e pró-apoptóticas (ex.: BCL-2 associated protein X [BAX]. OBJETIVO: O presente estudo objetivou avaliar a expressão de BCL-2 e BAX em tumores astrocíticos humanos. MATERIAL E MÉTODOS: Procedeu-se ao estudo imuno-histoquímico dessas proteínas utilizando-se o método da avidina-biotina-peroxidase em 55 astrocitomas (13 do grau I, 14 do II, sete do III e 21 do grau IV e cinco amostras de tecido cerebral não-tumoral (grupo controle. RESULTADOS: Os índices de positividade para BCL-2 e BAX demonstraram propensão ao acréscimo, de acordo com a gradação tumoral, com positividade geral de 43,26% e 24,67%, respectivamente. Essas proteínas não foram detectadas entre os espécimes não-tumorais. Os escores de marcação para BCL-2 apresentaram tendência ao aumento conforme a progressão histológica, enquanto os para BAX mostraram-se semelhantes nas diversas graduações. A análise conjunta dessas proteínas demonstrou significativa correlação com a gradação tumoral (p BACKGROUND: Astrocytomas represent the most frequent primary tumors of the central nervous system. Admittedly, part of tumor growth is due to inhibition of programmed cell death: the apoptosis. This phenomenon is basically regulated by the balance between anti-apoptotic (e.g.: B-cell lymphoma protein 2 [BCL-2] and pro-apoptotic (e.g.: BCL-2 associated protein X [BAX] molecules. OBJECTIVE: The present study aimed to evaluate the expression of BCL-2 and BAX in human astrocytic tumors of different histopathological grades. MATERIAL AND METHOD: An immunohistochemical study of those proteins using the avidin

  3. HIV-1-infected and immune-activated macrophages induce astrocytic differentiation of human cortical neural progenitor cells via the STAT3 pathway.

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    Hui Peng

    Full Text Available Diminished adult neurogenesis is considered a potential mechanism in the pathogenesis of HIV-1-associated dementia (HAD. In HAD, HIV-1-infected and immune-activated brain mononuclear phagocytes (MP; perivascular macrophages and microglia drive central nervous system (CNS inflammation and may alter normal neurogenesis. We previously demonstrated HIV-1-infected and lipopolysaccharide (LPS activated monocyte-derived macrophages (MDM inhibit human neural progenitor cell (NPC neurogenesis, while enhancing astrogliogenesis through the secretion of the inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α, in vitro and in vivo. Here we further test the hypothesis that HIV-1-infected/activated MDM promote NPC astrogliogenesis via activation of the transcription factor signal transducer and activator of transcription 3 (STAT3, a critical factor for astrogliogenesis. Our results show that LPS-activated MDM-conditioned medium (LPS-MCM and HIV-infected/LPS-activated MDM-conditioned medium (LPS+HIV-MCM induced Janus kinase 1 (Jak1 and STAT3 activation. Induction of the Jak-STAT3 activation correlated with increased glia fibrillary acidic protein (GFAP expression, demonstrating an induction of astrogliogenesis. Moreover, STAT3-targeting siRNA (siSTAT3 decreased MCM-induced STAT3 activation and NPC astrogliogenesis. Furthermore, inflammatory cytokines (including IL-6, IL-1β and TNF-α produced by LPS-activated and/or HIV-1-infected MDM may contribute to MCM-induced STAT3 activation and astrocytic differentiation. These observations were confirmed in severe combined immunodeficient (SCID mice with HIV-1 encephalitis (HIVE. In HIVE mice, siRNA control (without target sequence, sicon pre-transfected NPCs injected with HIV-1-infected MDM showed more astrocytic differentiation and less neuronal differentiation of NPCs as compared to NPC injection alone. siSTAT3 abrogated HIV-1-infected MDM-induced astrogliogenesis of injected NPCs. Collectively, these

  4. Thimerosal-Derived Ethylmercury Is a Mitochondrial Toxin in Human Astrocytes: Possible Role of Fenton Chemistry in the Oxidation and Breakage of mtDNA

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    Martyn A. Sharpe

    2012-01-01

    Full Text Available Thimerosal generates ethylmercury in aqueous solution and is widely used as preservative. We have investigated the toxicology of Thimerosal in normal human astrocytes, paying particular attention to mitochondrial function and the generation of specific oxidants. We find that ethylmercury not only inhibits mitochondrial respiration leading to a drop in the steady state membrane potential, but also concurrent with these phenomena increases the formation of superoxide, hydrogen peroxide, and Fenton/Haber-Weiss generated hydroxyl radical. These oxidants increase the levels of cellular aldehyde/ketones. Additionally, we find a five-fold increase in the levels of oxidant damaged mitochondrial DNA bases and increases in the levels of mtDNA nicks and blunt-ended breaks. Highly damaged mitochondria are characterized by having very low membrane potentials, increased superoxide/hydrogen peroxide production, and extensively damaged mtDNA and proteins. These mitochondria appear to have undergone a permeability transition, an observation supported by the five-fold increase in Caspase-3 activity observed after Thimerosal treatment.

  5. Human Rights Texts: Converting Human Rights Primary Source Documents into Data

    National Research Council Canada - National Science Library

    Fariss, Christopher J; Linder, Fridolin J; Jones, Zachary M; Crabtree, Charles D; Biek, Megan A; Ross, Ana-Sophia M; Kaur, Taranamol; Tsai, Michael

    2015-01-01

    We introduce and make publicly available a large corpus of digitized primary source human rights documents which are published annually by monitoring agencies that include Amnesty International, Human...

  6. Why are astrocytes important?

    Science.gov (United States)

    Verkhratsky, Alexei; Nedergaard, Maiken; Hertz, Leif

    2015-02-01

    Astrocytes, which populate the grey and white mater of the brain and the spinal cord are highly heterogeneous in their morphology and function. These cells are primarily responsible for homeostasis of the central nervous system (CNS). Most central synapses are surrounded by exceedingly thin astroglial perisynaptic processes, which act as "astroglial cradle" critical for genesis, maturation and maintenance of synaptic connectivity. The perisynaptic glial processes are densely packed with numerous transporters, which provide for homeostasis of ions and neurotransmitters in the synaptic cleft, for local metabolic support and for release of astroglial derived scavengers of reactive oxygen species. Through perivascular processes astrocytes contribute to blood-brain barrier and form "glymphatic" drainage system of the CNS. Furthermore astrocytes are indispensible for glutamatergic and γ-aminobutyrate-ergic synaptic transmission being the supplier of neurotransmitters precursor glutamine via an astrocytic/neuronal cycle. Pathogenesis of many neurological disorders, including neuropsychiatric and neurodegenerative diseases is defined by loss of homeostatic function (astroglial asthenia) or remodelling of astroglial homoeostatic capabilities. Astroglial cells further contribute to neuropathologies through mounting complex defensive programme generally known as reactive astrogliosis.

  7. Apolipoprotein E-specific innate immune response in astrocytes from targeted replacement mice

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    Montine Thomas J

    2006-04-01

    Full Text Available Abstract Background Inheritance of the three different alleles of the human apolipoprotein (apo E gene (APOE are associated with varying risk or clinical outcome from a variety of neurologic diseases. ApoE isoform-specific modulation of several pathogenic processes, in addition to amyloid β metabolism in Alzheimer's disease, have been proposed: one of these is innate immune response by glia. Previously we have shown that primary microglia cultures from targeted replacement (TR APOE mice have apoE isoform-dependent innate immune activation and paracrine damage to neurons that is greatest with TR by the ε4 allele (TR APOE4 and that derives from p38 mitogen-activated protein kinase (p38MAPK activity. Methods Primary cultures of TR APOE2, TR APOE3 and TR APOE4 astrocytes were stimulated with lipopolysaccharide (LPS. ApoE secretion, cytokine production, and nuclear factor-kappa B (NF-κB subunit activity were measured and compared. Results Here we showed that activation of primary astrocytes from TR APOE mice with LPS led to TR APOE-dependent differences in cytokine secretion that were greatest in TR APOE2 and that were associated with differences in NF-κB subunit activity. Conclusion Our results suggest that LPS activation of innate immune response in TR APOE glia results in opposing outcomes from microglia and astrocytes as a result of TR APOE-dependent activation of p38MAPK or NF-κB signaling in these two cell types.

  8. Knockdown of astrocyte elevated gene-1 inhibits tumor growth and modifies microRNAs expression profiles in human colorectal cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Sujun [East Department of Gastroenterology, Institute of Geriatrics, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080 (China); Southern Medical University, Guangzhou, Guangdong 510515 (China); Wu, Binwen, E-mail: wubinwengd@aliyun.com [East Department of Gastroenterology, Institute of Geriatrics, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080 (China); Li, Dongfeng; Zhou, Weihong; Deng, Gang; Zhang, Kaijun; Li, Youjia [East Department of Gastroenterology, Institute of Geriatrics, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080 (China)

    2014-02-14

    Highlights: • AEG-1 expression in CRC cell lines and down-regulation or upregulation of AEG-1 in vitro. • Knockdown of AEG-1 inhibits cell proliferation, colony formation and invasion. • Upregulation of AEG-1 enhances proliferation, invasion and colony formation. • Knockdown of AEG-1 accumulates G0/G1-phase cells and promotes apoptosis in CRC cells. • AEG-1 knockdown increases 5-FU cytotoxicity. - Abstract: Astrocyte elevated gene-1 (AEG-1), upregulated in various types of malignancies including colorectal cancer (CRC), has been reported to be associated with the carcinogenesis. MicroRNAs (miRNAs) are widely involved in the initiation and progression of cancer. However, the functional significance of AEG-1 and the relationship between AEG-1 and microRNAs in human CRC remains unclear. The aim of this study was to investigate whether AEG-1 could serve as a potential therapeutic target of human CRC and its possible mechanism. We adopted a strategy of ectopic overexpression or RNA interference to upregulate or downregulate expression of AEG-1 in CRC models. Their phenotypic changes were analyzed by Western blot, MTT and transwell matrix penetration assays. MicroRNAs expression profiles were performed using microarray analysis followed by validation using qRT-PCR. Knockdown of AEG-1 could significantly inhibit colon cancer cell proliferation, colony formation, invasion and promotes apoptosis. Conversely, upregulation of AEG-1 could significantly enhance cell proliferation, invasion and reduced apoptisis. AEG-1 directly contributes to resistance to chemotherapeutic drug. Targeted downregulation of AEG-1 might improve the expression of miR-181a-2{sup ∗}, -193b and -193a, and inversely inhibit miR-31 and -9{sup ∗}. Targeted inhibition of AEG-1 can lead to modification of key elemental characteristics, such as miRNAs, which may become a potential effective therapeutic strategy for CRC.

  9. Characterization of astrocytic and neuronal benzodiazepine receptors

    Energy Technology Data Exchange (ETDEWEB)

    Bender, A.S.

    1988-01-01

    Primary cultures of astrocytes and neurons express benzodiazepine receptors. Neuronal benzodiazepine receptors were of high-affinity, K{sub D} values were 7.5-43 nM and the densities of receptors (B{sub max}) were 924-4131 fmol/mg protein. Astrocytes posses a high-affinity benzodiazepine receptor, K{sub D} values were 6.6-13 nM. The B{sub max} values were 6,033-12,000 fmol/mg protein. The pharmacological profile of the neuronal benzodiazepine receptor was that of the central-type benzodiazepine receptor, where clonazepam has a high-affinity and Ro 5-4864 (4{prime}-chlorodiazepam) has a low-affinity. Whereas astrocytic benzoidazepine receptor was characteristic of the so called peripheral-type benzodiazepine receptors, which shows a high-affinity towards Ro 5-4863, and a low-affinity towards clonazepam. The astrocytic benzodiazepine receptors was functionally correlated with voltage dependent calcium channels, since dihydropyridines and benzodiazepines interacted with ({sup 3}H) diazepam and ({sup 3}H) nitrendipine receptors with the same rank order of potency, showing a statistically significant correlation. No such correlation was observed in neurons.

  10. Curcumin alleviates oxidative stress and mitochondrial dysfunction in astrocytes.

    Science.gov (United States)

    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. Astrocytic metabolic and inflammatory changes as a function of age.

    Science.gov (United States)

    Jiang, Tianyi; Cadenas, Enrique

    2014-12-01

    This study examines age-dependent metabolic-inflammatory axis in primary astrocytes isolated from brain cortices of 7-, 13-, and 18-month-old Sprague-Dawley male rats. Astrocytes showed an age-dependent increase in mitochondrial oxidative metabolism respiring on glucose and/or pyruvate substrates; this increase in mitochondrial oxidative metabolism was accompanied by increases in COX3/18SrDNA values, thus suggesting an enhanced mitochondrial biogenesis. Enhanced mitochondrial respiration in astrocytes limits the substrate supply from astrocytes to neurons; this may be viewed as an adaptive mechanism to altered cellular inflammatory-redox environment with age. These metabolic changes were associated with an age-dependent increase in hydrogen peroxide generation (largely ascribed to an enhanced expression of NOX2) and NFκB signaling in the cytosol as well as its translocation to the nucleus. Astrocytes also displayed augmented responses with age to inflammatory cytokines, IL-1β, and TNFα. Activation of NFκB signaling resulted in increased expression of nitric oxide synthase 2 (inducible nitric oxide synthase), leading to elevated nitric oxide production. IL-1β and TNFα treatment stimulated mitochondrial oxidative metabolism and mitochondrial biogenesis in astrocytes. It may be surmised that increased mitochondrial aerobic metabolism and inflammatory responses are interconnected and support the functionality switch of astrocytes, from neurotrophic to neurotoxic with age.

  12. Integrated Modular Teaching of Human Biology for Primary Care Practitioners

    Science.gov (United States)

    Glasgow, Michael S.

    1977-01-01

    Describes the use of integrated modular teaching of the human biology component of the Health Associate Program at Johns Hopkins University, where the goal is to develop an understanding of the sciences as applied to primary care. Discussion covers the module sequence, the human biology faculty, goals of the human biology faculty, laboratory…

  13. Sex differences in hypothalamic astrocyte response to estradiol stimulation

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    Kuo John

    2010-11-01

    Full Text Available Abstract Background Reproductive functions controlled by the hypothalamus are highly sexually differentiated. One of the most dramatic differences involves estrogen positive feedback, which leads to ovulation. A crucial feature of this positive feedback is the ability of estradiol to facilitate progesterone synthesis in female hypothalamic astrocytes. Conversely, estradiol fails to elevate hypothalamic progesterone levels in male rodents, which lack the estrogen positive feedback-induced luteinizing hormone (LH surge. To determine whether hypothalamic astrocytes are sexually differentiated, we examined the cellular responses of female and male astrocytes to estradiol stimulation. Methods Primary adult hypothalamic astrocyte cultures were established from wild type rats and mice, estrogen receptor-α knockout (ERKO mice, and four core genotype (FCG mice, with the sex determining region of the Y chromosome (Sry deleted and inserted into an autosome. Astrocytes were analyzed for Sry expression with reverse transcription PCR. Responses to estradiol stimulation were tested by measuring free cytoplasmic calcium concentration ([Ca2+]i with fluo-4 AM, and progesterone synthesis with column chromatography and radioimmunoassay. Membrane estrogen receptor-α (mERα levels were examined using surface biotinylation and western blotting. Results Estradiol stimulated both [Ca2+]i release and progesterone synthesis in hypothalamic astrocytes from adult female mice. Male astrocytes had a significantly elevated [Ca2+]i response but it was significantly lower than in females, and progesterone synthesis was not enhanced. Surface biotinylation demonstrated mERα in both female and male astrocytes, but only in female astrocytes did estradiol treatment increase insertion of the receptor into the membrane, a necessary step for maximal [Ca2+]i release. Regardless of the chromosomal sex, estradiol facilitated progesterone synthesis in astrocytes from mice with ovaries

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

    Science.gov (United States)

    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.

  15. Anti-thyroperoxidase antibodies from patients with Hashimoto's encephalopathy bind to cerebellar astrocytes.

    Science.gov (United States)

    Blanchin, Stéphanie; Coffin, Christine; Viader, Fausto; Ruf, Jean; Carayon, Pierre; Potier, Francette; Portier, Estelle; Comby, Elisabeth; Allouche, Stéphane; Ollivier, Yann; Reznik, Yves; Ballet, Jean Jacques

    2007-12-01

    A cohort of 10 Hashimoto's encephalopathy (HE) patients, 33 patients with unrelated neurological symptoms, 12 Hashimoto's thyroiditis patients and 4 healthy adult donors was studied to explore the neurological targets of anti-thyroperoxidase (TPO) autoantibodies (aAb) in HE. High levels of anti-TPO aAb were only detected in HE group's cerebrospinal fluids. In immunofluorescence assays on monkey brain cerebellum sections, both HE patients' sera and anti-TPO monoclonal antibodies (mAb) were able to bind cerebellar cells expressing glial fibrillary acid protein. Normal human astrocytes from primary cultures also reacted with anti-TPO mAb. Specific astrocyte binding of anti-TPO aAb suggests a role of these aAb in the HE pathogenesis.

  16. Dedifferentiation of neurons and astrocytes by oncogenes can induce gliomas in mice.

    Science.gov (United States)

    Friedmann-Morvinski, Dinorah; Bushong, Eric A; Ke, Eugene; Soda, Yasushi; Marumoto, Tomotoshi; Singer, Oded; Ellisman, Mark H; Verma, Inder M

    2012-11-23

    Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain tumor in humans. Here we show that gliomas can originate from differentiated cells in the central nervous system (CNS), including cortical neurons. Transduction by oncogenic lentiviral vectors of neural stem cells (NSCs), astrocytes, or even mature neurons in the brains of mice can give rise to malignant gliomas. All the tumors, irrespective of the site of lentiviral vector injection (the initiating population), shared common features of high expression of stem or progenitor markers and low expression of differentiation markers. Microarray analysis revealed that tumors of astrocytic and neuronal origin match the mesenchymal GBM subtype. We propose that most differentiated cells in the CNS upon defined genetic alterations undergo dedifferentiation to generate a NSC or progenitor state to initiate and maintain the tumor progression, as well as to give rise to the heterogeneous populations observed in malignant gliomas.

  17. Expression of the human isoform of glutamate dehydrogenase, hGDH2, augments TCA cycle capacity and oxidative metabolism of glutamate during glucose deprivation in astrocytes

    DEFF Research Database (Denmark)

    Nissen, Jakob D; Lykke, Kasper; Bryk, Jaroslaw

    2016-01-01

    including CO2 , respectively. We conclude that hGDH2 expression increases capacity for uptake and oxidative metabolism of glutamate, particularly during increased workload and aglycemia. Additionally, hGDH2 expression increased utilization of branched-chain amino acids (BCAA) during aglycemia and caused...... a general decrease in oxidative glucose metabolism. We speculate, that expression of hGDH2 allows astrocytes to spare glucose and utilize BCAAs during substrate shortages. These findings support the proposed role of hGDH2 in astrocytes as an important fail-safe during situations of intense glutamatergic...

  18. The effect of gallic acid on cytotoxicity, Ca(2+) homeostasis and ROS production in DBTRG-05MG human glioblastoma cells and CTX TNA2 rat astrocytes.

    Science.gov (United States)

    Hsu, Shu-Shong; Chou, Chiang-Ting; Liao, Wei-Chuan; Shieh, Pochuen; Kuo, Daih-Huang; Kuo, Chun-Chi; Jan, Chung-Ren; Liang, Wei-Zhe

    2016-05-25

    Gallic acid, a polyhydroxylphenolic compound, is widely distributed in various plants, fruits and foods. It has been shown that gallic acid passes into blood brain barrier and reaches the brain tissue of middle cerebral artery occlusion rats. However, the effect of gallic acid on Ca(2+) signaling in glia cells is unknown. This study explored whether gallic acid affected Ca(2+) homeostasis and induced Ca(2+)-associated cytotoxicity in DBTRG-05MG human glioblastoma cells and CTX TNA2 rat astrocytes. Gallic acid (20-40 μM) concentration-dependently induced cytotoxicity and intracellular Ca(2+) level ([Ca(2+)]i) increases in DBTRG-05MG cells but not in CTX TNA2 cells. In DBTRG-05MG cells, the Ca(2+) response was decreased by half by removal of extracellular Ca(2+). In Ca(2+)-containing medium, gallic acid-induced Ca(2+) entry was inhibited by store-operated Ca(2+) channel inhibitors (2-APB, econazole and SKF96365). In Ca(2+)-free medium, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin abolished gallic acid-induced [Ca(2+)]i increases. Conversely, incubation with gallic acid also abolished thapsigargin-induced [Ca(2+)]i increases. Inhibition of phospholipase C with U73122 abolished gallic acid-induced [Ca(2+)]i increases. Gallic acid significantly caused cytotoxicity in DBTRG-05MG cells, which was partially prevented by prechelating cytosolic Ca(2+) with BAPTA-AM. Moreover, gallic acid activated mitochondrial apoptotic pathways that involved ROS production. Together, in DBTRG-05MG cells but not in CTX TNA2 cells, gallic acid induced [Ca(2+)]i increases by causing Ca(2+) entry via 2-APB, econazole and SKF96365-sensitive store-operated Ca(2+) entry, and phospholipase C-dependent release from the endoplasmic reticulum. This Ca(2+) signal subsequently evoked mitochondrial pathways of apoptosis that involved ROS production.

  19. Small ubiquitin-like modifier 1-3 conjugation [corrected] is activated in human astrocytic brain tumors and is required for glioblastoma cell survival.

    Science.gov (United States)

    Yang, Wei; Wang, Liangli; Roehn, Gabriele; Pearlstein, Robert D; Ali-Osman, Francis; Pan, Hongjie; Goldbrunner, Roland; Krantz, Matthew; Harms, Christoph; Paschen, Wulf

    2013-01-01

    Small ubiquitin-like modifier (SUMO1-3) constitutes a group of proteins that conjugate to lysine residues of target proteins thereby modifying their activity, stability, and subcellular localization. A large number of SUMO target proteins are transcription factors and other nuclear proteins involved in gene expression. Furthermore, SUMO conjugation plays key roles in genome stability, quality control of newly synthesized proteins, proteasomal degradation of proteins, and DNA damage repair. Any marked increase in levels of SUMO-conjugated proteins is therefore expected to have a major impact on the fate of cells. We show here that SUMO conjugation is activated in human astrocytic brain tumors. Levels of both SUMO1- and SUMO2/3-conjugated proteins were markedly increased in tumor samples. The effect was least pronounced in low-grade astrocytoma (WHO Grade II) and most pronounced in glioblastoma multiforme (WHO Grade IV). We also found a marked rise in levels of Ubc9, the only SUMO conjugation enzyme identified so far. Blocking SUMO1-3 conjugation in glioblastoma cells by silencing their expression blocked DNA synthesis, cell growth, and clonogenic survival of cells. It also resulted in DNA-dependent protein kinase-induced phosphorylation of H2AX, indicative of DNA double-strand damage, and G(2) /M cell cycle arrest. Collectively, these findings highlight the pivotal role of SUMO conjugation in DNA damage repair processes and imply that the SUMO conjugation pathway could be a new target of therapeutic intervention aimed at increasing the sensitivity of glioblastomas to radiotherapy and chemotherapy.

  20. Dysbalance of astrocyte calcium under hyperammonemic conditions.

    Directory of Open Access Journals (Sweden)

    Nicole Haack

    Full Text Available Increased brain ammonium (NH4(+/NH3 plays a central role in the manifestation of hepatic encephalopathy (HE, a complex syndrome associated with neurological and psychiatric alterations, which is primarily a disorder of astrocytes. Here, we analysed the influence of NH4(+/NH3 on the calcium concentration of astrocytes in situ and studied the underlying mechanisms of NH4(+/NH3-evoked calcium changes, employing fluorescence imaging with Fura-2 in acute tissue slices derived from different regions of the mouse brain. In the hippocampal stratum radiatum, perfusion with 5 mM NH4(+/NH3 for 30 minutes caused a transient calcium increase in about 40% of astrocytes lasting about 10 minutes. Furthermore, the vast majority of astrocytes (∼ 90% experienced a persistent calcium increase by ∼ 50 nM. This persistent increase was already evoked at concentrations of 1-2 mM NH4(+/NH3, developed within 10-20 minutes and was maintained as long as the NH4(+/NH3 was present. Qualitatively similar changes were observed in astrocytes of different neocortical regions as well as in cerebellar Bergmann glia. Inhibition of glutamine synthetase resulted in significantly larger calcium increases in response to NH4(+/NH3, indicating that glutamine accumulation was not a primary cause. Calcium increases were not mimicked by changes in intracellular pH. Pharmacological inhibition of voltage-gated sodium channels, sodium-potassium-chloride-cotransporters (NKCC, the reverse mode of sodium/calcium exchange (NCX, AMPA- or mGluR5-receptors did not dampen NH4(+/NH3-induced calcium increases. They were, however, significantly reduced by inhibition of NMDA receptors and depletion of intracellular calcium stores. Taken together, our measurements show that sustained exposure to NH4(+/NH3 causes a sustained increase in intracellular calcium in astrocytes in situ, which is partly dependent on NMDA receptor activation and on release of calcium from intracellular stores. Our study

  1. The contribution of astrocytes and microglia to traumatic brain injury.

    Science.gov (United States)

    Karve, Ila P; Taylor, Juliet M; Crack, Peter J

    2016-02-01

    Traumatic brain injury (TBI) represents a major cause of death and disability in developed countries. Brain injuries are highly heterogeneous and can also trigger other neurological complications, including epilepsy, depression and dementia. The initial injury often leads to the development of secondary sequelae; cellular hyperexcitability, vasogenic and cytotoxic oedema, hypoxia-ischaemia, oxidative stress and inflammation, all of which influence expansion of the primary lesion. It is widely known that inflammatory events in the brain following TBI contribute to the widespread cell death and chronic tissue degeneration. Neuroinflammation is a multifaceted response involving a number of cell types, both within the CNS and in the peripheral circulation. Astrocytes and microglia, cells of the CNS, are considered key players in initiating an inflammatory response after injury. These cells are capable of secreting various cytokines, chemokines and growth factors, and following injury to the CNS, undergo changes in morphology. Ultimately, these changes can influence the local microenvironment and thus determine the extent of damage and subsequent repair. This review will focus on the roles of microglia and astrocytes following TBI, highlighting some of the key processes, pathways and mediators involved in this response. Additionally, both the beneficial and the detrimental aspects of these cellular responses will be examined using evidence from animal models and human post-mortem TBI studies. © 2015 The British Pharmacological Society.

  2. Long-term culture and expansion of primary human hepatocytes

    NARCIS (Netherlands)

    Levy, G.; Bomze, D.; Heinz, S.; Ramachandran, S.D.; Noerenberg, A.; Cohen, M.; Shibolet, O.; Sklan, E.; Braspenning, J.C.; Nahmias, Y.

    2015-01-01

    Hepatocytes have a critical role in metabolism, but their study is limited by the inability to expand primary hepatocytes in vitro while maintaining proliferative capacity and metabolic function. Here we describe the oncostatin M (OSM)-dependent expansion of primary human hepatocytes by low

  3. Long-term culture and expansion of primary human hepatocytes

    NARCIS (Netherlands)

    Levy, G.; Bomze, D.; Heinz, S.; Ramachandran, S.D.; Noerenberg, A.; Cohen, M.; Shibolet, O.; Sklan, E.; Braspenning, J.C.; Nahmias, Y.

    2015-01-01

    Hepatocytes have a critical role in metabolism, but their study is limited by the inability to expand primary hepatocytes in vitro while maintaining proliferative capacity and metabolic function. Here we describe the oncostatin M (OSM)-dependent expansion of primary human hepatocytes by low expressi

  4. Lentivirus-Mediated Knockdown of Astrocyte Elevated Gene-1 Inhibits Growth and Induces Apoptosis through MAPK Pathways in Human Retinoblastoma Cells.

    Directory of Open Access Journals (Sweden)

    Ying Chang

    Full Text Available To explore expression and function of astrocyte elevated gene-1 (AEG-1 in human retinoblastoma (RB.The expression of AEG-1 in histological sections of human RBs and in RB cell lines was examined using immunohistochemical staining and RT-PCR and Western blotting respectively. We knocked down AEG-1 gene levels by AEG-1-siRNA lentivirus transfection of human RB cell lines SO-RB50 and Y79, and using an MTT assay, we assessed the role of AEG-1 on RB cell proliferation. The biological significance of lentivirus transfection induced AEG-1 down-regulation was examined by assessing the apoptosis rate in the transfected RB cells by Annexin V-APC staining and flow cytometry. We additionally measured the expression of Bcl-2, Bax, cleaved-caspase-3 and caspase-3, and the phosphorylation and non-phosphorylation alternation of MAPKs.AEG-1 expression was detected to be strongly positive in the histological slides of 35 out of 54 (65% patients with RB. AEG-1 expression increased significantly (P<0.05 with tumor stage. In the RB cell lines SO-RB50, Y79 and WERI-RB1 as compared with retinal pigment epithelium cells, expression of AEG-1 mRNA and AEG-1 protein was significantly higher. In AEG-1-siRNA lentivirus transfected cell cultures as compared with negative control lentivirus transfected cell cultures, levels of AEG-1 mRNA and of AEG-1 protein (P<0.05 and cell growth rates (P<0.01 were significantly lower, and apoptosis rate (P<0.001, Bax/Bcl-2 ratio and cleaved-caspase-3 protein level were significantly increased. The P-ERK/ERK ratio was significantly decreased in the AEG-1-siRNA lentivirus transfected cell lines.Expression of AEG-1 was associated with RB, in histological slides of patients and in cell culture experiments. Lentivirus transfection induced knockdown of AEG-1 had a tumor suppressive effect, potentially by tumor cell apoptosis induction through inhibition of ERK.

  5. Inhibition or ablation of transglutaminase 2 impairs astrocyte migration.

    Science.gov (United States)

    Monteagudo, Alina; Ji, Changyi; Akbar, Abdullah; Keillor, Jeffrey W; Johnson, Gail V W

    2017-01-22

    Astrocytes play numerous complex roles that support and facilitate the function of neurons. Further, when there is an injury to the central nervous system (CNS) they can both facilitate or ameliorate functional recovery depending on the location and severity of the injury. When a CNS injury is relatively severe a glial scar is formed, which is primarily composed of astrocytes. The glial scar can be both beneficial, by limiting inflammation, and detrimental, by preventing neuronal projections, to functional recovery. Thus, understanding the processes and proteins that regulate astrocyte migration in response to injury is still of fundamental importance. One protein that is likely involved in astrocyte migration is transglutaminase 2 (TG2); a multifunctional protein expressed ubiquitously throughout the brain. Its functions include transamidation and GTPase activity, among others, and previous studies have implicated TG2 as a regulator of migration. Therefore, we examined the role of TG2 in primary astrocyte migration subsequent to injury. Using wild type or TG2(-/-) astrocytes, we manipulated the different functions and conformation of TG2 with novel irreversible inhibitors or mutant versions of the protein. Results showed that both inhibition and ablation of TG2 in primary astrocytes significantly inhibit migration. Additionally, we show that the deficiency in migration caused by deletion of TG2 can only be rescued with the native protein and not with mutants. Finally, the addition of TGFβ rescued the migration deficiency independent of TG2. Taken together, our study shows that transamidation and GTP/GDP-binding are necessary for inhibiting astrocyte migration and it is TGFβ independent. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. The Humanities in English Primary Schools: Struggling to Survive

    Science.gov (United States)

    Barnes, Jonathan; Scoffham, Stephen

    2017-01-01

    This article surveys the state of the humanities in English primary schools drawing on evidence from serving head teachers, current literature and policy documents. The findings suggest that whilst the humanities are highly valued in schools, there are serious challenges which threaten the "broad and balanced" curriculum. It is suggested…

  7. Unperturbed posttranscriptional regulatory Rev protein function and HIV-1 replication in astrocytes.

    Directory of Open Access Journals (Sweden)

    Ashok Chauhan

    Full Text Available Astrocytes protect neurons, but also evoke proinflammatory responses to injury and viral infections, including HIV. There is a prevailing notion that HIV-1 Rev protein function in astrocytes is perturbed, leading to restricted viral replication. In earlier studies, our finding of restricted viral entry into astrocytes led us to investigate whether there are any intracellular restrictions, including crippled Rev function, in astrocytes. Despite barely detectable levels of DDX3 (Rev-supporting RNA helicase and TRBP (anti-PKR in primary astrocytes compared to astrocytic cells, Rev function was unperturbed in wild-type, but not DDX3-ablated astrocytes. As in permissive cells, after HIV-1 entry bypass in astrocytes, viral-encoded Tat and Rev proteins had robust regulatory activities, leading to efficient viral replication. Productive HIV-1 infection in astrocytes persisted for several weeks. Our findings on HIV-1 entry bypass in astrocytes demonstrated that the intracellular environment is conducive to viral replication and that Tat and Rev functions are unperturbed.

  8. N-acetylcysteine prevents HIV gp 120-related damage of human cultured astrocytes: correlation with glutamine synthase dysfunction

    Directory of Open Access Journals (Sweden)

    Costa Nicola

    2007-12-01

    Full Text Available Abstract Background HIV envelope gp 120 glycoprotein is released during active HIV infection of brain macrophages thereby generating inflammation and oxidative stress which contribute to the development of the AIDS-Dementia Complex (ADC. Gp120 has also been found capable to generate excitotoxic effect on brain tissue via enhancement of glutamatergic neurotransmission, leading to neuronal and astroglial damage, though the mechanism is still to be better understood. Here we investigated on the effect of N-acetylcysteine (NAC, on gp120-induced damage in human cultured astroglial cells and the possible contribution of gp120-related reacting oxygen species (ROS in the imbalanced activity of glutamine synthase (GS, the enzyme that metabolizes glutamate into glutamine within astroglial cells playing a neuroprotective role in brain disorders. Results Incubation of Lipari human cultured astroglial cells with gp 120 (0.1–10 nM produced a significant reduction of astroglial cell viability and apoptosis as evaluated by TUNEL reaction and flow cytometric analysis (FACS. This effect was accompanied by lipid peroxidation as detected by means of malondialdehyde assay (MDA. In addition, gp 120 reduced both glutamine concentration in astroglial cell supernatants and GS expression as detected by immunocytochemistry and western blotting analysis. Pre-treatment of cells with NAC (0.5–5 mM, dose-dependently antagonised astroglial apoptotic cell death induced by gp 120, an effect accompanied by significant attenuation of MDA accumulation. Furthermore, both effects were closely associated with a significant recovery of glutamine levels in cell supernatants and by GS expression, thus suggesting that overproduction of free radicals might contribute in gp 120-related dysfunction of GS in astroglial cells. Conclusion In conclusion, the present experiments demonstrate that gp 120 is toxic to astroglial cells, an effect accompanied by lipid peroxidation and by altered

  9. Increasing tPA activity in astrocytes induced by multipotent mesenchymal stromal cells facilitate neurite outgrowth after stroke in the mouse.

    Directory of Open Access Journals (Sweden)

    Hongqi Xin

    Full Text Available We demonstrate that tissue plasminogen activator (tPA and its inhibitors contribute to neurite outgrowth in the central nervous system (CNS after treatment of stroke with multipotent mesenchymal stromal cells (MSCs. In vivo, administration of MSCs to mice subjected to middle cerebral artery occlusion (MCAo significantly increased activation of tPA and downregulated PAI-1 levels in the ischemic boundary zone (IBZ compared with control PBS treated mice, concurrently with increases of myelinated axons and synaptophysin. In vitro, MSCs significantly increased tPA levels and concomitantly reduced plasminogen activator inhibitor 1 (PAI-1 expression in astrocytes under normal and oxygen and glucose deprivation (OGD conditions. ELISA analysis of conditioned medium revealed that MSCs stimulated astrocytes to secrete tPA. When primary cortical neurons were cultured in the conditioned medium from MSC co-cultured astrocytes, these neurons exhibited a significant increase in neurite outgrowth compared to conditioned medium from astrocytes alone. Blockage of tPA with a neutralizing antibody or knock-down of tPA with siRNA significantly attenuated the effect of the conditioned medium on neurite outgrowth. Addition of recombinant human tPA into cortical neuronal cultures also substantially enhanced neurite outgrowth. Collectively, these in vivo and in vitro data suggest that the MSC mediated increased activation of tPA in astrocytes promotes neurite outgrowth after stroke.

  10. TNF-alpha/IFN-gamma-induced iNOS expression increased by prostaglandin E2 in rat primary astrocytes via EP2-evoked cAMP/PKA and intracellular calcium signaling.

    Science.gov (United States)

    Hsiao, Han-Yun; Mak, Oi-Tong; Yang, Chung-Shi; Liu, Yu-Peng; Fang, Kuan-Ming; Tzeng, Shun-Fen

    2007-01-15

    Astrocytes, the most abundant glia in the central nervous system (CNS), produce a large amount of prostaglandin E(2) (PGE(2)) in response to proinflammatory mediators after CNS injury. However, it is unclear whether PGE(2) has a regulatory role in astrocytic activity under the inflamed condition. In the present work, we showed that PGE(2) increased inducible nitric oxide synthase (iNOS) production by tumor necrosis factor-alpha and interferon-gamma (T/I) in astrocytes. Pharmacological and RNA interference approaches further indicated the involvement of the receptor EP2 in PGE(2)-induced iNOS upregulation in T/I-treated astrocytes. Quantitative real-time polymerase chain reaction and gel mobility shift assays also demonstrated that PGE(2) increased iNOS transcription through EP2-induced cAMP/protein kinase A (PKA)-dependent pathway. Consistently, the effect of EP2 was significantly attenuated by the PKA inhibitor KT-5720 and partially suppressed by the inhibitor (SB203580) of p38 mitogen-activated protein kinase (p38MAPK), which serves as one of the downstream components of the PKA-dependent pathway. Interestingly, EP2-mediated PKA signaling appeared to increase intracellular Ca(2+) release through inositol triphosphate (IP3) receptor activation, which might in turn stimulate protein kinase C (PKC) activation to promote iNOS production in T/I-primed astrocytes. By analyzing the expression of astrocytic glial fibrillary acidic protein (GFAP), we found that PGE(2) alone only triggered the EP2-induced cAMP/PKA/p38MAPK signaling pathway in astrocytes. Collectively, PGE(2) may enhance T/I-induced astrocytic activation by augmenting iNOS/NO production through EP2-mediated cross-talk between cAMP/PKA and IP3/Ca(2+) signaling pathways.

  11. The multisensory function of the human primary visual cortex.

    Science.gov (United States)

    Murray, Micah M; Thelen, Antonia; Thut, Gregor; Romei, Vincenzo; Martuzzi, Roberto; Matusz, Pawel J

    2016-03-01

    It has been nearly 10 years since Ghazanfar and Schroeder (2006) proposed that the neocortex is essentially multisensory in nature. However, it is only recently that sufficient and hard evidence that supports this proposal has accrued. We review evidence that activity within the human primary visual cortex plays an active role in multisensory processes and directly impacts behavioural outcome. This evidence emerges from a full pallet of human brain imaging and brain mapping methods with which multisensory processes are quantitatively assessed by taking advantage of particular strengths of each technique as well as advances in signal analyses. Several general conclusions about multisensory processes in primary visual cortex of humans are supported relatively solidly. First, haemodynamic methods (fMRI/PET) show that there is both convergence and integration occurring within primary visual cortex. Second, primary visual cortex is involved in multisensory processes during early post-stimulus stages (as revealed by EEG/ERP/ERFs as well as TMS). Third, multisensory effects in primary visual cortex directly impact behaviour and perception, as revealed by correlational (EEG/ERPs/ERFs) as well as more causal measures (TMS/tACS). While the provocative claim of Ghazanfar and Schroeder (2006) that the whole of neocortex is multisensory in function has yet to be demonstrated, this can now be considered established in the case of the human primary visual cortex.

  12. Connexin Hemichannels in Astrocytes

    DEFF Research Database (Denmark)

    Nielsen, Brian Skriver; Hansen, Daniel Bloch; Ransom, Bruce R.

    2017-01-01

    are reported to open the hemichannels and thereby create a permeation pathway through the plasma membrane. Cx30 and Cx43 have, in their hemichannel configuration, been proposed to act as ion channels and membrane pathways for different molecules, such as fluorescent dyes, ATP, prostaglandins, and glutamate......Astrocytes in the mammalian central nervous system are interconnected by gap junctions made from connexins of the subtypes Cx30 and Cx43. These proteins may exist as hemichannels in the plasma membrane in the absence of a ‘docked’ counterpart on the neighboring cell. A variety of stimuli...

  13. In vitro differentiation of human adipose-derived adult stromal cells into neuron-like cells in hippocampal astrocyte conditioned medium

    Institute of Scientific and Technical Information of China (English)

    Xinchun Ye; Hongjun He; Feng Yang; Kepeng Zhao; Jun Yao; Bin Liu

    2006-01-01

    BACKGROUND: At present, researches on differentiating from human adipose-derived adult stromal cells (hADASC) to neuron-like cells are focus on inducing by artificial-synthetic compound solution;however,hippocampal astrocyte conditioned medium(HCAM)can induce in vitro differentiation from hADASC to neuron-like cells is still unclear.OBJECTIVE:To observe whether HCAM can induce in vitro differentiation from hADASC to neuron-like cells.DESIGN:Randomized control study.SETTING:Department of Neurology,Taixing People's Hospital;Central Laboratory,North China Coal Medical College.MATERIALS:Donor of adipose tissue was donated by female volunteers suffering from caesarean section in the department of obstetrics & gynecology in our hospital and aged 20-35 years. Adipose tissue was collected from subcutaneous tissue of abdomen during the operation.In addition.8 male newborn Wistar rats within 24 hours with average body mass of 20 g were provided by Animal Institute of Chinese Academy of Medical Sciences.Rabbit-anti-human Nestin polyclonal antibody.Rabbit-anti-human glial fibriliary acidic protein (GFAP)polyclonal antibody, rabbit-anti-human neuro-specific enolase polyclonal antibody and mouse-anti-human microtubal associated protein 2(MAP-2)polyclonal antibody were provided by Wuhan Boster Company.METHODS:The experiment was carried out in the Central Laboratory of North China Coal Medical College from October 2004 to June 2005.hADASC was cultured with HCAM and its growth and morphological changes were observed under inverted phase contrast microscope.Immunocytochemistry.immunofluorescence and Western blotting were used to evaluate the expressions of Nestin,which was a specific sign of nerve precursor,neuro-specific enolase and MAP-2,which was a specific sign of nerve cell,and GFAP,which was a specific sign of neuroglial cells.MAIN OUTCOME MEASURES:Nestin,which was a specific sign of nerve precursor,neuro-specific enolase and MAP-2,which was a specific sign of nerve cell

  14. Preferential lentiviral targeting of astrocytes in the central nervous system.

    Directory of Open Access Journals (Sweden)

    Michael Fassler

    Full Text Available The ability to visualize and genetically manipulate specific cell populations of the central nervous system (CNS is fundamental to a better understanding of brain functions at the cellular and molecular levels. Tools to selectively target cells of the CNS include molecular genetics, imaging, and use of transgenic animals. However, these approaches are technically challenging, time consuming, and difficult to control. Viral-mediated targeting of cells in the CNS can be highly beneficial for studying and treating neurodegenerative diseases. Yet, despite specific marking of numerous cell types in the CNS, in vivo selective targeting of astrocytes has not been optimized. In this study, preferential targeting of astrocytes in the CNS was demonstrated using engineered lentiviruses that were pseudotyped with a modified Sindbis envelope and displayed anti-GLAST IgG on their surfaces as an attachment moiety. Viral tropism for astrocytes was initially verified in vitro in primary mixed glia cultures. When injected into the brains of mice, lentiviruses that displayed GLAST IgG on their surface, exhibited preferential astrocyte targeting, compared to pseudotyped lentiviruses that did not incorporate any IgG or that expressed a control isotype IgG. Overall, this approach is highly flexible and can be exploited to selectively target astrocytes or other cell types of the CNS. As such, it can open a window to visualize and genetically manipulate astrocytes or other cells of the CNS as means of research and treatment.

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

    Science.gov (United States)

    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.

  16. Ionizing radiation induces astrocyte gliosis through microglia activation.

    Science.gov (United States)

    Hwang, So-Young; Jung, Jae-Seob; Kim, Tae-Hyun; Lim, Soo-Jeong; Oh, Eok-Soo; Kim, Joo-Young; Ji, Kyung-Ae; Joe, Eun-Hye; Cho, Kwan-Ho; Han, Inn-Oc

    2006-03-01

    The aim of this study was to investigate the role of microglia in radiation-induced astrocyte gliosis. We found that a single dose of 15 Gy radiation to a whole rat brain increased immunostaining of glial fibrillary acidic protein in astrocytes 6 h later, and even more so 24 h later, indicating the initiation of gliosis. While irradiation of cultured rat astrocytes had little effect, irradiation of microglia-astrocyte mixed-cultures displayed altered astrocyte phenotype into more processed, which is another characteristic of gliosis. Experiments using microglia-conditioned media indicated this astrocyte change was due to factors released from irradiated microglia. Irradiation of cultured mouse microglial cells induced a dose-dependent increase in mRNA levels for cyclooxygenase-2 (COX-2), interleukin (IL)-1beta, IL-6, IL-18, tumor necrosis factor-alpha and interferon-gamma-inducible protein-10, which are usually associated with microglia activation. Consistent with these findings, irradiation of microglia activated NF-kappaB, a transcription factor that regulates microglial activation. Addition of prostaglandin E2 (PGE2: a metabolic product of the COX-2 enzyme) to primary cultured rat astrocytes resulted in phenotypic changes similar to those observed in mixed-culture experiments. Therefore, it appears that PGE(2) released from irradiated microglia is a key mediator of irradiation-induced gliosis or astrocyte phenotype change. These data suggest that radiation-induced microglial activation and resultant production of PGE2 seems to be associated with an underlying cause of inflammatory complications associated with radiation therapy for malignant gliomas.

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

    Science.gov (United States)

    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.

  18. Amyloid-β(25-35), an amyloid-β(1-42) surrogate, and proinflammatory cytokines stimulate VEGF-A secretion by cultured, early passage, normoxic adult human cerebral astrocytes.

    Science.gov (United States)

    Chiarini, Anna; Whitfield, James; Bonafini, Clara; Chakravarthy, Balu; Armato, Ubaldo; Dal Prà, Ilaria

    2010-01-01

    Cerebrovascular angiopathy affects late-onset Alzheimer's disease (LOAD) brains by possibly increasing vascular endothelial growth factor (VEGF). A expression, thereby stimulating endothelial cell proliferation and migration. Indeed, VEGF-A gene upregulation, with increased VEGF-A protein content of reactive astrocytes and microglia, occurs in LOAD brains, and neovascularization was observed one week after injecting amyloid-β (Aβ)(1-42) into rat hippocampus. We have now found, with cultured 'normoxic' normal adult human astrocytes (NAHAs), that fibrillar Aβ(25-35) (an active Aβ(1-42) fragment) or a cytokine mixture (the (CM)-trio (interleukin [IL]-1β+interferon [IFN]-γ+tumor necrosis factor [TNF]-α), or pair (IFN-γ+TNF-α) like those produced in LOAD brains) stimulates the nuclear translocation of stabilized hypoxia-inducible factor (HIF)-1α protein and its binding to VEGF-A hypoxia-response elements; the mRNA synthesis for three VEGF-A splice variants (121, 165, 189); and the secretion of VEGF-A165. The CM-trio was the most powerful stimulus, IFN-γ+TNF-α was less potent, and other cytokine pairs or single cytokines or Aβ(35-25) were ineffective. While Aβ(25-35) did not change HIF-1β protein levels, the CM-trio increased both HIF-1α and HIF-1β protein levels, thereby giving an earlier and stronger stimulus to VEGF-A secretion by NAHAs. Thus, increased VEGF-A secretion from astrocytes stimulated by Aβ(1-42) and by microglia-released cytokines might restore angiogenesis and Aβ(1-42) vascular clearance.

  19. 尼卡地平对谷氨酸诱导大鼠海马星形胶质细胞损伤的影响%Effects of Nicardipine on Glutamate Induced-injury of Primary Cultured Rats Hippocampal Astrocytes

    Institute of Scientific and Technical Information of China (English)

    秦成名; 李清; 赵邦娥; 陆江; 王贤裕

    2011-01-01

    Objective To investigate the effects of different concentrations nicardipine on glutamate induced-injury of primary cultured rats hippocampal astrocytes. Methods Astrocytes were taken from hippocampus of 2 ~ 3 days old SD rats and incubated for 3 weeks. The cells were randomly divided into six groups(n = 9) :normal control group were given Hanks solution (group C) , glutamate group (group C) were given glutamate till the final concentration to 500 pjnol/L, nicardipine group (group N) were given nicardipine till the final concentration to 10 (imol/L, glutamate combined nicardipine groups (group GN, ,GN2 ,GN3) were given glutamate till the final concentration to 500 μmol/L and then added nicardipine till the final concentration were 1,5,10 (imol/L 10 minutes later. After cultured for 30 minutes,the intracellular dissociated Ca2+ concentration ( [Ca2+ ]I) were determined,and then cultured for24 hours,the cells apoptosis was analyzed with flow cytometer, the contents of malondialdehyde( MDA) .activity of superoxide dismutase( SOD) and glutathione( GSH) intracellular were measured. Glial fibrillary acidic protein(GFAP) expression and the morphological changes were observed with immunofluo-rescence staining. Results Compared with group C,the apoptotic cells in group G and group GN, were the majority,the non-apoptotic astrocytes were hyperplasia and hypertrophy, and the GFAP expression, [ Ca2+ ] I, content of MDA were significantly increased,the activity of SOD and GSH were significantly decreased. Compared with group G,the cell apoptosis were significantly decreased in group GN2 and GN3, and the GFAP expression, [ Ca2+ ] I,content of MDA were significantly decreased, the activity of SOD and GSH were significantly increased. Conclusion Nicardipine could inhibit the glutamate induced injury of hippocampal astrocytes through decreasing the intracellular Ca2+ overload and lipid peroxidation,clearing oxygen free radcials.%目的:观察不同浓度尼卡地平对谷氨酸诱

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

    Science.gov (United States)

    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.

  1. Actin cytoskeleton remodeling governs aquaporin-4 localization in astrocytes.

    Science.gov (United States)

    Nicchia, Grazia Paola; Rossi, Andrea; Mola, Maria Grazia; Procino, Giuseppe; Frigeri, Antonio; Svelto, Maria

    2008-12-01

    Aquaporin-4 (AQP4) is constitutively concentrated in the plasma membrane of the perivascular glial processes, and its expression is altered in certain pathological conditions associated with brain edema or altered glial migration. When astrocytes are grown in culture, they lose their characteristic star-like shape and AQP4 continuous plasma membrane localization observed in vivo. In this study, we differentiated primary astrocyte cultures with cAMP and lovastatin, both able to induce glial stellation through a reorganization of F-actin cytoskeleton, and obtained AQP4 selectively localized on the cell plasma membrane associated with an increase in the plasma membrane water transport level, but only cAMP induced an increase in AQP4 total protein expression. Phosphorylation experiments indicated that AQP4 in astrocytes is neither phosphorylated nor a substrate of PKA. Depolymerization of F-actin cytoskeleton performed by cytochalasin-D suggested that F-actin cytoskeleton plays a primary role for AQP4 plasma membrane localization and during cell adhesion. Finally, AQP4 knockdown does not compromise the ability of astrocytes to stellate in the presence of cAMP, indicating that astrocyte stellation is independent of AQP4. Copyright 2008 Wiley-Liss, Inc.

  2. Co-culture of astrocytes with neurons from injured brain A time-dependent dichotomy

    Institute of Scientific and Technical Information of China (English)

    Xiaojing Xu; Min Wang; Jing Liu; Jingya Lv; Yanan Hu; Huanxiang Zhang

    2011-01-01

    As supportive cells for neuronal growth and development, much effort has been devoted to the role of astrocytes in the normal state. However, the effect of the astrocytes after injury remains elusive. In the present study, neurons isolated from the subventricular zone of injured neonatal rat brains were co-cultured with astrocytes. After 6 days, these astrocytes showed a mature neuron-like appearance and the number of survivingneurons, primary dendrites and total branches was significantly higher than those at 3 days. The neurons began to shrink at 9 days after co-culture with shorter and thinner processes and the number of primary dendrites and total branches was significantly reduced. These experimental findings indicate that astrocytes in the injured brain promote the development of neurons in the early stages of co-culture while these cells reversely inhibit neuronal growth and development at the later states.

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

    Science.gov (United States)

    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.

  4. Astrocytic expression of HIV-1 Nef impairs spatial and recognition memory.

    Science.gov (United States)

    Chompre, Gladys; Cruz, Emmanuel; Maldonado, Lucianette; Rivera-Amill, Vanessa; Porter, James T; Noel, Richard J

    2013-01-01

    Despite the widespread use of antiretroviral therapy that effectively limits viral replication, memory impairment remains a dilemma for HIV infected people. In the CNS, HIV infection of astrocytes leads to the production of the HIV-1 Nef protein without viral replication. Post mortem studies have found Nef expression in hippocampal astrocytes of people with HIV associated dementia suggesting that astrocytic Nef may contribute to HIV associated cognitive impairment even when viral replication is suppressed. To test whether astrocytic expression of Nef is sufficient to induce cognitive deficits, we examined the effect of implanting primary rat astrocytes expressing Nef into the hippocampus on spatial and recognition memory. Rats implanted unilaterally with astrocytes expressing Nef showed impaired novel location and novel object recognition in comparison with controls implanted with astrocytes expressing green fluorescent protein (GFP). This impairment was correlated with an increase in chemokine ligand 2 (CCL2) expression and the infiltration of peripheral macrophages into the hippocampus at the site of injection. Furthermore, the Nef exposed rats exhibited a bilateral loss of CA3 neurons. These results suggest that Nef protein expressed by the implanted astrocytes activates the immune system leading to neuronal damage and spatial and recognition memory deficits. Therefore, the continued expression of Nef by astrocytes in the absence of viral replication has the potential to contribute to HIV associated cognitive impairment.

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

    Directory of Open Access Journals (Sweden)

    Chao Zhang

    2016-10-01

    Full Text Available 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.

  6. Functional sex differences in human primary auditory cortex

    NARCIS (Netherlands)

    Ruytjens, Liesbet; Georgiadis, Janniko R.; Holstege, Gert; Wit, Hero P.; Albers, Frans W. J.; Willemsen, Antoon T. M.

    2007-01-01

    Background We used PET to study cortical activation during auditory stimulation and found sex differences in the human primary auditory cortex (PAC). Regional cerebral blood flow (rCBF) was measured in 10 male and 10 female volunteers while listening to sounds (music or white noise) and during a bas

  7. Functional sex differences in human primary auditory cortex

    NARCIS (Netherlands)

    Ruytjens, Liesbet; Georgiadis, Janniko R.; Holstege, Gert; Wit, Hero P.; Albers, Frans W. J.; Willemsen, Antoon T. M.

    2007-01-01

    Background We used PET to study cortical activation during auditory stimulation and found sex differences in the human primary auditory cortex (PAC). Regional cerebral blood flow (rCBF) was measured in 10 male and 10 female volunteers while listening to sounds (music or white noise) and during a

  8. Modulation of connexin expression and gap junction communication in astrocytes by the gram-positive bacterium S. aureus.

    Science.gov (United States)

    Esen, Nilufer; Shuffield, Debbie; Syed, Mohsin M D; Kielian, Tammy

    2007-01-01

    Gap junctions establish direct intercellular conduits between adjacent cells and are formed by the hexameric organization of protein subunits called connexins (Cx). It is unknown whether the proinflammatory milieu that ensues during CNS infection with S. aureus, one of the main etiologic agents of brain abscess in humans, is capable of eliciting regional changes in astrocyte homocellular gap junction communication (GJC) and, by extension, influencing neuron homeostasis at sites distant from the primary focus of infection. Here we investigated the effects of S. aureus and its cell wall product peptidoglycan (PGN) on Cx43, Cx30, and Cx26 expression, the main Cx isoforms found in astrocytes. Both bacterial stimuli led to a time-dependent decrease in Cx43 and Cx30 expression; however, Cx26 levels were elevated following bacterial exposure. Functional examination of dye coupling, as revealed by single-cell microinjections of Lucifer yellow, demonstrated that both S. aureus and PGN inhibited astrocyte GJC. Inhibition of protein synthesis with cyclohexamide (CHX) revealed that S. aureus directly modulates, in part, Cx43 and Cx30 expression, whereas Cx26 levels appear to be regulated by a factor(s) that requires de novo protein production; however, CHX did not alter the inhibitory effects of S. aureus on astrocyte GJC. The p38 MAPK inhibitor SB202190 was capable of partially restoring the S. aureus-mediated decrease in astrocyte GJC to that of unstimulated cells, suggesting the involvement of p38 MAPK-dependent pathway(s). These findings could have important implications for limiting the long-term detrimental effects of abscess formation in the brain which may include seizures and cognitive deficits. Copyright 2006 Wiley-Liss, Inc.

  9. Humanization policy in primary health care: a systematic review

    Science.gov (United States)

    Nora, Carlise Rigon Dalla; Junges, José Roque

    2013-01-01

    OBJECTIVE To analyze humanization practices in primary health care in the Brazilian Unified Health System according to the principles of the National Humanization Policy. METHODS A systematic review of the literature was carried out, followed by a meta-synthesis, using the following databases: BDENF (nursing database), BDTD (Brazilian digital library of theses and dissertations), CINAHL (Cumulative Index to nursing and allied health literature), LILACS (Latin American and Caribbean health care sciences literature), MedLine (International health care sciences literature), PAHO (Pan-American Health Care Organization Library) and SciELO (Scientific Electronic Library Online). The following descriptors were used: Humanization; Humanizing Health Care; Reception: Humanized care: Humanization in health care; Bonding; Family Health Care Program; Primary Care; Public Health and Sistema Único de Saúde (the Brazilian public health care system). Research articles, case studies, reports of experiences, dissertations, theses and chapters of books written in Portuguese, English or Spanish, published between 2003 and 2011, were included in the analysis. RESULTS Among the 4,127 publications found on the topic, 40 studies were evaluated and included in the analysis, producing three main categories: the first referring to the infrastructure and organization of the primary care service, made clear the dissatisfaction with the physical structure and equipment of the services and with the flow of attendance, which can facilitate or make difficult the access. The second, referring to the health work process, showed issues about the insufficient number of professionals, fragmentation of the work processes, the professional profile and responsibility. The third category, referring to the relational technologies, indicated the reception, bonding, listening, respect and dialog with the service users. CONCLUSIONS Although many practices were cited as humanizing they do not produce changes

  10. Truncated N-terminal huntingtin fragment with expanded-polyglutamine (htt552-100Q)suppresses brain-derived neurotrophic factor transcription in astrocytes

    Institute of Scientific and Technical Information of China (English)

    Linhui Wang; Fang Lin; Jin Wang; Junchao Wu; Rong Han; Lujia Zhu; Guoxing Zhang; Marian DiFiglia; Zhenghong Qin

    2012-01-01

    Although huntingtin (htt) can be cleaved at many sites by caspases,calpains,and aspartyl proteases,amino acid (aa) 552 was defined as a preferred site for cleavage in human Huntington disease (HD) brains in vivo.To date,the normal function of wild-type N-terminal htt fragment 1-552 aa (htt552) and its pathological roles of mutant htt552 are still unknown.Although mutant htt (mhtt) is also expressed in astrocytes,whether and how mhtt contributes to the neurodegeneration through astrocytes in HD remains largely unknown.In this study,a glia HD model,using an adenoviral vector to express wild-type htt552 (htt552-18Q) and its mutation (htt552-100Q) in rat primary cortical astrocytes,was generated to investigate the influence of htt552 on the transcription of brainderived neurotrophic factor (BDNF). Results from enzyme linked immunosorbent assay showed that the level of BDNF in astrocyte-conditioned medium was decreased in the astrocytes expressing htt552-100Q.Quantitative real-time polymerase chain reaction demonstrated that htt552-100Q reduced the transcripts of the BDNF Ⅲ and Ⅳ, hence, repressed the transcription of BDNF.Furthermore,immunofluorescence showed that aggregates formed by htt552-100Q entrapped transcription factors cAMP-response element-binding protein and stimulatory protein 1,which might account for the reduction of BDNF transcription.These findings suggest that mhtt552 reduces BDNF transcription in astrocytes,which might contribute to the neuronal dysfunction in HD.

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

    Science.gov (United States)

    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

  12. Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts

    DEFF Research Database (Denmark)

    Le Bihan, Marie-Catherine; Barrio, Inigo; Mortensen, Tenna Pavia;

    2015-01-01

    and the complex temporal protein dynamics accompanying the differentiation of primary human muscle cells remain poorly understood. Here, we demonstrate the advantages of applying a MS-based quantitative approach, stable isotope labeling by amino acids in cell culture (SILAC), for studying human myogenesis...... dynamic expression profiles during the course of myogenic differentiation and quantified 2240 proteins, 243 of which were regulated. These changes in protein expression occurred in sequential waves and underlined vast reprogramming in key processes governing cell fate decisions, i.e., cell cycle...

  13. Uptake of gold nanoparticles in primary human endothelial cells

    DEFF Research Database (Denmark)

    Klingberg, Henrik; Oddershede, Lene B.; Löschner, Katrin

    2015-01-01

    Gold nanoparticles (AuNPs) are relevant in nanomedicine for drug delivery in the vascular system, where endothelial cells are the first point of contact. We investigated the uptake of 80 nm AuNPs in primary human umbilical vein endothelial cells (HUVECs) by flow cytometry, 3D confocal microscopy....... Uptake of AuNPs in HUVECs occurred mainly by clathrin-mediated endocytosis and trafficking to membrane enclosures in the form of single particles and agglomerates of 2–3 particles....

  14. Pharmacological Activation Gi/o Protein Increases Glial Cell Line-Derived Neurotrophic Factor Production through Fibroblast Growth Factor Receptor and Extracellular Signal-Regulated Kinase Pathway in Primary Cultured Rat Cortical Astrocytes.

    Science.gov (United States)

    Hisaoka-Nakashima, Kazue; Matsumoto, Chie; Azuma, Honami; Taki, Sayaka; Takebayashi, Minoru; Nakata, Yoshihiro; Morioka, Norimitsu

    2017-01-01

    A significant reduction of glial cell line-derived neurotrophic factor (GDNF) has been identified in the pathophysiology of neurodegenerative and neuropsychiatric disorders. Thus, clarification of the mechanism of GDNF production, and modulating brain GDNF levels could be a novel therapeutic approach. A previous study demonstrated that antidepressant amitriptyline-induced GDNF production was significantly inhibited by pertussis toxin (PTX), a Gi/o protein inhibitor in astrocytes, the main source of GDNF in the brain. However, it is not known whether direct activation of Gi/o protein might induce GDNF expression, and what mechanisms might be involved after Gi/o protein activation. The current study investigated Gi/o protein-initiated GDNF production in rat cortical astrocytes using activators that directly activate Gi/o protein, mastoparan and compound48/80. Treatment of astrocytes with either mastoparan or compound48/80 increased GDNF mRNA expression at 3 and 6 h, and GDNF protein release at 24 h. Treatment of astrocyte with either mastoparan or compound48/80 increased brain-derived neurotrophic factor (BDNF) mRNA expression as well as GDNF. Mastoparan and compound48/80-induced GDNF mRNA expression were significantly inhibited by not only PTX, but also fibroblast growth factor receptor (FGFR) inhibitors, and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor. In fact, both FGFR substrate2α (FRS2α) and ERK phosphorylation were increased by treatment with either mastoparan or compound48/80, and these were significantly blocked by PTX. Thus, direct, receptor-independent Gi/o protein activation increases GDNF production through FGFR/ERK signaling pathway. The current results indicate a critical role of Gi/o signaling in the regulation of GDNF expression in astrocytes.

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

    Science.gov (United States)

    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.

  16. Medium-chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte-neuron lactate and ketone body shuttle systems.

    Science.gov (United States)

    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.

  17. [Characterization of epithelial primary culture from human conjunctiva].

    Science.gov (United States)

    Rivas, L; Blázquez, A; Muñoz-Negrete, F J; López, S; Rebolleda, G; Domínguez, F; Pérez-Esteban, A

    2014-01-01

    To evaluate primary cultures from human conjunctiva supplemented with fetal bovine serum, autologous serum, and platelet-rich autologous serum, over human amniotic membrane and lens anterior capsules. One-hundred and forty-eight human conjunctiva explants were cultured in CnT50(®) supplemented with 1, 2.5, 5 and 10% fetal bovine serum, autologous serum and platelet-rich autologous serum. Conjunctival samples were incubated at 37°C, 5% CO2 and 95% HR, for 3 weeks. The typical phenotype corresponding to conjunctival epithelial cells was present in all primary cultures. Conjunctival cultures had MUC5AC-positive secretory cells, K19-positive conjunctival cells, and MUC4-positive non-secretory conjunctival cells, but were not corneal phenotype (cytokeratin K3-negative) and fibroblasts (CD90-negative). Conjunctiva epithelial progenitor cells were preserved in all cultures; thus, a cell culture in CnT50(®) supplemented with 1 to 5% autologous serum over human amniotic membrane can provide better information of epithelial cell differentiation for the conjunctival surface reconstruction. Copyright © 2013 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

  18. Protein targeting to glycogen is a master regulator of glycogen synthesis in astrocytes

    KAUST Repository

    Ruchti, E.

    2016-10-08

    The storage and use of glycogen, the main energy reserve in the brain, is a metabolic feature of astrocytes. Glycogen synthesis is regulated by Protein Targeting to Glycogen (PTG), a member of specific glycogen-binding subunits of protein phosphatase-1 (PPP1). It positively regulates glycogen synthesis through de-phosphorylation of both glycogen synthase (activation) and glycogen phosphorylase (inactivation). In cultured astrocytes, PTG mRNA levels were previously shown to be enhanced by the neurotransmitter noradrenaline. To achieve further insight into the role of PTG in the regulation of astrocytic glycogen, its levels of expression were manipulated in primary cultures of mouse cortical astrocytes using adenovirus-mediated overexpression of tagged-PTG or siRNA to downregulate its expression. Infection of astrocytes with adenovirus led to a strong increase in PTG expression and was associated with massive glycogen accumulation (>100 fold), demonstrating that increased PTG expression is sufficient to induce glycogen synthesis and accumulation. In contrast, siRNA-mediated downregulation of PTG resulted in a 2-fold decrease in glycogen levels. Interestingly, PTG downregulation strongly impaired long-term astrocytic glycogen synthesis induced by insulin or noradrenaline. Finally, these effects of PTG downregulation on glycogen metabolism could also be observed in cultured astrocytes isolated from PTG-KO mice. Collectively, these observations point to a major role of PTG in the regulation of glycogen synthesis in astrocytes and indicate that conditions leading to changes in PTG expression will directly impact glycogen levels in this cell type.

  19. Reactive Transformation and Increased BDNF Signaling by Hippocampal Astrocytes in Response to MK-801.

    Directory of Open Access Journals (Sweden)

    Wenjuan Yu

    Full Text Available MK-801, also known as dizocilpine, is a noncompetitive N-methyl-D-aspartic acid (NMDA receptor antagonist that induces schizophrenia-like symptoms. While astrocytes have been implicated in the pathophysiology of psychiatric disorders, including schizophrenia, astrocytic responses to MK-801 and their significance to schizotypic symptoms are unclear. Changes in the expression levels of glial fibrillary acid protein (GFAP, a marker of astrocyte activation in response to a variety of pathogenic stimuli, were examined in the hippocampus of rats treated with the repeated MK-801 injection (0.5 mg/10 ml/kg body weight for 6 days and in primary cultured hippocampal astrocytes incubated with MK-801 (5 or 20 μM for 24 h. Moreover, the expression levels of BDNF and its receptors TrkB and p75 were examined in MK-801-treated astrocyte cultures. MK-801 treatment enhanced GFAP expression in the rat hippocampus and also increased the levels of GFAP protein and mRNA in hippocampal astrocytes in vitro. Treatment of cultured hippocampal astrocytes with MK-801 enhanced protein and mRNA levels of BDNF, TrkB, and p75. Collectively, our results suggest that hippocampal astrocytes may contribute to the pathophysiology of schizophrenia symptoms associated with NMDA receptor hypofunction by reactive transformation and altered BDNF signaling.

  20. Astrocytic modulation of neuronal excitability through K(+) spatial buffering.

    Science.gov (United States)

    Bellot-Saez, Alba; Kékesi, Orsolya; Morley, John W; Buskila, Yossi

    2017-03-06

    The human brain contains two major cell populations, neurons and glia. While neurons are electrically excitable and capable of discharging short voltage pulses known as action potentials, glial cells are not. However, astrocytes, the prevailing subtype of glia in the cortex, are highly connected and can modulate the excitability of neurons by changing the concentration of potassium ions in the extracellular environment, a process called K(+) clearance. During the past decade, astrocytes have been the focus of much research, mainly due to their close association with synapses and their modulatory impact on neuronal activity. It has been shown that astrocytes play an essential role in normal brain function including: nitrosative regulation of synaptic release in the neocortex, synaptogenesis, synaptic transmission and plasticity. Here, we discuss the role of astrocytes in network modulation through their K(+) clearance capabilities, a theory that was first raised 50 years ago by Orkand and Kuffler. We will discuss the functional alterations in astrocytic activity that leads to aberrant modulation of network oscillations and synchronous activity.

  1. Intermediate filament transcription in astrocytes is repressed by proteasome inhibition

    Science.gov (United States)

    Middeldorp, Jinte; Kamphuis, Willem; Sluijs, Jacqueline A.; Achoui, Dalila; Leenaars, Cathalijn H. C.; Feenstra, Matthijs G. P.; van Tijn, Paula; Fischer, David F.; Berkers, Celia; Ovaa, Huib; Quinlan, Roy A.; Hol, Elly M.

    2009-01-01

    Increased expression of the astrocytic intermediate filament protein glial fibrillary acidic protein (GFAP) is a characteristic of astrogliosis. This process occurs in the brain during aging and neurodegeneration and coincides with impairment of the ubiquitin proteasome system. Inhibition of the proteasome impairs protein degradation; therefore, we hypothesized that the increase in GFAP may be the result of impaired proteasomal activity in astrocytes. We investigated the effect of proteasome inhibitors on GFAP expression and other intermediate filament proteins in human astrocytoma cells and in a rat brain model for astrogliosis. Extensive quantitative RT-PCR, immunocytochemistry, and Western blot analysis resulted unexpectedly in a strong decrease of GFAP mRNA to Hol, E. M. Intermediate filament transcription in astrocytes is repressed by proteasome inhibition. PMID:19332645

  2. Chronic infusion of SOD1(G93A) astrocyte-secreted factors induces spinal motoneuron degeneration and neuromuscular dysfunction in healthy rats.

    Science.gov (United States)

    Ramírez-Jarquín, Uri N; Rojas, Fabiola; van Zundert, Brigitte; Tapia, Ricardo

    2017-01-27

    Amyotrophic lateral sclerosis is a fatal neurodegenerative disease and studies in vitro show that motoneuron degeneration is triggered by non-cell-autonomous mechanisms. However, whether soluble toxic factor(s) released by mutant superoxide dismutase 1 (SOD1) expressing astrocytes induces death of motoneurons and leads to motor dysfunction in vivo is not known. To directly test this, healthy adult rats were treated with conditioned media derived from primary mouse astrocytes (ACM) that express human (h) SOD1(G93A) (ACM-hG93A) via chronic osmotic pump infusion in the lumbar spinal cord. Controls included ACM derived from transgenic mice expressing hSOD1(WT) (ACM-hWT) or non-transgenic mouse SOD1(WT) (ACM-WT) astrocytes. Rats chronically infused with ACM-hG93A started to develop motor dysfunction at 8 days, as measured by rotarod performance. Additionally, immunohistochemical analyses at day 16 revealed reactive astrogliosis and significant loss of motoneurons in the ventral horn of the infused region. Controls did not show significant motor behavior alterations or neuronal damage. Thus, we demonstrate that factors released in vitro from astrocytes derived from ALS mice cause spinal motoneuron death and consequent neuromuscular dysfunction in vivo.

  3. In vitro methods to culture primary human breast epithelial cells.

    Science.gov (United States)

    Raouf, Afshin; Sun, Yu Jia

    2013-01-01

    Current evidence suggests that much like leukemia, breast tumors are maintained by a small subpopulation of tumor cells that have stem cell properties. These cancer stem cells are envisaged to be responsible for tumor formation and relapse. Therefore, knowledge about their nature will provide a platform to develop therapies to eliminate these breast cancer stem cells. This concept highlights the need to understand the mechanisms that regulate the normal functions of the breast stem cells and their immediate progeny as alterations to these same mechanisms can cause these primitive cells to act as cancer stem cells. The study of the primitive cell functions relies on the ability to isolate them from primary sources of breast tissue. This chapter describes processing of discarded tissue from reduction mammoplasty samples as sources of normal primary human breast epithelial cells and describes cell culture systems to grow single-cell suspensions prepared from these reduction samples in vitro.

  4. Pulpotomies with portland cement in human primary molars

    Directory of Open Access Journals (Sweden)

    Taísa Regina Conti

    2009-02-01

    Full Text Available Two clinical cases in which Portland cement (PC was applied as a medicament after pulpotomy of mandibular primary molars in children are presented. Pulpotomy using PC was carried out in two mandibular first molars and one mandibular second molar, which were further followed-up. At the 3, 6 and 12-month follow-up appointments, clinical and radiographic examinations of the pulpotomized teeth and their periradicular area revealed that the treatments were successful in maintaining the teeth asymptomatic and preserving pulpal vitality. Additionally, the formation of a dentin bridge immediately below the PC could be observed in the three molars treated. PC may be considered as an effective alternative for primary molar pulpotomies, at least in a short-term period. Randomized clinical trials with human teeth are required in order to determine the suitability of PC before unlimited clinical use can be recommended.

  5. Neuromyelitis optica IgG stimulates an immunological response in rat astrocyte cultures

    Institute of Scientific and Technical Information of China (English)

    Howe CL; Kaptzan T; Magaa SM; Ayers-Ringler JR; LaFrance-Corey RG; Lucchinetti CF

    2014-01-01

    Neuromyelitis optica (NMO) is a primary astrocyte disease associated with central nervous system inflammation, demyelination, and tissue injury. Brain lesions are frequently observed in regions enriched in expression of the aquaporin-4 (AQP4) water channel, an antigenic target of the NMO IgG serologic marker. Based on observations of disease reversibility and careful characterization of NMO lesion development, we propose that the NMO IgG may induce a dynamic immunological response in astrocytes. Using primary rat astrocyte-enriched cultures and treatment with NMO patient-derived serum or purified IgG, we observed a robust pattern of gene expression changes consistent with the induction of a reactive and inflammatory phenotype in astrocytes. The reactive astrocyte factor lipocalin-2 and a broad spectrum of chemokines, cytokines, and stress response factors were induced by either NMO patient serum or purified IgG. Treatment with IgG from healthy controls had no effect. The effect is disease-specific, as serum from patients with relapsing-remitting multiple sclerosis, Sj gren's, or systemic lupus erythematosus did not induce a response in the cultures. We hypothesize that binding of the NMO IgG to AQP4 induces a cellular response that results in transcriptional and translational events within the astrocyte that are consistent with a reactive and inflammatory phenotype. Strategies aimed at reducing the inflammatory response of astrocytes may short circuit an amplification loop associated with NMO lesion development.

  6. Effect of fatty acids isolated from edible oils like mustard, linseed or coconut on astrocytes maturation.

    Science.gov (United States)

    Joardar, Anindita; Das, Sumantra

    2007-12-01

    The omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA, 22:6n-3) has been previously shown to facilitate some of the vital functions of astrocytes. Since some dietary oils contain alpha-linolenic acid (ALA, 18:3n-3), which is a precursor of DHA, we examined their effect on astrocyte development. Fatty acids (FAs) were isolated from commonly used oils and their compositions were determined by GLC. FAs from three oils, viz. coconut, mustard and linseed were studied for their effect on astrocyte morphology. Parallel studies were conducted with FAs from the same oils after heating for 72 h. Unlike coconut oil, FAs from mustard and linseed, both heated and raw, caused significant morphogenesis of astrocytes in culture. ss-AR binding was also substantially increased in astrocytes treated with FAs from raw mustard and linseed oils as compared to astrocytes grown in normal medium. The expression profile of the isoforms of GFAP showed that astrocyte maturation by FAs of mustard and linseed oil was associated with appearance of acidic variants of GFAP and disappearance of some neutral isoforms similar to that observed in cultures grown in serum containing medium or in the presence of DHA. Taken together, the study highlights the contribution of specific dietary oils in facilitating astrocyte development that can have potential impact on human health.

  7. Acute death of astrocytes in blast-exposed rat organotypic hippocampal slice cultures

    Science.gov (United States)

    Miller, Anna P.; Shah, Alok S.; Aperi, Brandy V.; Kurpad, Shekar N.; Stemper, Brian D.; Glavaski-Joksimovic, Aleksandra

    2017-01-01

    Blast traumatic brain injury (bTBI) affects civilians, soldiers, and veterans worldwide and presents significant health concerns. The mechanisms of neurodegeneration following bTBI remain elusive and current therapies are largely ineffective. It is important to better characterize blast-evoked cellular changes and underlying mechanisms in order to develop more effective therapies. In the present study, our group utilized rat organotypic hippocampal slice cultures (OHCs) as an in vitro system to model bTBI. OHCs were exposed to either 138 ± 22 kPa (low) or 273 ± 23 kPa (high) overpressures using an open-ended helium-driven shock tube, or were assigned to sham control group. At 2 hours (h) following injury, we have characterized the astrocytic response to a blast overpressure. Immunostaining against the astrocytic marker glial fibrillary acidic protein (GFAP) revealed acute shearing and morphological changes in astrocytes, including clasmatodendrosis. Moreover, overlap of GFAP immunostaining and propidium iodide (PI) indicated astrocytic death. Quantification of the number of dead astrocytes per counting area in the hippocampal cornu Ammonis 1 region (CA1), demonstrated a significant increase in dead astrocytes in the low- and high-blast, compared to sham control OHCs. However only a small number of GFAP-expressing astrocytes were co-labeled with the apoptotic marker Annexin V, suggesting necrosis as the primary type of cell death in the acute phase following blast exposure. Moreover, western blot analyses revealed calpain mediated breakdown of GFAP. The dextran exclusion additionally indicated membrane disruption as a potential mechanism of acute astrocytic death. Furthermore, although blast exposure did not evoke significant changes in glutamate transporter 1 (GLT-1) expression, loss of GLT-1-expressing astrocytes suggests dysregulation of glutamate uptake following injury. Our data illustrate the profound effect of blast overpressure on astrocytes in OHCs at 2 h

  8. Telomerase contributes to fludarabine resistance in primary human leukemic lymphocytes.

    Science.gov (United States)

    Shawi, May; Chu, Tsz Wai; Martinez-Marignac, Veronica; Yu, Y; Gryaznov, Sergei M; Johnston, James B; Lees-Miller, Susan P; Assouline, Sarit E; Autexier, Chantal; Aloyz, Raquel

    2013-01-01

    We report that Imetelstat, a telomerase inhibitor that binds to the RNA component of telomerase (hTR), can sensitize primary CLL lymphocytes to fludarabine in vitro. This effect was observed in lymphocytes from clinically resistant cases and with cytogenetic abnormalities associated with bad prognosis. Imetelstat mediated-sensitization to fludarabine was not associated with telomerase activity, but with the basal expression of Ku80. Since both Imetelstat and Ku80 bind hTR, we assessed 1) if Ku80 and Imetelstat alter each other's binding to hTR in vitro and 2) the effect of an oligonucleotide complementary to the Ku binding site in hTR (Ku oligo) on the survival of primary CLL lymphocytes exposed to fludarabine. We show that Imetelstat interferes with the binding of Ku70/80 (Ku) to hTR and that the Ku oligo can sensitize CLL lymphocytes to FLU. Our results suggest that Ku binding to hTR may contribute to fludarabine resistance in CLL lmphocytes. This is the first report highlighting the potentially broad effectiveness of Imetelstat in CLL, and the potential biological and clinical implications of a functional interaction between Ku and hTR in primary human cancer cells.

  9. Telomerase contributes to fludarabine resistance in primary human leukemic lymphocytes.

    Directory of Open Access Journals (Sweden)

    May Shawi

    Full Text Available We report that Imetelstat, a telomerase inhibitor that binds to the RNA component of telomerase (hTR, can sensitize primary CLL lymphocytes to fludarabine in vitro. This effect was observed in lymphocytes from clinically resistant cases and with cytogenetic abnormalities associated with bad prognosis. Imetelstat mediated-sensitization to fludarabine was not associated with telomerase activity, but with the basal expression of Ku80. Since both Imetelstat and Ku80 bind hTR, we assessed 1 if Ku80 and Imetelstat alter each other's binding to hTR in vitro and 2 the effect of an oligonucleotide complementary to the Ku binding site in hTR (Ku oligo on the survival of primary CLL lymphocytes exposed to fludarabine. We show that Imetelstat interferes with the binding of Ku70/80 (Ku to hTR and that the Ku oligo can sensitize CLL lymphocytes to FLU. Our results suggest that Ku binding to hTR may contribute to fludarabine resistance in CLL lmphocytes. This is the first report highlighting the potentially broad effectiveness of Imetelstat in CLL, and the potential biological and clinical implications of a functional interaction between Ku and hTR in primary human cancer cells.

  10. NT2 derived neuronal and astrocytic network signalling.

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    Eric J Hill

    Full Text Available A major focus of stem cell research is the generation of neurons that may then be implanted to treat neurodegenerative diseases. However, a picture is emerging where astrocytes are partners to neurons in sustaining and modulating brain function. We therefore investigated the functional properties of NT2 derived astrocytes and neurons using electrophysiological and calcium imaging approaches. NT2 neurons (NT2Ns expressed sodium dependent action potentials, as well as responses to depolarisation and the neurotransmitter glutamate. NT2Ns exhibited spontaneous and coordinated calcium elevations in clusters and in extended processes, indicating local and long distance signalling. Tetrodotoxin sensitive network activity could also be evoked by electrical stimulation. Similarly, NT2 astrocytes (NT2As exhibited morphology and functional properties consistent with this glial cell type. NT2As responded to neuronal activity and to exogenously applied neurotransmitters with calcium elevations, and in contrast to neurons, also exhibited spontaneous rhythmic calcium oscillations. NT2As also generated propagating calcium waves that were gap junction and purinergic signalling dependent. Our results show that NT2 derived astrocytes exhibit appropriate functionality and that NT2N networks interact with NT2A networks in co-culture. These findings underline the utility of such cultures to investigate human brain cell type signalling under controlled conditions. Furthermore, since stem cell derived neuron function and survival is of great importance therapeutically, our findings suggest that the presence of complementary astrocytes may be valuable in supporting stem cell derived neuronal networks. Indeed, this also supports the intriguing possibility of selective therapeutic replacement of astrocytes in diseases where these cells are either lost or lose functionality.

  11. Astrocyte-Synapse Structural Plasticity

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    Yann Bernardinelli

    2014-01-01

    Full Text Available The function and efficacy of synaptic transmission are determined not only by the composition and activity of pre- and postsynaptic components but also by the environment in which a synapse is embedded. Glial cells constitute an important part of this environment and participate in several aspects of synaptic functions. Among the glial cell family, the roles played by astrocytes at the synaptic level are particularly important, ranging from the trophic support to the fine-tuning of transmission. Astrocytic structures are frequently observed in close association with glutamatergic synapses, providing a morphological entity for bidirectional interactions with synapses. Experimental evidence indicates that astrocytes sense neuronal activity by elevating their intracellular calcium in response to neurotransmitters and may communicate with neurons. The precise role of astrocytes in regulating synaptic properties, function, and plasticity remains however a subject of intense debate and many aspects of their interactions with neurons remain to be investigated. A particularly intriguing aspect is their ability to rapidly restructure their processes and modify their coverage of the synaptic elements. The present review summarizes some of these findings with a particular focus on the mechanisms driving this form of structural plasticity and its possible impact on synaptic structure and function.

  12. Astrocytic glycogenolysis: mechanisms and functions.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  14. RANK and RANK ligand expression in primary human osteosarcoma

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    Daniel Branstetter

    2015-09-01

    Our results demonstrate RANKL expression was observed in the tumor element in 68% of human OS using IHC. However, the staining intensity was relatively low and only 37% (29/79 of samples exhibited≥10% RANKL positive tumor cells. RANK expression was not observed in OS tumor cells. In contrast, RANK expression was clearly observed in other cells within OS samples, including the myeloid osteoclast precursor compartment, osteoclasts and in giant osteoclast cells. The intensity and frequency of RANKL and RANK staining in OS samples were substantially less than that observed in GCTB samples. The observation that RANKL is expressed in OS cells themselves suggests that these tumors may mediate an osteoclastic response, and anti-RANKL therapy may potentially be protective against bone pathologies in OS. However, the absence of RANK expression in primary human OS cells suggests that any autocrine RANKL/RANK signaling in human OS tumor cells is not operative, and anti-RANKL therapy would not directly affect the tumor.

  15. Formaldehyde stimulates Mrp1-mediated glutathione deprivation of cultured astrocytes.

    Science.gov (United States)

    Tulpule, Ketki; Dringen, Ralf

    2011-02-01

    Formaldehyde (Fal) is an environmental neurotoxin that is also endogenously produced in brain. Since the tripeptide glutathione (GSH) plays an important role in detoxification processes in brain cells, we have investigated the consequences of a Fal exposure on the GSH metabolism of brain cells, using astrocyte-rich primary cultures as model system. Treatment of these cultures with Fal resulted in a rapid time- and concentration-dependent depletion of cellular GSH and a matching increase in the extracellular GSH content. Exposure of astrocytes to 1mm Fal for 3h did not compromise cell viability but almost completely deprived the cells of GSH. Half-maximal deprivation of cellular GSH was observed after application of 0.3mm Fal. This effect was rather specific for Fal, since methanol, formate or acetaldehyde did not affect cellular GSH levels. The Fal-stimulated GSH loss from viable astrocytes was completely prevented by semicarbazide-mediated chemical removal of Fal or by the application of MK571, an inhibitor of the multidrug resistance protein 1. These data demonstrate that Fal deprives astrocytes of cellular GSH by a multidrug resistance protein 1-mediated process.

  16. Evaluation of the importance of astrocytes when screening for acute toxicity in neuronal cell systems.

    Science.gov (United States)

    Woehrling, E K; Hill, E J; Coleman, M D

    2010-02-01

    Reliable, high throughput, in vitro preliminary screening batteries have the potential to greatly accelerate the rate at which regulatory neurotoxicity data is generated. This study evaluated the importance of astrocytes when predicting acute toxic potential using a neuronal screening battery of pure neuronal (NT2.N) and astrocytic (NT2.A) and integrated neuronal/astrocytic (NT2.N/A) cell systems derived from the human NT2.D1 cell line, using biochemical endpoints (mitochondrial membrane potential (MMP) depolarisation and ATP and GSH depletion). Following exposure for 72 h, the known acute human neurotoxicants trimethyltin-chloride, chloroquine and 6-hydroxydopamine were frequently capable of disrupting biochemical processes in all of the cell systems at non-cytotoxic concentrations. Astrocytes provide key metabolic and protective support to neurons during toxic challenge in vivo and generally the astrocyte containing cell systems showed increased tolerance to toxicant insult compared with the NT2.N mono-culture in vitro. Whilst there was no consistent relationship between MMP, ATP and GSH log IC(50) values for the NT2.N/A and NT2.A cell systems, these data did provide preliminary evidence of modulation of the acute neuronal toxic response by astrocytes. In conclusion, the suitability of NT2 neurons and astrocytes as cell systems for acute toxicity screening deserves further investigation.

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

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    Lorenzo Di Cesare Mannelli

    2015-01-01

    Full Text Available 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.

  18. Central role of maladapted astrocytic plasticity in ischemic brain edema formation

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    Yu-Feng eWang

    2016-05-01

    Full Text Available 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 ensuing reperfusion injury play primary roles. In response to the ischemic insult, the neurovascular unit experiences both intracellular and extracellular edemas; the two processes are interactive closely under the driving of maladapted astrocytic plasticity. The astrocytic plasticity includes both morphologic and functional plasticity. The former involves a reactive gliosis and the ensuing 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. 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 and water channel protein aquaporin 4 to form functional networks, regulate hydromineral balance across cell membranes and maintain the integrity of the blood-brain barrier. 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 blood-brain barrier, but also lead 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.

  19. Astrocytic Expression of CTMP Following an Excitotoxic Lesion in the Mouse Hippocampus

    Science.gov (United States)

    Shin, Nara; Yi, Min-Hee; Kim, Sena; Baek, Hyunjung; Triantafillu, Ursula L.

    2017-01-01

    Akt (also known as protein kinase B, PKB) has been seen to play a role in astrocyte activation of neuroprotection; however, the underlying mechanism on deregulation of Akt signaling in brain injuries is not fully understood. We investigated the role of carboxy-terminal modulator protein (CTMP), an endogenous Akt inhibitor, in brain injury following kainic acid (KA)-induced neurodegeneration of mouse hippocampus. In control mice, there was a weak signal for CTMP in the hippocampus, but CTMP was markedly increased in the astrocytes 3 days after KA treatment. To further investigate the effectiveness of Akt signaling, the phosphorylation of CTMP was examined. KA treatment induced an increased p-CTMP expression in the astrocytes of hippocampus at 1 day. LPS/IFN-γ-treatment on primary astrocytes promoted the p-CTMP was followed by phosphorylation of Akt and finally upregulation of CTMP and p-CREB. Time-dependent expression of p-CTMP, p-Akt, p-CREB, and CTMP indicate that LPS/IFN-γ-induced phosphorylation of CTMP can activate Akt/CREB signaling, whereas lately emerging enhancement of CTMP can inhibit it. These results suggest that elevation of CTMP in the astrocytes may suppress Akt activity and ultimately negatively affect the outcome of astrocyte activation (astroglisiois). Early time point enhancers of phosphorylation of CTMP and/or late time inhibitors specifically targeting CTMP may be beneficial in astrocyte activation for neuroprotection within treatment in neuroinflammatory conditions.

  20. AMPK Activation Affects Glutamate Metabolism in Astrocytes

    DEFF Research Database (Denmark)

    Voss, Caroline Marie; Pajęcka, Kamilla; Stridh, Malin H

    2015-01-01

    on glutamate metabolism in astrocytes was studied using primary cultures of these cells from mouse cerebral cortex during incubation in media containing 2.5 mM glucose and 100 µM [U-(13)C]glutamate. The metabolism of glutamate including a detailed analysis of its metabolic pathways involving the tricarboxylic...... acid (TCA) cycle was studied using high-performance liquid chromatography analysis supplemented with gas chromatography-mass spectrometry technology. It was found that AMPK activation had profound effects on the pathways involved in glutamate metabolism since the entrance of the glutamate carbon...... affected by a reduction of the flux of glutamate derived carbon through the malic enzyme and pyruvate carboxylase catalyzed reactions. Finally, it was found that in the presence of glutamate as an additional substrate, glucose metabolism monitored by the use of tritiated deoxyglucose was unaffected by AMPK...

  1. Adenosine Receptors Differentially Regulate the Expression of Regulators of G-Protein Signalling (RGS 2, 3 and 4 in Astrocyte-Like Cells.

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    Till Nicolas Eusemann

    Full Text Available The "regulators of g-protein signalling" (RGS comprise a large family of proteins that limit by virtue of their GTPase accelerating protein domain the signal transduction of G-protein coupled receptors. RGS proteins have been implicated in various neuropsychiatric diseases such as schizophrenia, drug abuse, depression and anxiety and aggressive behaviour. Since conditions associated with a large increase of adenosine in the brain such as seizures or ischemia were reported to modify the expression of some RGS proteins we hypothesized that adenosine might regulate RGS expression in neural cells. We measured the expression of RGS-2,-3, and -4 in both transformed glia cells (human U373 MG astrocytoma cells and in primary rat astrocyte cultures stimulated with adenosine agonists. Expression of RGS-2 mRNA as well as RGS2 protein was increased up to 30-fold by adenosine agonists in astrocytes. The order of potency of agonists and the blockade by the adenosine A2B-antagonist MRS1706 indicated that this effect was largely mediated by adenosine A2B receptors. However, a smaller effect was observed due to activation of adenosine A2A receptors. In astrocytoma cells adenosine agonists elicited an increase in RGS-2 expression solely mediated by A2B receptors. Expression of RGS-3 was inhibited by adenosine agonists in both astrocytoma cells and astrocytes. However while this effect was mediated by A2B receptors in astrocytoma cells it was mediated by A2A receptors in astrocytes as assessed by the order of potency of agonists and selective blockade by the specific antagonists MRS1706 and ZM241385 respectively. RGS-4 expression was inhibited in astrocytoma cells but enhanced in astrocytes by adenosine agonists.

  2. Rapamycin increases neuronal survival, reduces inflammation and astrocyte proliferation after spinal cord injury.

    Science.gov (United States)

    Goldshmit, Yona; Kanner, Sivan; Zacs, Maria; Frisca, Frisca; Pinto, Alexander R; Currie, Peter D; Pinkas-Kramarski, Ronit

    2015-09-01

    Spinal cord injury (SCI) frequently leads to a permanent functional impairment as a result of the initial injury followed by secondary injury mechanism, which is characterised by increased inflammation, glial scarring and neuronal cell death. Finding drugs that may reduce inflammatory cell invasion and activation to reduce glial scarring and increase neuronal survival is of major importance for improving the outcome after SCI. In the present study, we examined the effect of rapamycin, an mTORC1 inhibitor and an inducer of autophagy, on recovery from spinal cord injury. Autophagy, a process that facilitates the degradation of cytoplasmic proteins, is also important for maintenance of neuronal homeostasis and plays a major role in neurodegeneration after neurotrauma. We examined rapamycin effects on the inflammatory response, glial scar formation, neuronal survival and regeneration in vivo using spinal cord hemisection model in mice, and in vitro using primary cortical neurons and human astrocytes. We show that a single injection of rapamycin, inhibited p62/SQSTM1, a marker of autophagy, inhibited mTORC1 downstream effector p70S6K, reduced macrophage/neutrophil infiltration into the lesion site, microglia activation and secretion of TNFα. Rapamycin inhibited astrocyte proliferation and reduced the number of GFAP expressing cells at the lesion site. Finally, it increased neuronal survival and axonogenesis towards the lesion site. Our study shows that rapamycin treatment increased significantly p-Akt levels at the lesion site following SCI. Similarly, rapamycin treatment of neurons and astrocytes induced p-Akt elevation under stress conditions. Together, these findings indicate that rapamycin is a promising candidate for treatment of acute SCI condition and may be a useful therapeutic agent.

  3. Effect of silver nanoparticles on human primary keratinocytes.

    Science.gov (United States)

    Szmyd, Radoslaw; Goralczyk, Anna Grazyna; Skalniak, Lukasz; Cierniak, Agnieszka; Lipert, Barbara; Filon, Francesca Larese; Crosera, Matteo; Borowczyk, Julia; Laczna, Eliza; Drukala, Justyna; Klein, Andrzej; Jura, Jolanta

    2013-01-01

    Silver nanoparticles (AgNPs) have many biological applications in biomedicine, biotechnology and other life sciences. Depending on the size, shape and the type of carrier, AgNPs demonstrate different physical and chemical properties. AgNPs have strong antimicrobial, antiviral and antifungal activity, thus they are used extensively in a range of medical settings, particularly in wound dressings but also in cosmetics. This study was undertaken to examine the potential toxic effects of 15 nm polyvinylpyrrolidone-coated AgNPs on primary normal human epidermal keratinocytes (NHEK). Cells were treated with different concentrations of AgNPs and then cell viability, metabolic activity and other biological and biochemical aspects of keratinocytes functioning were studied. We observed that AgNPs decrease keratinocyte viability, metabolism and also proliferatory and migratory potential of these cells. Moreover, longer exposure resulted in activation of caspase 3/7 and DNA damage. Our studies show for the first time, that AgNPs may present possible danger for primary keratinocytes, concerning activation of genotoxic and cytotoxic processes depending on the concentration.

  4. Dopamine Receptor Activation Increases HIV Entry into Primary Human Macrophages

    Science.gov (United States)

    Gaskill, Peter J.; Yano, Hideaki H.; Kalpana, Ganjam V.; Javitch, Jonathan A.; Berman, Joan W.

    2014-01-01

    Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers. PMID:25268786

  5. Dopamine receptor activation increases HIV entry into primary human macrophages.

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    Peter J Gaskill

    Full Text Available Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers.

  6. The cutaneous rabbit illusion affects human primary sensory cortex somatotopically.

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    Felix Blankenburg

    2006-03-01

    Full Text Available We used functional magnetic resonance imaging (fMRI to study neural correlates of a robust somatosensory illusion that can dissociate tactile perception from physical stimulation. Repeated rapid stimulation at the wrist, then near the elbow, can create the illusion of touches at intervening locations along the arm, as if a rabbit hopped along it. We examined brain activity in humans using fMRI, with improved spatial resolution, during this version of the classic cutaneous rabbit illusion. As compared with control stimulation at the same skin sites (but in a different order that did not induce the illusion, illusory sequences activated contralateral primary somatosensory cortex, at a somatotopic location corresponding to the filled-in illusory perception on the forearm. Moreover, the amplitude of this somatosensory activation was comparable to that for veridical stimulation including the intervening position on the arm. The illusion additionally activated areas of premotor and prefrontal cortex. These results provide direct evidence that illusory somatosensory percepts can affect primary somatosensory cortex in a manner that corresponds somatotopically to the illusory percept.

  7. Steroid synthesis by primary human keratinocytes; implications for skin disease

    Energy Technology Data Exchange (ETDEWEB)

    Hannen, Rosalind F., E-mail: r.f.hannen@qmul.ac.uk [Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AT (United Kingdom); Michael, Anthony E. [Centre for Developmental and Endocrine Signalling, Academic Section of Obstetrics and Gynaecology, Division of Clinical Developmental Sciences, 3rd Floor, Lanesborough Wing, St. George' s, University of London, Cranmer Terrace, Tooting, London SW17 0RE (United Kingdom); Jaulim, Adil [Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AT (United Kingdom); Bhogal, Ranjit [Life Science, Unilever R and D Colworth House, Sharnbrook, Bedfordshire MK44 1LQ (United Kingdom); Burrin, Jacky M. [Centre for Endocrinology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ (United Kingdom); Philpott, Michael P. [Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AT (United Kingdom)

    2011-01-07

    Research highlights: {yields} Primary keratinocytes express the steroid enzymes required for cortisol synthesis. {yields} Normal primary human keratinocytes can synthesise cortisol. {yields} Steroidogenic regulators, StAR and MLN64, are expressed in normal epidermis. {yields} StAR expression is down regulated in eczema and psoriatic epidermis. -- Abstract: Cortisol-based therapy is one of the most potent anti-inflammatory treatments available for skin conditions including psoriasis and atopic dermatitis. Previous studies have investigated the steroidogenic capabilities of keratinocytes, though none have demonstrated that these skin cells, which form up to 90% of the epidermis are able to synthesise cortisol. Here we demonstrate that primary human keratinocytes (PHK) express all the elements required for cortisol steroidogenesis and metabolise pregnenolone through each intermediate steroid to cortisol. We show that normal epidermis and cultured PHK express each of the enzymes (CYP11A1, CYP17A1, 3{beta}HSD1, CYP21 and CYP11B1) that are required for cortisol synthesis. These enzymes were shown to be metabolically active for cortisol synthesis since radiometric conversion assays traced the metabolism of [7-{sup 3}H]-pregnenolone through each steroid intermediate to [7-{sup 3}H]-cortisol in cultured PHK. Trilostane (a 3{beta}HSD1 inhibitor) and ketoconazole (a CYP17A1 inhibitor) blocked the metabolism of both pregnenolone and progesterone. Finally, we show that normal skin expresses two cholesterol transporters, steroidogenic acute regulatory protein (StAR), regarded as the rate-determining protein for steroid synthesis, and metastatic lymph node 64 (MLN64) whose function has been linked to cholesterol transport in steroidogenesis. The expression of StAR and MLN64 was aberrant in two skin disorders, psoriasis and atopic dermatitis, that are commonly treated with cortisol, suggesting dysregulation of epidermal steroid synthesis in these patients. Collectively these data

  8. Human Rights Texts: Converting Human Rights Primary Source Documents into Data.

    Science.gov (United States)

    Fariss, Christopher J; Linder, Fridolin J; Jones, Zachary M; Crabtree, Charles D; Biek, Megan A; Ross, Ana-Sophia M; Kaur, Taranamol; Tsai, Michael

    2015-01-01

    We introduce and make publicly available a large corpus of digitized primary source human rights documents which are published annually by monitoring agencies that include Amnesty International, Human Rights Watch, the Lawyers Committee for Human Rights, and the United States Department of State. In addition to the digitized text, we also make available and describe document-term matrices, which are datasets that systematically organize the word counts from each unique document by each unique term within the corpus of human rights documents. To contextualize the importance of this corpus, we describe the development of coding procedures in the human rights community and several existing categorical indicators that have been created by human coding of the human rights documents contained in the corpus. We then discuss how the new human rights corpus and the existing human rights datasets can be used with a variety of statistical analyses and machine learning algorithms to help scholars understand how human rights practices and reporting have evolved over time. We close with a discussion of our plans for dataset maintenance, updating, and availability.

  9. Astrocyte-neuron co-culture on microchips based on the model of SOD mutation to mimic ALS.

    Science.gov (United States)

    Kunze, Anja; Lengacher, Sylvain; Dirren, Elisabeth; Aebischer, Patrick; Magistretti, Pierre J; Renaud, Philippe

    2013-07-24

    Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease. ALS is believed to be a non-cell autonomous condition, as other cell types, including astrocytes, have been implicated in disease pathogenesis. Hence, to facilitate the development of therapeutics against ALS, it is crucial to better understand the interactions between astrocytes and neural cells. Furthermore, cell culture assays are needed that mimic the complexity of cell to cell communication at the same time as they provide control over the different microenvironmental parameters. Here, we aim to validate a previously developed microfluidic system for an astrocyte-neuron cell culture platform, in which astrocytes have been genetically modified to overexpress either a human wild-type (WT) or a mutated form of the super oxide dismutase enzyme 1 (SOD1). Cortical neural cells were co-cultured with infected astrocytes and studied for up to two weeks. Using our microfluidic device that prevents direct cell to cell contact, we could evaluate neural cell response in the vicinity of astrocytes. We showed that neuronal cell density was reduced by about 45% when neurons were co-cultured with SOD-mutant astrocytes. Moreover, we demonstrated that SOD-WT overexpressing astrocytes reduced oxidative stress on cortical neurons that were in close metabolic contact. In contrast, cortical neurons in metabolic contact with SOD-mutant astrocytes lost their synapsin protein expression after severe glutamate treatment, an indication of the toxicity potentiating effect of the SOD-mutant enzyme.

  10. Interhemispheric interactions between the human primary somatosensory cortices.

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    Patrick Ragert

    Full Text Available In the somatosensory domain it is still unclear at which processing stage information reaches the opposite hemispheres. Due to dense transcallosal connections, the secondary somatosensory cortex (S2 has been proposed to be the key candidate for interhemispheric information transfer. However, recent animal studies showed that the primary somatosensory cortex (S1 might as well account for interhemispheric information transfer. Using paired median nerve somatosensory evoked potential recordings in humans we tested the hypothesis that interhemispheric inhibitory interactions in the somatosensory system occur already in an early cortical processing stage such as S1. Conditioning right S1 by electrical median nerve (MN stimulation of the left MN (CS resulted in a significant reduction of the N20 response in the target (left S1 relative to a test stimulus (TS to the right MN alone when the interstimulus interval between CS and TS was between 20 and 25 ms. No such changes were observed for later cortical components such as the N20/P25, N30, P40 and N60 amplitude. Additionally, the subcortically generated P14 response in left S1 was also not affected. These results document the existence of interhemispheric inhibitory interactions between S1 in human subjects in the critical time interval of 20-25 ms after median nerve stimulation.

  11. Proliferative Effects of Histamine on Primary Human Pterygium Fibroblasts

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    Zhenwei Qin

    2016-01-01

    Full Text Available Purpose. It has been confirmed that inflammatory cytokines are involved in the progression of pterygium. Histamine can enhance proliferation and migration of many cells. Therefore, we intend to investigate the proliferative and migratory effects of histamine on primary culture of human pterygium fibroblasts (HPFs. Methods. Pterygium and conjunctiva samples were obtained from surgery, and toluidine blue staining was used to identify mast cells. 3-[4, 5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT was performed to evaluate the proliferative rate of HPFs and human conjunctival fibroblasts (HCFs; ki67 expression was also measured by immunofluorescence analysis. Histamine receptor-1 (H1R antagonist (Diphenhydramine Hydrochloride and histamine receptor-2 (H2R antagonist (Nizatidine were added to figure out which receptor was involved. Wound healing model was used to evaluate the migratory ability of HPFs. Results. The numbers of total mast cells and degranulated mast cells were both higher in pterygium than in conjunctiva. Histamine had a proliferative effect on both HPFs and HCFs, the effective concentration (10 μmol/L on HPFs was lower than on HCFs (100 μmol/L, and the effect could be blocked by H1R antagonist. Histamine showed no migratory effect on HPFs. Conclusion. Histamine may play an important role in the proliferation of HPFs and act through H1R.

  12. Effect of matrine on primary human hepatocytes in vitro.

    Science.gov (United States)

    Gong, Xiaobing; Gao, Yuan; Guo, Guoqing; Vondran, Florian W R; Schwartlander, Ruth; Efimova, Ekaterina; Pless, Gesine; Sauera, Igor M; Neuhaus, Peter

    2015-03-01

    Matrine is a bioactive component of the traditional Chinese medical herb Sophora flavescens that has been used in China to treat various kinds of diseases including virus hepatitis. However, the molecular mechanisms underlying its hepatoprotective effects remains elusive. In the present study, primary human hepatocytes were employed to elucidate the protective effects and molecular mechanisms of matrine. We observed that low concentrations of matrine had no significant impact on albumin secretion, but high concentrations (>140 mg/L) of matrine decreased the albumin secretion in hepatocytes. Western blot data indicated that matrine at 140 mg/L at 72 h induced protein expression of CYP2A6, CYP2B6 and CYP3A4. Furthermore, high concentrations of matrine reduced LDH and AST levels and were cytotoxic to hepatocytes, leading to a decreased cell viability and total protein amount. Moreover, low concentrations of matrine, enhanced the ECOD activity and decreased the level of NO2 (-) induced by cytokines in human hepatocytes. Taken together, the present study sheds novel light on the molecular mechanisms of matrine and potential application of matrine in hepatic diseases.

  13. Transcranial focused ultrasound stimulation of human primary visual cortex

    Science.gov (United States)

    Lee, Wonhye; Kim, Hyun-Chul; Jung, Yujin; Chung, Yong An; Song, In-Uk; Lee, Jong-Hwan; Yoo, Seung-Schik

    2016-09-01

    Transcranial focused ultrasound (FUS) is making progress as a new non-invasive mode of regional brain stimulation. Current evidence of FUS-mediated neurostimulation for humans has been limited to the observation of subjective sensory manifestations and electrophysiological responses, thus warranting the identification of stimulated brain regions. Here, we report FUS sonication of the primary visual cortex (V1) in humans, resulting in elicited activation not only from the sonicated brain area, but also from the network of regions involved in visual and higher-order cognitive processes (as revealed by simultaneous acquisition of blood-oxygenation-level-dependent functional magnetic resonance imaging). Accompanying phosphene perception was also reported. The electroencephalo graphic (EEG) responses showed distinct peaks associated with the stimulation. None of the participants showed any adverse effects from the sonication based on neuroimaging and neurological examinations. Retrospective numerical simulation of the acoustic profile showed the presence of individual variability in terms of the location and intensity of the acoustic focus. With exquisite spatial selectivity and capability for depth penetration, FUS may confer a unique utility in providing non-invasive stimulation of region-specific brain circuits for neuroscientific and therapeutic applications.

  14. Helicobacter pylori phagosome maturation in primary human macrophages

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    Borlace Glenn N

    2011-03-01

    Full Text Available Abstract Background Helicobacter pylori (H. pylori is a micro-aerophilic, spiral-shaped, motile bacterium that is the principal cause of gastric and duodenal ulcers in humans and is a major risk factor for the development of gastric cancer. Despite provoking a strong innate and adaptive immune response in the host, H. pylori persists in the gastric mucosa, avoiding eradication by macrophages and other phagocytic cells, which are recruited to the site of infection. Here we have characterised the critical degradative process of phagosome maturation in primary human macrophages for five genotypically and phenotypically distinct clinical strains of H. pylori. Results All of the H. pylori strains examined showed some disruption to the phagosome maturation process, when compared to control E. coli. The early endosome marker EEA1 and late endosome marker Rab7 were retained on H. pylori phagosomes, while the late endosome-lysosome markers CD63, LAMP-1 and LAMP-2 were acquired in an apparently normal manner. Acquisition of EEA1 by H. pylori phagosomes appeared to occur by two distinct, strain specific modes. H. pylori strains that were negative for the cancer associated virulence factor CagA were detected in phagosomes that recruited large amounts of EEA1 relative to Rab5, compared to CagA positive strains. There were also strain specific differences in the timing of Rab7 acquisition which correlated with differences in the rate of intracellular trafficking of phagosomes and the timing of megasome formation. Megasomes were observed for all of the H. pylori strains examined. Conclusions H. pylori appeared to disrupt the normal process of phagosome maturation in primary human macrophages, appearing to block endosome fission. This resulted in the formation of a hybrid phagosome-endosome-lysosome compartment, which we propose has reduced degradative capacity. Reduced killing by phagocytes is consistent with the persistence of H. pylori in the host, and would

  15. Genetic control of astrocyte function in neural circuits

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    Hannah Maria Jahn

    2015-08-01

    Full Text Available During the last two decades numerous genetic approaches affecting cell function in vivo have been developed. Current state-of-the-art technology permits the selective switching of gene function in distinct cell populations within the complex organization of a given tissue parenchyma. The tamoxifen-inducible Cre/loxP gene recombination and the doxycycline-dependent modulation of gene expression are probably the most popular genetic paradigms.Here, we will review applications of these two strategies while focussing on the interactions of astrocytes and neurons in the central nervous system (CNS and their impact for the whole organism. Abolishing glial sensing of neuronal activity by selective deletion of glial transmitter receptors demonstrated the impact of astrocytes for higher cognitive functions such as learning and memory, or the more basic body control of muscle coordination. Interestingly, also interfering with glial output, i.e. the release of gliotransmitters can drastically change animal’s physiology like sleeping behavior. Furthermore, such genetic approaches have also been used to restore astrocyte function. In these studies two alternatives were employed to achieve proper genetic targeting of astrocytes: transgenes using the promoter of the human glial fibrillary acidic protein (GFAP or homologous recombination into the glutamate-aspartate transporter (GLAST locus. We will highlight their specific properties that could be relevant for their use.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Andrej Korenić

    Full Text Available 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.

  18. Astrocyte-Synapse Structural Plasticity

    OpenAIRE

    2014-01-01

    The function and efficacy of synaptic transmission are determined not only by the composition and activity of pre- and postsynaptic components but also by the environment in which a synapse is embedded. Glial cells constitute an important part of this environment and participate in several aspects of synaptic functions. Among the glial cell family, the roles played by astrocytes at the synaptic level are particularly important, ranging from the trophic support to the fine-tuning of transmissi...

  19. Astrocyte calcium signaling: the third wave.

    Science.gov (United States)

    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.

  20. Triptolide protects astrocytes from hypoxia/ reoxygenation injury

    Institute of Scientific and Technical Information of China (English)

    Minfang Guo; Hongcui Fan; Jiezhong Yu; Ning Ji; Yongsheng Sun; Liyun Liang; Baoguo Xiao; Cungen Ma

    2011-01-01

    Astrocytes in an in vitro murine astrocyte model of oxygen and glucose deprivation/hypoxia and reoxygenation were treated with different concentrations of triptolide (250, 500, 1 000 ng/mL) in a broader attempt to elucidate the protection and mechanism underlying triptolide treatment on astrocytes exposed to hypoxia/reoxygenation injury. The results showed that the matrix metalloproteinase-9, interleukin-1β, tumor necrosis factor α and interleukin-6 expressions were significantly decreased after triptolide treatment in the astrocytes exposed to hypoxia/ reoxygenation injury, while interleukin-10 expression was upregulated. In addition, the vitality of the injured astrocytes was enhanced, the triptolide's effect was apparent at 500 ng/mL. These experimental findings indicate that triptolide treatment could protect astrocytes against hypoxia/ reoxygenation injury through the inhibition of inflammatory response and the reduction of matrix metalloproteinase-9 expression.

  1. Astrocyte-derived vascular endothelial growth factor stabilizes vessels in the developing retinal vasculature.

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    Andrew Scott

    Full Text Available Vascular endothelial growth factor (VEGF plays a critical role in normal development as well as retinal vasculature disease. During retinal vascularization, VEGF is most strongly expressed by not yet vascularized retinal astrocytes, but also by retinal astrocytes within the developing vascular plexus, suggesting a role for retinal astrocyte-derived VEGF in angiogenesis and vessel network maturation. To test the role of astrocyte-derived VEGF, we used Cre-lox technology in mice to delete VEGF in retinal astrocytes during development. Surprisingly, this only had a minor impact on retinal vasculature development, with only small decreases in plexus spreading, endothelial cell proliferation and survival observed. In contrast, astrocyte VEGF deletion had more pronounced effects on hyperoxia-induced vaso-obliteration and led to the regression of smooth muscle cell-coated radial arteries and veins, which are usually resistant to the vessel-collapsing effects of hyperoxia. These results suggest that VEGF production from retinal astrocytes is relatively dispensable during development, but performs vessel stabilizing functions in the retinal vasculature and might be relevant for retinopathy of prematurity in humans.

  2. RTTN Mutations Cause Primary Microcephaly and Primordial Dwarfism in Humans.

    Science.gov (United States)

    Shamseldin, Hanan; Alazami, Anas M; Manning, Melanie; Hashem, Amal; Caluseiu, Oana; Tabarki, Brahim; Esplin, Edward; Schelley, Susan; Innes, A Micheil; Parboosingh, Jillian S; Lamont, Ryan; Majewski, Jacek; Bernier, Francois P; Alkuraya, Fowzan S

    2015-12-03

    Primary microcephaly is a developmental brain anomaly that results from defective proliferation of neuroprogenitors in the germinal periventricular zone. More than a dozen genes are known to be mutated in autosomal-recessive primary microcephaly in isolation or in association with a more generalized growth deficiency (microcephalic primordial dwarfism), but the genetic heterogeneity is probably more extensive. In a research protocol involving autozygome mapping and exome sequencing, we recruited a multiplex consanguineous family who is affected by severe microcephalic primordial dwarfism and tested negative on clinical exome sequencing. Two candidate autozygous intervals were identified, and the second round of exome sequencing revealed a single intronic variant therein (c.2885+8A>G [p.Ser963(∗)] in RTTN exon 23). RT-PCR confirmed that this change creates a cryptic splice donor and thus causes retention of the intervening 7 bp of the intron and leads to premature truncation. On the basis of this finding, we reanalyzed the exome file of a second consanguineous family affected by a similar phenotype and identified another homozygous change in RTTN as the likely causal mutation. Combined linkage analysis of the two families confirmed that RTTN maps to the only significant linkage peak. Finally, through international collaboration, a Canadian multiplex family affected by microcephalic primordial dwarfism and biallelic mutation of RTTN was identified. Our results expand the phenotype of RTTN-related disorders, hitherto limited to polymicrogyria, to include microcephalic primordial dwarfism with a complex brain phenotype involving simplified gyration. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  3. Knockdown of astrocyte elevated gene-1 inhibits proliferation and enhancing chemo-sensitivity to cisplatin or doxorubicin in neuroblastoma cells

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    Xie Li

    2009-02-01

    Full Text Available Abstract Background Astrocyte elevated gene-1 (AEG-1 was originally characterized as a HIV-1-inducible gene in primary human fetal astrocyte. Recent studies highlight a potential role of AEG-1 in promoting tumor progression and metastasis. The aim of this study was to investigate if AEG-1 serves as a potential therapeutic target of human neuroblastoma. Methods We employed RNA interference to reduce AEG-1 expression in human neuroblastoma cell lines and analyzed their phenotypic changes. Results We found that the knockdown of AEG-1 expression in human neuroblastoma cells significantly inhibited cell proliferation and apoptosis. The specific downregulation induced cell arrest in the G0/G1 phase of cell cycle. In the present study, we also observed a significant enhancement of chemo-sensitivity to cisplatin and doxorubicin by knockdown of AEG-1. Conclusion Our study suggests that overexpressed AEG-1 enhance the tumorogenic properties of neuroblastoma cells. The inhibition of AEG-1 expression could be a new adjuvant therapy for neuroblastoma.

  4. Self-contained induction of neurons from human embryonic stem cells.

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    Tsuyoshi Okuno

    Full Text Available BACKGROUND: Neurons and glial cells can be efficiently induced from mouse embryonic stem (ES cells in a conditioned medium collected from rat primary-cultured astrocytes (P-ACM. However, the use of rodent primary cells for clinical applications may be hampered by limited supply and risk of contamination with xeno-proteins. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an alternative method for unimpeded production of human neurons under xeno-free conditions. Initially, neural stem cells in sphere-like clusters were induced from human ES (hES cells after being cultured in P-ACM under free-floating conditions. The resultant neural stem cells could circumferentially proliferate under subsequent adhesive culture, and selectively differentiate into neurons or astrocytes by changing the medium to P-ACM or G5, respectively. These hES cell-derived neurons and astrocytes could procure functions similar to those of primary cells. Interestingly, a conditioned medium obtained from the hES cell-derived astrocytes (ES-ACM could successfully be used to substitute P-ACM for induction of neurons. Neurons made by this method could survive in mice brain after xeno-transplantation. CONCLUSION/SIGNIFICANCE: By inducing astrocytes from hES cells in a chemically defined medium, we could produce human neurons without the use of P-ACM. This self-serving method provides an unlimited source of human neural cells and may facilitate clinical applications of hES cells for neurological diseases.

  5. Relaxin protects astrocytes from hypoxia in vitro.

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    Jordan M Willcox

    Full Text Available The peptide relaxin has recently been shown to protect brain tissues from the detrimental effects of ischemia. To date, the mechanisms for this remain unclear. In order to investigate the neuroprotective mechanisms by which relaxin may protect the brain, we investigated the possibility that relaxin protects astrocytes from hypoxia or oxygen/glucose deprivation (OGD. Cultured astrocytes were pre-treated with either relaxin-2 or relaxin-3 and exposed to OGD for 24 or 48 hours. Following OGD exposure, viability assays showed that relaxin-treated cells exhibited a higher viability when compared to astrocytes that experienced OGD-alone. Next, to test whether relaxin reduced the production of reactive oxygen species (ROS astrocytes were exposed to the same conditions as the previous experiment and a commercially available ROS detection kit was used to detect ROS production. Astrocytes that were treated with relaxin-2 and relaxin-3 showed a marked decrease in ROS production when compared to control astrocytes that were exposed only to OGD. Finally, experiments were performed to determine whether or not the mitochondrial membrane potential was affected by relaxin treatment during 24 hour OGD. Mitochondrial membrane potential was higher in astrocytes that were treated with relaxin-2 and relaxin-3 compared to untreated OGD-alone astrocytes. Taken together, these data present novel findings that show relaxin protects astrocytes from ischemic conditions through the reduction of ROS production and the maintenance of mitochondrial membrane potential.

  6. Alteration of astrocytes and Wnt/β-catenin signaling in the frontal cortex of autistic subjects

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    Cao Fujiang

    2012-09-01

    Full Text Available Abstract Background Autism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors. To date the etiology of this disorder is poorly understood. Studies suggest that astrocytes play critical roles in neural plasticity by detecting neuronal activity and modulating neuronal networks. Recently, a number of studies suggested that an abnormal function of glia/astrocytes may be involved in the development of autism. However, there is yet no direct evidence showing how astrocytes develop in the brain of autistic individuals. Methods Study subjects include brain tissue from autistic subjects, BTBR T + tfJ (BTBR and Neuroligin (NL-3 knock-down mice. Western blot analysis, Immunohistochemistry and confocal microscopy studies have be used to examine the density and morphology of astrocytes, as well as Wnt and β-catenin protein expression. Results In this study, we demonstrate that the astrocytes in autisitcsubjects exhibit significantly reduced branching processes, total branching length and cell body sizes. We also detected an astrocytosis in the frontal cortex of autistic subjects. In addition, we found that the astrocytes in the brain of an NL3 knockdown mouse exhibited similar alterations to what we found in the autistic brain. Furthermore, we detected that both Wnt and β-catenin proteins are decreased in the frontal cortex of autistic subjects. Wnt/β-catenin pathway has been suggested to be involved in the regulation of astrocyte development. Conclusions Our findings imply that defects in astrocytes could impair neuronal plasticity and partially contribute to the development of autistic-like behaviors in both humans and mice. The alteration of Wnt/β-catenin pathway in the brain of autistic subjects may contribute to the changes of astrocytes.

  7. Inhibitory effect of the antidepressant imipramine on NF-κB-dependent CXCL1 expression in TNFα-exposed astrocytes.

    Science.gov (United States)

    Lee, Young Han; Kim, Se Hyun; Kim, Yeni; Lim, Yoongho; Ha, Kyooseob; Shin, Soon Young

    2012-04-01

    Neuroinflammation is associated with the pathophysiology of various neurodegenerative diseases. Emerging evidence indicates that imipramine, a tricyclic antidepressant commonly used in depressive disorders, exhibits neuroprotective activity partly through anti-inflammatory effects. However, the molecular mechanisms underlying imipramine-mediated anti-inflammatory response are poorly understood. In this study, rat primary cultured astrocytes were used to elucidate the effect of the imipramine on TNFα-induced inflammatory responses. The results clearly demonstrated that imipramine reduced TNFα-induced CXCL1 expression through suppression of NF-κB-dependent CXCL1 promoter activity in primary astrocytes. In addition, we found that imipramine suppressed TNFα-induced phosphorylation of inhibitor of κBα (IκBα) and p65/RelA nuclear factor-κB (NF-κB), as well as the nuclear translocation of p65/RelA in primary cultured astrocytes. Chemotaxis assay demonstrated that astrocyte-derived CXCL1 contributed to migration of BV2 microglial cells toward astrocytes. This response was significantly blocked by treatment of astrocytes with imipramine or NF-κB inhibitor BAY11-7082. This study indicates that the antidepressant imipramine inhibits TNFα-induced CXCL1 expression via down-regulation of NF-κB signaling pathway in astrocytes and suggests that imipramine has a potential as an anti-inflammatory drug.

  8. Noradrenaline release and the pathophysiology of primary human hypertension

    Energy Technology Data Exchange (ETDEWEB)

    Esler, M.; Jennings, G.; Lambert, G.

    1989-03-01

    Measurements of the overflow of norepinephrine to plasma from individual organs (using radiotracer methodology) were used to delineate the pattern of sympathetic nervous system activation present in primary human hypertension. Mean total norepinephrine (NE) spillover in hypertensive patients was 418 ng/min, 42% (124 ng/min) higher than in subjects with normal blood pressure (BP)(P less than .05). Norepinephrine spillover among hypertensive patients was a function of age, only being elevated in patients under 40 years of age. Half of the excess in total norepinephrine release in hypertensive patients was accounted for by increased cardiorenal spillover. Mean renal norepinephrine spillover was 120 ng/min, compared with 69 ng/min in healthy subjects (P less than .02). Renal spillover was highest in younger patients. Corresponding cardiac norepinephrine spillover values were 12.6 ng/min and 5.1 ng/min (P less than .01). The balance of the excess total norepinephrine spillover comes from undetermined sites, but not the lungs or hepatomesenteric circulation. These measurements of regional norepinephrine overflow suggest that sympathetic nervous outflow to the kidneys and heart is selectively activated in early hypertension. 21 references.

  9. Irreversible electroporation of human primary uveal melanoma in enucleated eyes.

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    Yossi Mandel

    Full Text Available Uveal melanoma (UM is the most common primary intraocular tumor in adults and is characterized by high rates of metastatic disease. Although brachytherapy is the most common globe-sparing treatment option for small- and medium-sized tumors, the treatment is associated with severe adverse reactions and does not lead to increased survival rates as compared to enucleation. The use of irreversible electroporation (IRE for tumor ablation has potential advantages in the treatment of tumors in complex organs such as the eye. Following previous theoretical work, herein we evaluate the use of IRE for uveal tumor ablation in human ex vivo eye model. Enucleated eyes of patients with uveal melanoma were treated with short electric pulses (50-100 µs, 1000-2000 V/cm using a customized electrode design. Tumor bioimpedance was measured before and after treatment and was followed by histopathological evaluation. We found that IRE caused tumor ablation characterized by cell membrane disruption while sparing the non-cellular sclera. Membrane disruption and loss of cellular capacitance were also associated with significant reduction in total tumor impedance and loss of impedance frequency dependence. The effect was more pronounced near the pulsing electrodes and was dependent on time from treatment to fixation. Future studies should further evaluate the potential of IRE as an alternative method of uveal melanoma treatment.

  10. 3D Cultivation Techniques for Primary Human Hepatocytes

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    Anastasia Bachmann

    2015-02-01

    Full Text Available One of the main challenges in drug development is the prediction of in vivo toxicity based on in vitro data. The standard cultivation system for primary human hepatocytes is based on monolayer cultures, even if it is known that these conditions result in a loss of hepatocyte morphology and of liver-specific functions, such as drug-metabolizing enzymes and transporters. As it has been demonstrated that hepatocytes embedded between two sheets of collagen maintain their function, various hydrogels and scaffolds for the 3D cultivation of hepatocytes have been developed. To further improve or maintain hepatic functions, 3D cultivation has been combined with perfusion. In this manuscript, we discuss the benefits and drawbacks of different 3D microfluidic devices. For most systems that are currently available, the main issues are the requirement of large cell numbers, the low throughput, and expensive equipment, which render these devices unattractive for research and the drug-developing industry. A higher acceptance of these devices could be achieved by their simplification and their compatibility with high-throughput, as both aspects are of major importance for a user-friendly device.

  11. Functional sex differences in human primary auditory cortex

    Energy Technology Data Exchange (ETDEWEB)

    Ruytjens, Liesbet [University Medical Center Groningen, Department of Otorhinolaryngology, Groningen (Netherlands); University Medical Center Utrecht, Department Otorhinolaryngology, P.O. Box 85500, Utrecht (Netherlands); Georgiadis, Janniko R. [University of Groningen, University Medical Center Groningen, Department of Anatomy and Embryology, Groningen (Netherlands); Holstege, Gert [University of Groningen, University Medical Center Groningen, Center for Uroneurology, Groningen (Netherlands); Wit, Hero P. [University Medical Center Groningen, Department of Otorhinolaryngology, Groningen (Netherlands); Albers, Frans W.J. [University Medical Center Utrecht, Department Otorhinolaryngology, P.O. Box 85500, Utrecht (Netherlands); Willemsen, Antoon T.M. [University Medical Center Groningen, Department of Nuclear Medicine and Molecular Imaging, Groningen (Netherlands)

    2007-12-15

    We used PET to study cortical activation during auditory stimulation and found sex differences in the human primary auditory cortex (PAC). Regional cerebral blood flow (rCBF) was measured in 10 male and 10 female volunteers while listening to sounds (music or white noise) and during a baseline (no auditory stimulation). We found a sex difference in activation of the left and right PAC when comparing music to noise. The PAC was more activated by music than by noise in both men and women. But this difference between the two stimuli was significantly higher in men than in women. To investigate whether this difference could be attributed to either music or noise, we compared both stimuli with the baseline and revealed that noise gave a significantly higher activation in the female PAC than in the male PAC. Moreover, the male group showed a deactivation in the right prefrontal cortex when comparing noise to the baseline, which was not present in the female group. Interestingly, the auditory and prefrontal regions are anatomically and functionally linked and the prefrontal cortex is known to be engaged in auditory tasks that involve sustained or selective auditory attention. Thus we hypothesize that differences in attention result in a different deactivation of the right prefrontal cortex, which in turn modulates the activation of the PAC and thus explains the sex differences found in the activation of the PAC. Our results suggest that sex is an important factor in auditory brain studies. (orig.)

  12. A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

    Science.gov (United States)

    Anderl, Janet L; Redpath, Stella; Ball, Andrew J

    2009-01-01

    -culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling βIII-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery. PMID:19417729

  13. A neuronal and astrocyte co-culture assay for high content analysis of neurotoxicity.

    Science.gov (United States)

    Anderl, Janet L; Redpath, Stella; Ball, Andrew J

    2009-05-05

    -culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling betaIII-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

  14. Astrocyte loss and astrogliosis in neuroinflammatory disorders

    NARCIS (Netherlands)

    Hostenbach, Stephanie; Cambron, Melissa; D'haeseleer, Miguel; Kooijman, Ron; De Keyser, Jacques

    2014-01-01

    Neuroinflammation can lead to either damage of astrocytes or astrogliosis. Astrocyte loss may be caused by cytotoxic T cells as seen in Rasmussen encephalitis, auto-antibodies such as in neuromyelitis optica (aquaporin-4 antibodies), or cytokines such as TNF-alpha in major depressive disorder. Inter

  15. Sodium benzoate, a metabolite of cinnamon and a food additive, upregulates ciliary neurotrophic factor in astrocytes and oligodendrocytes

    Science.gov (United States)

    Modi, Khushbu K.; Jana, Malabendu; Mondal, Susanta; Pahan, Kalipada

    2015-01-01

    Ciliary neurotrophic factor (CNTF) is a promyelinating trophic factor that plays an important role in multiple sclerosis (MS). However, mechanisms by which CNTF expression could be increased in the brain are poorly understood. Recently we have discovered anti-inflammatory and immunomodulatory activities of sodium benzoate (NaB), a metabolite of cinnamon and a widely-used food additive. Here, we delineate that NaB is also capable of increasing the mRNA and protein expression of CNTF in primary mouse astrocytes and oligodendrocytes and primary human astrocytes. Accordingly, oral administration of NaB and cinnamon led to the upregulation of astroglial and oligodendroglial CNTF in vivo in mouse brain. Induction of experimental allergic encephalomyelitis (EAE), an animal model of MS, reduced the level of CNTF in the brain, which was restored by oral administration of cinnamon. While investigating underlying mechanisms, we observed that NaB induced the activation of protein kinase A (PKA) and H-89, an inhibitor of PKA, abrogated NaB-induced expression of CNTF. The activation of cAMP response element binding (CREB) protein by NaB, the recruitment of CREB and CREB-binding protein to the CNTF promoter by NaB and the abrogation of NaB-induced expression of CNTF in astrocytes by siRNA knockdown of CREB suggest that NaB increases the expression of CNTF via the activation of CREB. These results highlight a novel myelinogenic property of NaB and cinnamon, which may be of benefit for MS and other demyelinating disorders. PMID:26399250

  16. Expression of muscarinic binding sites in primary human brain tumors.

    Science.gov (United States)

    Gurwitz, D; Razon, N; Sokolovsky, M; Soreq, H

    1984-05-01

    The expression of muscarinic binding sites was examined in a collection of primary brain tumors of different cellular origins and various degrees of dedifferentiation, as compared to control specimens. Eleven gliogenous tumors were examined, all of which contained substantial amounts of muscarinic binding sites. Most of the other tumor types examined did not display detectable binding of [3H]N-methyl-4-piperidyl benzilate ([3H]4NMPB). Scatchard analysis indicated the existence of homogeneous antagonist sites in both normal forebrain and glioblastoma multiforme, with Kd values of 1.2 nM and 0.9 nM, respectively. The density of muscarinic binding sites varied between tumors from different patients, and also between specimens prelevated from different areas of the same tumor. This variability, as well as the average density of binding sites, appeared to be larger in highly malignant tumors than in less malignant ones. In contrast, the density of muscarinic receptors from control specimens was invariably high, but within the same order of magnitude. To test whether the muscarinic binding activity in the brain tumors is correlated to other cholinoceptive properties, cholinesterase activity was also examined. Individual data for density of [3H]4NMPB binding sites were then plotted against corresponding values of cholinesterase activity. The pattern of distribution of these values was clearly different in tumor specimens, when compared to that observed in samples derived from non-malignant brain. Our observations indicate that human brain cells of gliogenous origin are capable of expressing muscarinic binding sites, and that, if a correlation exists between muscarinic receptors and cholinesterase levels in gliogenous tumors, it differs from that of non-malignant brain tissue.

  17. Saturated long-chain fatty acids activate inflammatory signaling in astrocytes.

    Science.gov (United States)

    Gupta, Sunita; Knight, Alecia G; Gupta, Shruti; Keller, Jeffrey N; Bruce-Keller, Annadora J

    2012-03-01

    This study describes the effects of long-chain fatty acids on inflammatory signaling in cultured astrocytes. Data show that the saturated fatty acid palmitic acid, as well as lauric acid and stearic acid, trigger the release of TNFα and IL-6 from astrocytes. Unsaturated fatty acids were unable to induce cytokine release from cultured astrocytes. Furthermore, the effects of palmitic acid on cytokine release require Toll-like receptor 4 rather than CD36 or Toll-like receptor 2, and do not depend on palmitic acid metabolism to palmitoyl-CoA. Inhibitor studies revealed that pharmacologic inhibition of p38 or p42/44 MAPK pathways prevents the pro-inflammatory effects of palmitic acid, whereas JNK and PI3K inhibition does not affect cytokine release. Depletion of microglia from primary astrocyte cultures using the lysosomotropic agent l-leucine methyl ester revealed that the ability of palmitic acid to trigger cytokine release is not dependent on the presence of microglia. Finally, data show that the essential ω-3 fatty acid docosahexaenoic acid acts in a dose-dependent manner to prevent the actions of palmitic acid on inflammatory signaling in astrocytes. Collectively, these data demonstrate the ability of saturated fatty acids to induce astrocyte inflammation in vitro. These data thus raise the possibility that high levels of circulating saturated fatty acids could cause reactive gliosis and brain inflammation in vivo, and could potentially participate in the reported adverse neurologic consequences of obesity and metabolic syndrome.

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

    Science.gov (United States)

    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. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Deimination level and peptidyl arginine deiminase 2 expression are elevated in astrocytes with increased incubation temperature.

    Science.gov (United States)

    Enriquez-Algeciras, Mabel; Bhattacharya, Sanjoy K; Serra, Horacio M

    2015-09-01

    Astrocytes respond to environmental cues, including changes in temperatures. Increased deimination, observed in many progressive neurological diseases, is thought to be contributed by astrocytes. We determined the level of deimination and expression of peptidyl arginine deiminase 2 (PAD2) in isolated primary astrocytes in response to changes on either side (31°C and 41°C) of the optimal temperature (37°C). We investigated changes in the astrocytes by using a number of established markers and accounted for cell death with the CellTiter-Blue assay. We found increased expression of glial fibrillary acidic protein, ALDH1L1, and J1-31, resulting from increased incubation temperature and increased expression of TSP1, S100β, and AQP4, resulting from decreased incubation temperature vs. optimal temperature, suggesting activation of different biochemical pathways in astrocytes associated with different incubation temperatures. Mass spectrometric analyses support such trends. The PAD2 level was increased only as a result of increased incubation temperature with a commensurate increased level of deimination. Actin cytoskeleton and iso[4]LGE, a lipid peroxidase modification, also showed an increase with higher incubation temperature. Altogether, these results suggest that temperature, as an environmental cue, activates astrocytes in a different manner on either side of the optimal temperature and that increase in deimination is associated only with the higher temperature side of the spectrum.

  20. Manipulation of Human Primary Endothelial Cell and Osteoblast Coculture Ratios to Augment Vasculogenesis and Mineralization

    Science.gov (United States)

    2014-06-01

    has not been determined for human primary cells. Human umbilical vein ECs were cultured with normal human primary OBs in different EC/OB ratios...MATERIALS AND METHODS Cell Culture Human umbilical vein ECs (Invitrogen, Carlsbad, CA) were cultured in EC media (Lifeline, Walkersville, MD) and used after...into the membrane insert. At each time point, phase contrast microscopy (Nikon, Melville, NY) was used to image the OBs on each well at 10 (n 4

  1. Nitric Oxide in Astrocyte-Neuron Signaling

    Energy Technology Data Exchange (ETDEWEB)

    Li, Nianzhen [Iowa State Univ., Ames, IA (United States)

    2002-01-01

    Astrocytes, a subtype of glial cell, have recently been shown to exhibit Ca2+ elevations in response to neurotransmitters. A Ca2+ elevation can propagate to adjacent astrocytes as a Ca2+ wave, which allows an astrocyte to communicate with its neighbors. Additionally, glutamate can be released from astrocytes via a Ca2+-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 Ca2+ signaling by imaging NO in purified murine cortical astrocyte cultures. Physiological concentrations of a natural messenger, ATP, caused a Ca2+-dependent NO production. To test the roles of NO in astrocytic Ca2+ 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 Ca2+, possibly through store-operated Ca2+ channels. The NO-induced Ca2+ signaling is cGMP-independent since 8-Br-cGMP, an agonistic analog of cGMP, did not induce a detectable Ca2+ change. The consequence of this NO-induced Ca2+ influx was assessed by simultaneously monitoring of cytosolic and internal store Ca2+ using fluorescent Ca2+ 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 Ca2+ release, suggesting that NO modulates internal store refilling. Furthermore, locally photo-release of NO to a single astrocyte led to a Ca2+ elevation in the stimulated astrocyte and a subsequent Ca2+ wave to neighbors. Finally, the author tested the role of NO inglutamate-mediated astrocyte-neuron signaling by

  2. Nitric Oxide in Astrocyte-Neuron Signaling

    Energy Technology Data Exchange (ETDEWEB)

    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

  3. Cdh1 inhibits reactive astrocyte proliferation after oxygen-glucose deprivation and reperfusion.

    Science.gov (United States)

    Qiu, Jin; Zhang, Chuanhan; Lv, Youyou; Zhang, Yue; Zhu, Chang; Wang, Xueren; Yao, Wenlong

    2013-08-01

    Anaphase-promoting complex (APC) and its co-activator Cdh1 are required for cell cycle regulation in proliferating cells. Recent studies have defined diverse functions of APC-Cdh1 in nervous system development and injury. Our previous studies have demonstrated the activity of APC-Cdh1 is down-regulated in hippocampus after global cerebral ischemia. But the detailed mechanisms of APC-Cdh1 in ischemic nervous injury are unclear. It is known that astrocyte proliferation is an important pathophysiological process following cerebral ischemia. However, the role of APC-Cdh1 in reactive astrocyte proliferation is not determined yet. In the present study, we cultured primary cerebral astrocytes and set up in vitro oxygen-glucose deprivation and reperfusion model. Our results showed that the expression of Cdh1 was decreased while Skp2 (the downstream substrate of APC-Cdh1) was increased in astrocytes after 1h oxygen-glucose deprivation and reperfusion. The down-regulation of APC-Cdh1 was coupled with reactive astrocyte proliferation. By constructing Cdh1 expressing lentivirus system, we also found exogenous Cdh1 can down-regulate Skp2 and inhibit reactive astrocyte proliferation induced by oxygen-glucose deprivation and reperfusion. Moreover, Western blot showed that other downstream proteins of APC-Cdh1, PFK-1 and SnoN, were decreased in the inhibition of reactive astrocyte proliferation with Cdh1 expressing lentivirus treatment. These results suggest that Cdh1 plays an important role in the regulation of reactive astrocyte proliferation induced by oxygen-glucose deprivation and reperfusion.

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

    Science.gov (United States)

    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.

  5. Neuroinflammation leads to region-dependent alterations in astrocyte gap junction communication and hemichannel activity.

    Science.gov (United States)

    Karpuk, Nikolay; Burkovetskaya, Maria; Fritz, Teresa; Angle, Amanda; Kielian, Tammy

    2011-01-12

    Inflammation attenuates gap junction (GJ) communication in cultured astrocytes. Here we used a well-characterized model of experimental brain abscess as a tool to query effects of the CNS inflammatory milieu on astrocyte GJ communication and electrophysiological properties. Whole-cell patch-clamp recordings were performed on green fluorescent protein (GFP)-positive astrocytes in acute brain slices from glial fibrillary acidic protein-GFP mice at 3 or 7 d after Staphylococcus aureus infection in the striatum. Astrocyte GJ communication was significantly attenuated in regions immediately surrounding the abscess margins and progressively increased to levels typical of uninfected brain with increasing distance from the abscess proper. Conversely, astrocytes bordering the abscess demonstrated hemichannel activity as evident by enhanced ethidium bromide (EtBr) uptake that could be blocked by several pharmacological inhibitors, including the connexin 43 (Cx43) mimetic peptide Gap26, carbenoxolone, the pannexin1 (Panx1) mimetic peptide (10)Panx1, and probenecid. However, hemichannel opening was transient with astrocytic EtBr uptake observed near the abscess at day 3 but not day 7 after infection. The region-dependent pattern of hemichannel activity at day 3 directly correlated with increases in Cx43, Cx30, Panx1, and glutamate transporter expression (glial L-glutamate transporter and L-glutamate/L-aspartate transporter) along the abscess margins. Changes in astrocyte resting membrane potential and input conductance correlated with the observed changes in GJ communication and hemichannel activity. Collectively, these findings indicate that astrocyte coupling and electrical properties are most dramatically affected near the primary inflammatory site and reveal an opposing relationship between the open states of GJ channels versus hemichannels during acute infection. This relationship may extend to other CNS diseases typified with an inflammatory component.

  6. Primary Adult Human Retinal Pigment Epithelial Cell Cultures on Human Amniotic Membranes

    Directory of Open Access Journals (Sweden)

    Singhal Shweta

    2005-01-01

    Full Text Available Purpose: Retinal pigment epithelial (RPE cells grow well on surfaces that provide an extracellular matrix. Our aim was to establish primary adult human RPE cell cultures that retain their epithelial morphology in vitro using human amniotic membrane (hAM as substrate. Materials and Methods: Human cadaver eyeballs (16 were obtained from the eye bank after corneal trephination. RPE cells were harvested by a mechanical dissection of the inner choroid surface (10, group 1 or by b enzymatic digestion using 0.25% Trypsin/0.02% EDTA (6, group 2. The cells were explanted onto de-epithelialized hAM, nourished using DMEM/HAMS F-12 media and monitored for growth under the phase contrast microscope. Cell cultures were characterised by whole mount studies and paraffin sections. Growth data in the two groups were compared using the students′ ′t′ test. Results: Eleven samples (68.75% showed positive cultures with small, hexagonal cells arising from around the explant which formed a confluent and progressively pigmented monolayer. Whole mounts showed closely placed polygonal cells with heavily pigmented cytoplasm and indistinct nuclei. The histologic sections showed monolayers of cuboidal epithelium with variable pigmentation within the cytoplasm. Growth was seen by day 6-23 (average 11.5 days in the mechanical group, significantly earlier ( P Conclusions: Primary adult human RPE cell cultures retain epithelial morphology in vitro when cultured on human amniotic membranes . Mechanical dissection of the inner choroid surface appears to be an effective method of isolating RPE cells and yields earlier growth in cultures as compared to isolation by enzymatic digestion

  7. Decreased Motor Neuron Support by SMA Astrocytes due to Diminished MCP1 Secretion.

    Science.gov (United States)

    Martin, Jasmin E; Nguyen, TrangKimberly T; Grunseich, Christopher; Nofziger, Jonathan H; Lee, Philip R; Fields, Douglas; Fischbeck, Kenneth H; Foran, Emily

    2017-05-24

    Spinal muscular atrophy (SMA) is an autosomal-recessive disorder characterized by severe, often fatal muscle weakness due to loss of motor neurons. SMA patients have deletions and other mutations of the survival of motor neuron 1 (SMN1) gene, resulting in decreased SMN protein. Astrocytes are the primary support cells of the CNS and are responsible for glutamate clearance, metabolic support, response to injury, and regulation of signal transmission. Astrocytes have been implicated in SMA as in in other neurodegenerative disorders. Astrocyte-specific rescue of SMN protein levels has been shown to mitigate disease manifestations in mice. However, the mechanism by which SMN deficiency in astrocytes may contribute to SMA is unclear and what aspect of astrocyte activity is lacking is unknown. Therefore, it is worthwhile to identify defects in SMN-deficient astrocytes that compromise normal function. We show here that SMA astrocyte cultures derived from mouse spinal cord of both sexes are deficient in supporting both WT and SMN-deficient motor neurons derived from male, female, and mixed-sex sources and that this deficiency may be mitigated with secreted factors. In particular, SMN-deficient astrocytes have decreased levels of monocyte chemoactive protein 1 (MCP1) secretion compared with controls and MCP1 restoration stimulates outgrowth of neurites from cultured motor neurons. Correction of MCP1 deficiency may thus be a new therapeutic approach to SMA.SIGNIFICANCE STATEMENT Spinal muscular atrophy (SMA) is caused by the loss of motor neurons, but astrocyte dysfunction also contributes to the disease in mouse models. Monocyte chemoactive protein 1 (MCP1) has been shown to be neuroprotective and is released by astrocytes. Here, we report that MCP1 levels are decreased in SMA mice and that replacement of deficient MCP1 increases differentiation and neurite length of WT and SMN-deficient motor-neuron-like cells in cell culture. This study reveals a novel aspect of astrocyte

  8. Astrocyte Elevated Gene-1 (AEG-1) Contributes to Non-thyroidal Illness Syndrome (NTIS) Associated with Hepatocellular Carcinoma (HCC)*

    OpenAIRE

    Srivastava, Jyoti; Robertson, Chadia L.; Gredler, Rachel; Siddiq, Ayesha; Rajasekaran, Devaraja; Akiel, Maaged A; Emdad, Luni; Mas, Valeria; Mukhopadhyay, Nitai D.; FISHER, PAUL B.; Sarkar, Devanand

    2015-01-01

    Background: Astrocyte elevated gene-1 (AEG-1) inhibits retinoid X receptor (RXR) function and is overexpressed in human hepatocellular carcinoma (HCC), which is associated with non-thyroidal illness syndrome (NTIS).

  9. Distinct Contributions of Astrocytes and Pericytes to Neuroinflammation Identified in a 3D Human Blood-Brain Barrier on a Chip

    NARCIS (Netherlands)

    Herland, Anna; van der Meer, Andries Dirk; Fitzgerald, Edward A.; Park, Tae-Eun; Sleeboom, Jelle J.F.; Ingber, Donald E.

    2016-01-01

    Neurovascular inflammation is a major contributor to many neurological disorders, but modeling these processes in vitro has proven to be difficult. Here, we microengineered a three-dimensional (3D) model of the human blood-brain barrier (BBB) within a microfluidic chip by creating a cylindrical

  10. Attachment of human primary osteoblast cells to modified polyethylene surfaces.

    Science.gov (United States)

    Poulsson, Alexandra H C; Mitchell, Stephen A; Davidson, Marcus R; Johnstone, Alan J; Emmison, Neil; Bradley, Robert H

    2009-04-09

    Ultra-high-molecular-weight polyethylene (UHMWPE) has a long history of use in medical devices, primarily for articulating surfaces due to its inherent low surface energy which limits tissue integration. To widen the applications of UHMWPE, the surface energy can be increased. The increase in surface energy would improve the adsorption of proteins and attachment of cells to allow tissue integration, thereby allowing UHMWPE to potentially be used for a wider range of implants. The attachment and function of human primary osteoblast-like (HOB) cells to surfaces of UHMWPE with various levels of incorporated surface oxygen have been investigated. The surface modification of the UHMWPE was produced by exposure to a UV/ozone treatment. The resulting surface chemistry was studied using X-ray photoelectron spectroscopy (XPS), and the topography and surface structure were probed by atomic force microscopy (AFM) and scanning electron microscopy (SEM), which showed an increase in surface oxygen from 11 to 26 atom % with no significant change to the surface topography. The absolute root mean square roughness of both untreated and UV/ozone-treated surfaces was within 350-450 nm, and the water contact angles decreased with increasing oxygen incorporation, i.e., showing an increase in surface hydrophilicity. Cell attachment and functionality were assessed over a 21 day period for each cell-surface combination studied; these were performed using SEM and the alamarBlue assay to study cell attachment and proliferation and energy-dispersive X-ray (EDX) analysis to confirm extracellular mineral deposits, and total protein assay to examine the intra- and extracellular protein expressed by the cells. HOB cells cultured for 21 days on the modified UHMWPE surfaces with 19 and 26 atom % oxygen incorporated showed significantly higher cell densities compared to cells cultured on tissue culture polystyrene (TCPS) from day 3 onward. This indicated that the cells attached and proliferated more

  11. Genome-wide binding and transcriptome analysis of human farnesoid X receptor in primary human hepatocytes.

    Directory of Open Access Journals (Sweden)

    Le Zhan

    Full Text Available Farnesoid X receptor (FXR, NR1H4 is a ligand-activated transcription factor, belonging to the nuclear receptor superfamily. FXR is highly expressed in the liver and is essential in regulating bile acid homeostasis. FXR deficiency is implicated in numerous liver diseases and mice with modulation of FXR have been used as animal models to study liver physiology and pathology. We have reported genome-wide binding of FXR in mice by chromatin immunoprecipitation - deep sequencing (ChIP-seq, with results indicating that FXR may be involved in regulating diverse pathways in liver. However, limited information exists for the functions of human FXR and the suitability of using murine models to study human FXR functions.In the current study, we performed ChIP-seq in primary human hepatocytes (PHHs treated with a synthetic FXR agonist, GW4064 or DMSO control. In parallel, RNA deep sequencing (RNA-seq and RNA microarray were performed for GW4064 or control treated PHHs and wild type mouse livers, respectively.ChIP-seq showed similar profiles of genome-wide FXR binding in humans and mice in terms of motif analysis and pathway prediction. However, RNA-seq and microarray showed more different transcriptome profiles between PHHs and mouse livers upon GW4064 treatment.In summary, we have established genome-wide human FXR binding and transcriptome profiles. These results will aid in determining the human FXR functions, as well as judging to what level the mouse models could be used to study human FXR functions.

  12. Profilin isoforms modulate astrocytic morphology and the motility of astrocytic processes.

    Directory of Open Access Journals (Sweden)

    Stefanie K Schweinhuber

    Full Text Available The morphology of astrocytic processes determines their close structural association with synapses referred to as the 'tripartite synapse'. Concerted morphological plasticity processes at tripartite synapses are supposed to shape neuronal communication. Morphological changes in astrocytes as well as the motility of astrocytic processes require remodeling of the actin cytoskeleton. Among the regulators of fast timescale actin-based motility, the actin binding protein profilin 1 has recently been shown to control the activity-dependent outgrowth of astrocytic processes. Here, we demonstrate that cultured murine astrocytes in addition to the ubiquitous profilin 1 also express the neuronal isoform profilin 2a. To analyze the cellular function of both profilins in astrocytes, we took advantage of a shRNA mediated isoform-specific downregulation. Interestingly, consistent with earlier results in neurons, we found redundant as well as isoform-specific functions of both profilins in modulating cellular physiology. The knockdown of either profilin 1 or profilin 2a led to a significant decrease in cell spreading of astrocytes. In contrast, solely the knockdown of profilin 2a resulted in a significantly reduced morphological complexity of astrocytes in both dissociated and slice culture astrocytes. Moreover, both isoforms proved to be crucial for forskolin-induced astrocytic stellation. Furthermore, forskolin treatment resulted in isoform-specific changes in the phosphorylation level of profilin 1 and profilin 2a, leading to a PKA-dependent phosphorylation of profilin 2a. In addition, transwell assays revealed an involvement of both isoforms in the motility of astrocytic processes, while FRAP analysis displayed an isoform-specific role of profilin 1 in the regulation of actin dynamics in peripheral astrocytic processes. Taken together, we suggest profilin isoforms to be important modulators of astrocytic morphology and motility with overlapping as well as

  13. Major histocompatibility complex class I molecules protect motor neurons from astrocyte-induced toxicity in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Song, SungWon; Miranda, Carlos J; Braun, Lyndsey; Meyer, Kathrin; Frakes, Ashley E; Ferraiuolo, Laura; Likhite, Shibi; Bevan, Adam K; Foust, Kevin D; McConnell, Michael J; Walker, Christopher M; Kaspar, Brian K

    2016-04-01

    Astrocytes isolated from individuals with amyotrophic lateral sclerosis (ALS) are toxic to motor neurons (MNs) and play a non-cell autonomous role in disease pathogenesis. The mechanisms underlying the susceptibility of MNs to cell death remain unclear. Here we report that astrocytes derived from either mice bearing mutations in genes associated with ALS or human subjects with ALS reduce the expression of major histocompatibility complex class I (MHCI) molecules on MNs; reduced MHCI expression makes these MNs susceptible to astrocyte-induced cell death. Increasing MHCI expression on MNs increases survival and motor performance in a mouse model of ALS and protects MNs against astrocyte toxicity. Overexpression of a single MHCI molecule, HLA-F, protects human MNs from ALS astrocyte-mediated toxicity, whereas knockdown of its receptor, the killer cell immunoglobulin-like receptor KIR3DL2, on human astrocytes results in enhanced MN death. Thus, our data indicate that, in ALS, loss of MHCI expression on MNs renders them more vulnerable to astrocyte-mediated toxicity.

  14. Validation of an original incubator set-up for the exposure of human astrocyte cells to X-band microwaves in a GTEM-chamber.

    Science.gov (United States)

    Pérez-Bruzón, R N; Del Moral, A; Pérez-Castejón, C; Llorente, M; Vera, A; Azanza, M J

    2011-09-01

    A current concern about the biological effects of electromagnetic fields (EMF) is increasing with the wide spread use of X-band microwaves (MW, 8-10 GHz range). Gigahertz transverse electromagnetic (GTEM) field flat transmission lines are currently being used for experimental exposure of biological samples to high frequency EMF. Experiments carried out on human cells in culture require optimal growing temperature conditions, i.e. 37 °C, 5% CO2 in a humidified atmosphere. The aim of our work has been: i) to built up an original incubator set-up, the so called GTEM-incubator, for exposure of human cells in culture to MW inside a GTEM-chamber, under optimal growing physical conditions; ii) to make the validation of the GTEM-incubator by growing cell samples inside the non-energized GTEM-chamber (test sample) comparing the results with the ones obtained from cell samples grown inside a standard incubator (control samples). The features for comparison were: cell morphology, expression and distribution of cytoskeleton proteins, genotoxicity, viability and cell cycle progression. Any variation in any of the studied parameters would allow for detecting any possible failure or misconception in our GTEM-incubator working test. The results obtained in control and test incubators showed non-significant differences in the development of both cell populations for any of the studied parameters. Thereby our GTEM-incubator is considered valid for our purposes of human cell exposures to X-band MW.

  15. Genes involved in the astrocyte-neuron lactate shuttle (ANLS) are specifcally regulated in cortical astrocytes following sleep deprivation in mice

    KAUST Repository

    Petit, Jean Marie

    2013-10-01

    Study Objectives: There is growing evidence indicating that in order to meet the neuronal energy demands, astrocytes provide lactate as an energy substrate for neurons through a mechanism called "astrocyte-neuron lactate shuttle" (ANLS). Since neuronal activity changes dramatically during vigilance states, we hypothesized that the ANLS may be regulated during the sleep-wake cycle. To test this hypothesis we investigated the expression of genes associated with the ANLS specifcally in astrocytes following sleep deprivation. Astrocytes were purifed by fuorescence-activated cell sorting from transgenic mice expressing the green fuorescent protein (GFP) under the control of the human astrocytic GFAP-promoter. Design: 6-hour instrumental sleep deprivation (TSD). Setting: Animal sleep research laboratory. Participants: Young (P23-P27) FVB/N-Tg (GFAP-GFP) 14Mes/J (Tg) mice of both sexes and 7-8 week male Tg and FVB/Nj mice. Interventions: Basal sleep recordings and sleep deprivation achieved using a modifed cage where animals were gently forced to move. Measurements and Results: Since Tg and FVB/Nj mice displayed a similar sleep-wake pattern, we performed a TSD in young Tg mice. Total RNA was extracted from the GFP-positive and GFP-negative cells sorted from cerebral cortex. Quantitative RT-PCR analysis showed that levels of Glut1, a-2-Na/K pump, Glt1, and Ldha mRNAs were signifcantly increased following TSD in GFP-positive cells. In GFP-negative cells, a tendency to increase, although not signifcant, was observed for Ldha, Mct2, and α-3-Na/K pump mRNAs. Conclusions: This study shows that TSD induces the expression of genes associated with ANLS specifcally in astrocytes, underlying the important role of astrocytes in the maintenance of the neuro-metabolic coupling across the sleep-wake cycle.

  16. Genes involved in the astrocyte-neuron lactate shuttle (ANLS) are specifically regulated in cortical astrocytes following sleep deprivation in mice.

    Science.gov (United States)

    Petit, Jean-Marie; Gyger, Joël; Burlet-Godinot, Sophie; Fiumelli, Hubert; Martin, Jean-Luc; Magistretti, Pierre J

    2013-10-01

    There is growing evidence indicating that in order to meet the neuronal energy demands, astrocytes provide lactate as an energy substrate for neurons through a mechanism called "astrocyte-neuron lactate shuttle" (ANLS). Since neuronal activity changes dramatically during vigilance states, we hypothesized that the ANLS may be regulated during the sleep-wake cycle. To test this hypothesis we investigated the expression of genes associated with the ANLS specifically in astrocytes following sleep deprivation. Astrocytes were purified by fluorescence-activated cell sorting from transgenic mice expressing the green fluorescent protein (GFP) under the control of the human astrocytic GFAP-promoter. 6-hour instrumental sleep deprivation (TSD). Animal sleep research laboratory. Young (P23-P27) FVB/N-Tg (GFAP-GFP) 14Mes/J (Tg) mice of both sexes and 7-8 week male Tg and FVB/Nj mice. Basal sleep recordings and sleep deprivation achieved using a modified cage where animals were gently forced to move. Since Tg and FVB/Nj mice displayed a similar sleep-wake pattern, we performed a TSD in young Tg mice. Total RNA was extracted from the GFP-positive and GFP-negative cells sorted from cerebral cortex. Quantitative RT-PCR analysis showed that levels of Glut1, α-2-Na/K pump, Glt1, and Ldha mRNAs were significantly increased following TSD in GFP-positive cells. In GFP-negative cells, a tendency to increase, although not significant, was observed for Ldha, Mct2, and α-3-Na/K pump mRNAs. This study shows that TSD induces the expression of genes associated with ANLS specifically in astrocytes, underlying the important role of astrocytes in the maintenance of the neuro-metabolic coupling across the sleep-wake cycle.

  17. Inhibition of a SNARE-sensitive pathway in astrocytes attenuates damage following stroke.

    Science.gov (United States)

    Hines, Dustin J; Haydon, Philip G

    2013-03-06

    A strong body of research has defined the role of excitotoxic glutamate in animal models of brain ischemia and stroke; however, clinical trials of glutamate receptor antagonists have demonstrated their limited capacity to prevent brain damage following ischemia. We propose that astrocyte-neuron signaling represents an important modulatory target that may be useful in mediating damage following stroke. To assess the impact of astrocyte signaling on damage following stroke, we have used the astrocyte-specific dominant-negative SNARE mouse model (dnSNARE). Recent findings have shown that the astrocytic SNARE signaling pathway can affect neuronal excitability by regulating the surface expression of NMDA receptors. Using focal photothrombosis via the Rose Bengal method, as well as excitotoxic NMDA lesions, we show that dnSNARE animals exhibited a sparing of damaged tissue quantified using Nissl and NeuN staining. At the same time point, animals were also tested in behavioral tasks that probe the functional integrity of stroke- or lesion-damaged motor and somatosensory areas. We found that dnSNARE mice performed significantly better than littermate controls on rung walk and adhesive dot removal tasks following lesion. Together, our results demonstrate the important role of astrocytic signaling under ischemic conditions. Drugs targeting astrocyte signaling have a potential benefit for the outcome of stroke in human patients by limiting the spread of damage.

  18. Transplantation of stem cell-derived astrocytes for thetreatment of amyotrophic lateral sclerosis and spinal cordinjury

    Institute of Scientific and Technical Information of China (English)

    Charles Nicaise; Dinko Mitrecic; Aditi Falnikar; Angelo C Lepore

    2015-01-01

    Neglected for years, astrocytes are now recognized tofulfill and support many, if not all, homeostatic functionsof the healthy central nervous system (CNS). Duringneurodegenerative diseases such as amyotrophiclateral sclerosis (ALS) and spinal cord injury (SCI),astrocytes in the vicinity of degenerating areasundergo both morphological and functional changesthat might compromise their intrinsic properties.Evidence from human and animal studies show thatdeficient astrocyte functions or loss-of-astrocytes largelycontribute to increased susceptibility to cell death forneurons, oligodendrocytes and axons during ALS andSCI disease progression. Despite exciting advances inexperimental CNS repair, most of current approachesthat are translated into clinical trials focus on thereplacement or support of spinal neurons throughstem cell transplantation, while none focus on thespecific replacement of astroglial populations. Knowingthe important functions carried out by astrocytesin the CNS, astrocyte replacement-based therapiesmight be a promising approach to alleviate overallastrocyte dysfunction, deliver neurotrophic support todegenerating spinal tissue and stimulate endogenousCNS repair abilities. Enclosed in this review, we gatheredexperimental evidence that argue in favor of astrocytetransplantation during ALS and SCI. Based on theirintrinsic properties and according to the cell typetransplanted, astrocyte precursors or stem cell-derivedastrocytes promote axonal growth, support mechanismsand cells involved in myelination, are able to modulatethe host immune response, deliver neurotrophic factorsand provide protective molecules against oxidative orexcitotoxic insults, amongst many possible benefits.Embryonic or adult stem cells can even be geneticallyengineered in order to deliver missing gene productsand therefore maximize the chance of neuroprotectionand functional recovery. However, before broad clinicaltranslation, further preclinical data on safety

  19. Apoptosis of human primary gastric carcinoma cells induced by genistein

    Institute of Scientific and Technical Information of China (English)

    Hai-Bo Zhou; Juan-Juan Chen; Wen-Xia Wang; Jian-Ting Cai; Qin Du

    2004-01-01

    AIM: To investigate the apoptosis in primary gastric cancer cells induced by genistein, and the relationship between this apoptosis and expression of bcl-2 and bax.METHODS: MTT assay was used to determine the cell growth inhibitory rate in vitro. Transmission electron microscope and TUNEL staining were used to quantitatively and qualitatively detect the apoptosis of primary gastric cancer cells before and after genistein treatment. Immunohistochemical staining and RT-PCR were used to detect the expression of apoptosisassociated genes bcl-2 and bax.RESULTS: Genistein inhibited the growth of primary gastric cancer cells in dose-and time-dependent manner. Genistein induced primary gastric cancer cells to undergo apoptosis with typically apoptotic characteristics. TUNEL assay showed that after the treatment of primary gastric cancer cells with genistein for 24 to 96 h, the apoptotic rates of primary gastric cancer cells increased time-dependently. Immunohistochemical staining showed that after the treatment of primary gastric cancer cells with genistein for 24 to 96 h, the positivity rates of Bcl-2 proteins were apparently reduced with time and the positivity rates of Bax proteins were apparently increased with time. After exposed to genistein at 20 μmol/L for 24,48, 72 and 96 respectively, the density of bcl-2 mRNA decreased progressively and the density of bax mRNA increased progressively with elongation of time.CONCLUSION: Genistein is able to induce the apoptosis in primary gastric cancer cells. This apoptosis may be mediated by down-regulating the apoptosis- associated bcl-2 gene and up-regulating the expression of apoptosis-associated bax gene.

  20. Trajectories in human domination of ecosystems : Human appropriation of net primary production in the Philippines during the 20th century

    NARCIS (Netherlands)

    Kastner, Thomas

    2009-01-01

    The "human appropriation of net primary production" (HANPP) has been suggested as a comprehensive indicator to measure impacts of human land use on ecosystems. It accounts for (a) human-induced changes in biological productivity and (b) biomass harvest. This study presents an analysis of aboveground

  1. GABA(A) Increases Calcium in Subventricular Zone Astrocyte-Like Cells Through L- and T-Type Voltage-Gated Calcium Channels

    DEFF Research Database (Denmark)

    Young, Stephanie Z; Platel, Jean-Claude; Nielsen, Jakob V;

    2010-01-01

    intracellular Ca(2+) dynamics in SVZ astrocytes. To monitor Ca(2+) activity selectively in astrocyte-like cells, we used two lines of transgenic mice expressing either GFP fused to a Gq-coupled receptor or DsRed under the human glial fibrillary acidic protein (hGFAP) promoter. GABA(A) receptor activation......-like cells to 75%, suggesting that the majority of SVZ astrocytes express functional VGCCs. SVZ astrocytes also displayed spontaneous Ca(2+) activity, the frequency of which was regulated by tonic GABA(A) receptor activation. These data support a role for ambient GABA in tonically regulating intracellular Ca...

  2. Artificial astrocytes improve neural network performance.

    Science.gov (United States)

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

    2011-04-19

    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.

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

    Science.gov (United States)

    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.

  4. Fatty acid oxidation and ketogenesis in astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Auestad, N.

    1988-01-01

    Astrocytes were derived from cortex of two-day-old rat brain and grown in primary culture to confluence. The metabolism of the fatty acids, octanoate and palmitate, to CO{sub 2} in oxidative respiration and to the formation of ketone bodies was examined by radiolabeled tracer methodology. The net production of acetoacetate was also determined by measurement of its mass. The enzymes in the ketogenic pathway were examined by measuring enzymic activity and/or by immunoblot analyses. Labeled CO{sub 2} and labeled ketone bodies were produced from the oxidation of fatty acids labeled at carboxy- and {omega}-terminal carbons, indicating that fatty acids were oxidized by {beta}-oxidation. The results from the radiolabeled tracer studies also indicated that a substantial proportion of the {omega}-terminal 4-carbon unit of the fatty acids bypassed the {beta}-ketothiolase step of the {beta}-oxidation pathway. The ({sup 14}C)acetoacetate formed from the (1-{sup 14}C)labeled fatty acids, obligated to pass through the acetyl-CoA pool, contained 50% of the label at carbon 3 and 50% at carbon 1. In contrast, the ({sup 14}C)acetoacetate formed from the ({omega}-1)labeled fatty acids contained 90% of the label at carbon 3 and 10% at carbon 1.

  5. Astrocytes Produce IL-19 in Response to Bacterial Challenge and are Sensitive to the Immunosuppressive Effects of this IL-10 Family Member

    Science.gov (United States)

    Cooley, Ian D.; Chauhan, Vinita S.; Donneyz, Miguel A.; Marriott, Ian

    2014-01-01

    There is growing appreciation that resident glial cells can initiate and/or regulate inflammation following trauma or infection in the central nervous system (CNS). We have previously demonstrated the ability of microglia and astrocytes to respond to bacterial pathogens or their products by rapid production of inflammatory mediators, followed by the production of the immunosuppressive cytokine interleukin (IL)210. IL-19, another member of the IL-10 family of cytokines, has been studied in the context of a number of inflammatory conditions in the periphery and is known to modulate immune cell activity. In the present study, we demonstrate the constitutive and/or inducible expression of IL-19 and its cognate receptor subunits, IL-19Rα and IL-19Rβ (also known as IL-20R1 and IL-20R2, and IL-20RA and IL-20RB), in mouse brain tissue, and by primary murine and human astrocytes. We also provide evidence for the presence of a novel truncated IL-19Rα transcript variant in mouse brain tissue, but not glial cells, that shows reduced expression following bacterial infection. Importantly, IL-19R functionality in GLIA is indicated by the ability of IL-19 to regulate signaling component expression in these cells. Furthermore, while IL-19 itself had no effect on glial cytokine production, IL-19 treatment of bacterially infected or Toll-like receptor ligand stimulated astrocytes significantly attenuated pro-inflammatory cytokine production. The bacterially induced production of IL-19 by these resident CNS cells, the constitutive expression of its cognate receptor subunits, and the immunomodulatory effects of this cytokine, suggest a novel mechanism by which astrocytes can regulate CNS inflammation. PMID:24677051

  6. Efficient replication of the novel human betacoronavirus EMC on primary human epithelium highlights its zoonotic potential.

    Science.gov (United States)

    Kindler, Eveline; Jónsdóttir, Hulda R; Muth, Doreen; Hamming, Ole J; Hartmann, Rune; Rodriguez, Regulo; Geffers, Robert; Fouchier, Ron A M; Drosten, Christian; Müller, Marcel A; Dijkman, Ronald; Thiel, Volker

    2013-02-19

    The recent emergence of a novel human coronavirus (HCoV-EMC) in the Middle East raised considerable concerns, as it is associated with severe acute pneumonia, renal failure, and fatal outcome and thus resembles the clinical presentation of severe acute respiratory syndrome (SARS) observed in 2002 and 2003. Like SARS-CoV, HCoV-EMC is of zoonotic origin and closely related to bat coronaviruses. The human airway epithelium (HAE) represents the entry point and primary target tissue for respiratory viruses and is highly relevant for assessing the zoonotic potential of emerging respiratory viruses, such as HCoV-EMC. Here, we show that pseudostratified HAE cultures derived from different donors are highly permissive to HCoV-EMC infection, and by using reverse transcription (RT)-PCR and RNAseq data, we experimentally determined the identity of seven HCoV-EMC subgenomic mRNAs. Although the HAE cells were readily responsive to type I and type III interferon (IFN), we observed neither a pronounced inflammatory cytokine nor any detectable IFN responses following HCoV-EMC, SARS-CoV, or HCoV-229E infection, suggesting that innate immune evasion mechanisms and putative IFN antagonists of HCoV-EMC are operational in the new host. Importantly, however, we demonstrate that both type I and type III IFN can efficiently reduce HCoV-EMC replication in HAE cultures, providing a possible treatment option in cases of suspected HCoV-EMC infection. IMPORTANCE A novel human coronavirus, HCoV-EMC, has recently been described to be associated with severe respiratory tract infection and fatalities, similar to severe acute respiratory syndrome (SARS) observed during the 2002-2003 epidemic. Closely related coronaviruses replicate in bats, suggesting that, like SARS-CoV, HCoV-EMC is of zoonotic origin. Since the animal reservoir and circumstances of zoonotic transmission are yet elusive, it is critically important to assess potential species barriers of HCoV-EMC infection. An important first

  7. Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery

    DEFF Research Database (Denmark)

    Kiprilov, Enko N; Awan, Aashir; Desprat, Romain

    2008-01-01

    Human embryonic stem cells (hESCs) are potential therapeutic tools and models of human development. With a growing interest in primary cilia in signal transduction pathways that are crucial for embryological development and tissue differentiation and interest in mechanisms regulating human h...

  8. Serodiagnosis of primary infections with human parvovirus 4, Finland.

    Science.gov (United States)

    Lahtinen, Anne; Kivelä, Pia; Hedman, Lea; Kumar, Arun; Kantele, Anu; Lappalainen, Maija; Liitsola, Kirsi; Ristola, Matti; Delwart, Eric; Sharp, Colin; Simmonds, Peter; Söderlund-Venermo, Maria; Hedman, Klaus

    2011-01-01

    To determine the prevalence of parvovirus 4 infection and its clinical and sociodemographic correlations in Finland, we used virus-like particle-based serodiagnostic procedures (immunoglobulin [Ig] G, IgM, and IgG avidity) and PCR. We found 2 persons with parvovirus 4 primary infection who had mild or asymptomatic clinical features among hepatitis C virus-infected injection drug users.

  9. Primary cell culture of human adenocarcinomas--practical considerations.

    Science.gov (United States)

    Lerescu, Lucian; Tucureanu, Cătălin; Caraş, Iuliana; Neagu, Stefan; Melinceanu, Laura; Sălăgeanu, Aurora

    2008-01-01

    Cell culture is one of the major tools for oncology research, being an excellent system in which to study the biochemistry and molecular biology associated with individual cancer types and to understand cancer cell physiology. Progress in understanding the biology of any type of carcinoma has been impeded by the inability to culture adequately malignant cells from most epithelial tissues. The ultimate in vitro tumor model would completely reflect the in vivo tumor microenvironment in function and mechanism. Unfortunately, such a model does not currently exist. Homogeneous cell lines that can be continuously propagated on plastic surfaces have been extensively used as a surrogate for tumor environment; however they are very different from the in vivo tumor cells. Model systems involving primary culture represent the situation most closely related to the original tissue although they have a number of disadvantages over cell lines, such as the limited ability to repeat studies with a well characterized culture system that can be used in multiple laboratories. The primary culture may contain many types of stromal and infiltrating cell types potentially complicating the interpretation of data. Yet, their properties better reflect the cellular interactions present in intact tissue. The present article reviews the critical steps in obtaining, routine maintenance and cryopreservation of primary tumor cell cultures, based on information from literature and personal experience on the subject. The article also includes an updated protocol for primary tumor cell isolation and culture.

  10. Astrocytic LRP1 Mediates Brain Aβ Clearance and Impacts Amyloid Deposition.

    Science.gov (United States)

    Liu, Chia-Chen; Hu, Jin; Zhao, Na; Wang, Jian; Na, Wang; Cirrito, John R; Kanekiyo, Takahisa; Holtzman, David M; Bu, Guojun

    2017-03-08

    Accumulation and deposition of amyloid-β (Aβ) in the brain represents an early and perhaps necessary step in the pathogenesis of Alzheimer's disease (AD). Aβ accumulation leads to the formation of Aβ aggregates which may directly and indirectly lead to eventual neurodegeneration. While Aβ production is accelerated in many familial forms of early-onset AD, increasing evidence indicates that impaired clearance of Aβ is more evident in late-onset AD. To uncover the mechanisms underlying impaired Aβ clearance in AD, we examined the role of low-density lipoprotein receptor-related protein 1 (LRP1) in astrocytes. Although LRP1 has been shown to play critical roles in brain Aβ metabolism in neurons and vascular mural cells, its role in astrocytes, the most abundant cell type in the brain responsible for maintaining neuronal homeostasis, remains unclear. Here, we show that astrocytic LRP1 plays a critical role in brain Aβ clearance. LRP1 knockdown in primary astrocytes resulted in decreased cellular Aβ uptake and degradation. In addition, silencing of LRP1 in astrocytes led to down-regulation of several major Aβ-degrading enzymes, including matrix metalloproteases MMP2, MMP9 and insulin-degrading enzyme (IDE). More important, conditional knockout of the Lrp1 gene in astrocytes in the background of APP/PS1 mice impaired brain Aβ clearance, exacerbated Aβ accumulation and accelerated amyloid plaque deposition without affecting its production. Together, our results demonstrate that astrocytic LRP1 plays an important role in Aβ metabolism and that restoring LRP1 expression and function in the brain could be an effective strategy to facilitate Aβ clearance and counter amyloid pathology in AD.SIGNIFICANCE STATEMENTAstrocytes represent a major cell type regulating brain homeostasis; however, their roles in brain clearance of amyloid-β (Aβ) and underlying mechanism are not clear. In this study, we used both cellular models and conditional knockout mouse models to

  11. Astrocytes generate Na+-mediated metabolic waves.

    Science.gov (United States)

    Bernardinelli, Yann; Magistretti, Pierre J; Chatton, Jean-Yves

    2004-10-12

    Glutamate-evoked Na+ increase in astrocytes has been identified as a signal coupling synaptic activity to glucose consumption. Astrocytes participate in multicellular signaling by transmitting intercellular Ca2+ waves. Here we show that intercellular Na+ waves are also evoked by activation of single cultured cortical mouse astrocytes in parallel with Ca2+ waves; however, there are spatial and temporal differences. Indeed, maneuvers that inhibit Ca2+ waves also inhibit Na+ waves; however, inhibition of the Na+/glutamate cotransporters or enzymatic degradation of extracellular glutamate selectively inhibit the Na+ wave. Thus, glutamate released by a Ca2+ wave-dependent mechanism is taken up by the Na+/glutamate cotransporters, resulting in a regenerative propagation of cytosolic Na+ increases. The Na+ wave gives rise to a spatially correlated increase in glucose uptake, which is prevented by glutamate transporter inhibition. Therefore, astrocytes appear to function as a network for concerted neurometabolic coupling through the generation of intercellular Na+ and metabolic waves.

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

    Science.gov (United States)

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

    2014-11-07

    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.

  13. Age, environment, object recognition and morphological diversity of GFAP-immunolabeled astrocytes.

    Science.gov (United States)

    Diniz, Daniel Guerreiro; de Oliveira, Marcus Augusto; de Lima, Camila Mendes; Fôro, César Augusto Raiol; Sosthenes, Marcia Consentino Kronka; Bento-Torres, João; da Costa Vasconcelos, Pedro Fernando; Anthony, Daniel Clive; Diniz, Cristovam Wanderley Picanço

    2016-10-10

    Few studies have explored the glial response to a standard environment and how the response may be associated with age-related cognitive decline in learning and memory. Here we investigated aging and environmental influences on hippocampal-dependent tasks and on the morphology of an unbiased selected population of astrocytes from the molecular layer of dentate gyrus, which is the main target of perforant pathway. Six and twenty-month-old female, albino Swiss mice were housed, from weaning, in a standard or enriched environment, including running wheels for exercise and tested for object recognition and contextual memories. Young adult and aged subjects, independent of environment, were able to distinguish familiar from novel objects. All experimental groups, except aged mice from standard environment, distinguish stationary from displaced objects. Young adult but not aged mice, independent of environment, were able to distinguish older from recent objects. Only young mice from an enriched environment were able to distinguish novel from familiar contexts. Unbiased selected astrocytes from the molecular layer of the dentate gyrus were reconstructed in three-dimensions and classified using hierarchical cluster analysis of bimodal or multimodal morphological features. We found two morphological phenotypes of astrocytes and we designated type I the astrocytes that exhibited significantly higher values of morphological complexity as compared with type II. Complexity = [Sum of the terminal orders + Number of terminals] × [Total branch length/Number of primary branches]. On average, type I morphological complexity seems to be much more sensitive to age and environmental influences than that of type II. Indeed, aging and environmental impoverishment interact and reduce the morphological complexity of type I astrocytes at a point that they could not be distinguished anymore from type II. We suggest these two types of astrocytes may have different physiological roles

  14. Selective stimulation of dendrite outgrowth from identified corticospinal neurons by homotopic astrocytes

    NARCIS (Netherlands)

    Gispen, W.H.; Dijkstra, S.; Bar, P.R.; Joosten, E.A.

    1999-01-01

    Corticospinal neurons were identified in primary cultures of cortical neurons established from rats that had been injected with a fluorescent tracer to retrogradely label the corticospinal tract. We measured neurite outgrowth from corticospinal neurons after they had been co-cultured with astrocytes

  15. Gamma oscillations in human primary somatosensory cortex reflect pain perception.

    Directory of Open Access Journals (Sweden)

    Joachim Gross

    2007-05-01

    Full Text Available Successful behavior requires selection and preferred processing of relevant sensory information. The cortical representation of relevant sensory information has been related to neuronal oscillations in the gamma frequency band. Pain is of invariably high behavioral relevance and, thus, nociceptive stimuli receive preferred processing. Here, by using magnetoencephalography, we show that selective nociceptive stimuli induce gamma oscillations between 60 and 95 Hz in primary somatosensory cortex. Amplitudes of pain-induced gamma oscillations vary with objective stimulus intensity and subjective pain intensity. However, around pain threshold, perceived stimuli yielded stronger gamma oscillations than unperceived stimuli of equal stimulus intensity. These results show that pain induces gamma oscillations in primary somatosensory cortex that are particularly related to the subjective perception of pain. Our findings support the hypothesis that gamma oscillations are related to the internal representation of behaviorally relevant stimuli that should receive preferred processing.

  16. Excessive astrocyte-derived neurotrophin-3 contributes to the abnormal neuronal dendritic development in a mouse model of fragile X syndrome.

    Directory of Open Access Journals (Sweden)

    Qi Yang

    Full Text Available Fragile X syndrome (FXS is a form of inherited mental retardation in humans that results from expansion of a CGG repeat in the Fmr1 gene. Recent studies suggest a role of astrocytes in neuronal development. However, the mechanisms involved in the regulation process of astrocytes from FXS remain unclear. In this study, we found that astrocytes derived from a Fragile X model, the Fmr1 knockout (KO mouse which lacks FMRP expression, inhibited the proper elaboration of dendritic processes of neurons in vitro. Furthermore, astrocytic conditioned medium (ACM from KO astrocytes inhibited proper dendritic growth of both wild-type (WT and KO neurons. Inducing expression of FMRP by transfection of FMRP vectors in KO astrocytes restored dendritic morphology and levels of synaptic proteins. Further experiments revealed elevated levels of the neurotrophin-3 (NT-3 in KO ACM and the prefrontal cortex of Fmr1 KO mice. However, the levels of nerve growth factor (NGF, brain-derived neurotrophic factor (BDNF, glial cell-derived neurotrophic factor (GDNF, and ciliary neurotrophic factor (CNTF were normal. FMRP has multiple RNA-binding motifs and is involved in translational regulation. RNA-binding protein immunoprecipitation (RIP showed the NT-3 mRNA interacted with FMRP in WT astrocytes. Addition of high concentrations of exogenous NT-3 to culture medium reduced the dendrites of neurons and synaptic protein levels, whereas these measures were ameliorated by neutralizing antibody to NT-3 or knockdown of NT-3 expression in KO astrocytes through short hairpin RNAs (shRNAs. Prefrontal cortex microinjection of WT astrocytes or NT-3 shRNA infected KO astrocytes rescued the deficit of trace fear memory in KO mice, concomitantly decreased the NT-3 levels in the prefrontal cortex. This study indicates that excessive NT-3 from astrocytes contributes to the abnormal neuronal dendritic development and that astrocytes could be a potential therapeutic target for FXS.

  17. Excessive astrocyte-derived neurotrophin-3 contributes to the abnormal neuronal dendritic development in a mouse model of fragile X syndrome.

    Science.gov (United States)

    Yang, Qi; Feng, Bin; Zhang, Kun; Guo, Yan-yan; Liu, Shui-bing; Wu, Yu-mei; Li, Xiao-qiang; Zhao, Ming-gao

    2012-01-01

    Fragile X syndrome (FXS) is a form of inherited mental retardation in humans that results from expansion of a CGG repeat in the Fmr1 gene. Recent studies suggest a role of astrocytes in neuronal development. However, the mechanisms involved in the regulation process of astrocytes from FXS remain unclear. In this study, we found that astrocytes derived from a Fragile X model, the Fmr1 knockout (KO) mouse which lacks FMRP expression, inhibited the proper elaboration of dendritic processes of neurons in vitro. Furthermore, astrocytic conditioned medium (ACM) from KO astrocytes inhibited proper dendritic growth of both wild-type (WT) and KO neurons. Inducing expression of FMRP by transfection of FMRP vectors in KO astrocytes restored dendritic morphology and levels of synaptic proteins. Further experiments revealed elevated levels of the neurotrophin-3 (NT-3) in KO ACM and the prefrontal cortex of Fmr1 KO mice. However, the levels of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF) were normal. FMRP has multiple RNA-binding motifs and is involved in translational regulation. RNA-binding protein immunoprecipitation (RIP) showed the NT-3 mRNA interacted with FMRP in WT astrocytes. Addition of high concentrations of exogenous NT-3 to culture medium reduced the dendrites of neurons and synaptic protein levels, whereas these measures were ameliorated by neutralizing antibody to NT-3 or knockdown of NT-3 expression in KO astrocytes through short hairpin RNAs (shRNAs). Prefrontal cortex microinjection of WT astrocytes or NT-3 shRNA infected KO astrocytes rescued the deficit of trace fear memory in KO mice, concomitantly decreased the NT-3 levels in the prefrontal cortex. This study indicates that excessive NT-3 from astrocytes contributes to the abnormal neuronal dendritic development and that astrocytes could be a potential therapeutic target for FXS.

  18. Tetrachromacy of human vision: spectral channels and primary colors

    Science.gov (United States)

    Gavrik, Vitali V.

    2002-06-01

    Full-color imaging requires four channels as, in contrast to a colorimeter, can add no primary to matched scene colors themselves. An ideal imaging channel should have the same spectral sensitivity of scene recording as a retinal receptor and evoke the same primary color sensation. The alternating matching functions of a triad of real primaries are inconsistent with the three cones but explicable of two pairs of independent opponent receptors with their alternating blue-yellow and green-red chromatic axes in the color space. Much other controversy of trichromatic approach can also be explained with the recently proposed intra- receptor processes in the photopic rod and cone, respectively. Each of their four primary sensations, unmixed around 465, 495, 575, and 650 nm, is evoked within a different spectral region. The current trichromatic photographic systems have been found separately to approximate the blue and red receptors, as well as their spectral opponency against the respective yellow and blue- green receptors simulated with a single middle-wave imaging channel. The channel sensitivities are delimited by the neutral points of rod and cone and cannot simulate the necessary overlap of non-opponent channels for properly to render some mixed colors. The yellow and cyan positive dyes closely control the brightness of blue and red sensations, respectively. Those red and blue respectively to control the yellow and blue-green sensations on brightness scales are replaced by magenta dye, controlling them together. Accurate rendering of natural saturation metameric colors, problematic blue-green, purple-red, and low-illumination colors requires to replace the hybrid 'green' channel with the blue-green and yellow channels.

  19. Astrocytic Vesicle Mobility in Health and Disease

    Directory of Open Access Journals (Sweden)

    Robert Zorec

    2013-05-01

    Full Text Available 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.

  20. Astrocytic vesicle mobility in health and disease.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  2. TRPV1 on astrocytes rescues nigral dopamine neurons in Parkinson’s disease via CNTF

    Science.gov (United States)

    Nam, Jin H.; Park, Eun S.; Won, So-Yoon; Lee, Yu A.; Kim, Kyoung I.; Jeong, Jae Y.; Baek, Jeong Y.; Cho, Eun J.; Jin, Minyoung; Chung, Young C.; Lee, Byoung D.; Kim, Sung Hyun; Kim, Eung-Gook; Byun, Kyunghee; Lee, Bonghee; Woo, Dong Ho; Lee, C. Justin; Kim, Sang R.; Bok, Eugene; Kim, Yoon-Seong; Ahn, Tae-Beom; Ko, Hyuk Wan; Brahmachari, Saurav; Pletinkova, Olga; Troconso, Juan C.; Dawson, Valina L.; Dawson, Ted M.

    2015-01-01

    Currently there is no neuroprotective or neurorestorative therapy for Parkinson’s disease. Here we report that transient receptor potential vanilloid 1 (TRPV1) on astrocytes mediates endogenous production of ciliary neurotrophic factor (CNTF), which prevents the active degeneration of dopamine neurons and leads to behavioural recovery through CNTF receptor alpha (CNTFRα) on nigral dopamine neurons in both the MPP+-lesioned or adeno-associated virus α-synuclein rat models of Parkinson’s disease. Western blot and immunohistochemical analysis of human post-mortem substantia nigra from Parkinson’s disease suggests that this endogenous neuroprotective system (TRPV1 and CNTF on astrocytes, and CNTFRα on dopamine neurons) might have relevance to human Parkinson’s disease. Our results suggest that activation of astrocytic TRPV1 activates endogenous neuroprotective machinery in vivo and that it is a novel therapeutic target for the treatment of Parkinson’s disease. PMID:26490328

  3. Xanthohumol induces different cytotoxicity and apoptotic pathways in malignant and normal astrocytes.

    Science.gov (United States)

    Zajc, I; Filipič, M; Lah, T T

    2012-11-01

    Cytotoxicity and the mechanisms of cell death induced by xanthohumol (XN) were compared in normal and cancerous human cells as the differences may be relevant for the potential use of XN in cancer therapy. The cancer cells seemed to be more susceptible to the cytotoxicity of XN than normal cells, but a significant difference was observed only in astrocytic cells. XN induced a higher rate of apoptosis in glioblastoma cells than in normal astrocytes, which was associated with activation of p53 and an elevated Bax/Bcl-2 ratio in glioblastoma cells, indicating an intrinsic caspase-dependent apoptotic pathway. In contrast, a reduced Bax/Bcl-2 ratio was observed in normal human astrocytes. This was also associated with higher expression of the cell cycle inhibitor, p21, in glioblastoma cells than in normal astrocytes. In addition, at a lower, non-cytotoxic concentration, XN partially inhibited the invasiveness of glioblastoma cells. Due to the selective sensitivity of astrocytic cells to XN, this compound should be studied further as a candidate for adjuvant therapy in the treatment of glioma.

  4. Astrocyte Apoptosis and HIV Replication Are Modulated in Host Cells Coinfected with Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    Javier M. Urquiza

    2017-08-01

    Full Text Available The protozoan Trypanosoma cruzi is the etiological agent of Chagas disease. In immunosuppressed individuals, as it occurs in the coinfection with human immunodeficiency virus (HIV, the central nervous system may be affected. In this regard, reactivation of Chagas disease is severe and often lethal, and it accounts for meningoencephalitis. Astrocytes play a crucial role in the environment maintenance of healthy neurons; however, they can host HIV and T. cruzi. In this report, human astrocytes were infected in vitro with both genetically modified-pathogens to express alternative fluorophore. As evidenced by fluorescence microscopy and flow cytometry, HIV and T. cruzi coexist in the same astrocyte, likely favoring reciprocal interactions. In this context, lower rates of cell death were observed in both T. cruzi monoinfected-astrocytes and HIV-T. cruzi coinfection in comparison with those infected only with HIV. The level of HIV replication is significantly diminished under T. cruzi coinfection, but without affecting the infectivity of the HIV progeny. This interference with viral replication appears to be related to the T. cruzi multiplication rate or its increased intracellular presence but does not require their intracellular cohabitation or infected cell-to-cell contact. Among several Th1/Th2/Th17 profile-related cytokines, only IL-6 was overexpressed in HIV-T. cruzi coinfection exhibiting its cytoprotective role. This study demonstrates that T. cruzi and HIV are able to coinfect astrocytes thus altering viral replication and apoptosis.

  5. Enamel matrix derivative promote primary human pulp cell differentiation and mineralization

    National Research Council Canada - National Science Library

    Riksen, Elisabeth Aurstad; Landin, Maria A; Reppe, Sjur; Nakamura, Yukio; Lyngstadaas, Ståle Petter; Reseland, Janne E

    2014-01-01

    ...; however the molecular mechanisms involved are unclear. The effect of EMD (5-50 μg/mL) on primary human pulp cells were compared to untreated cells and cells incubated with 10⁻⁸ M dexamethasone (DEX...

  6. Reprogramming of HUVECs into Induced Pluripotent Stem Cells (HiPSCs), Generation and Characterization of HiPSC-Derived Neurons and Astrocytes

    Science.gov (United States)

    Boakye, Paul A.; Baker, Glen; Smith, Peter A.; Murray, Allan G.; Giuliani, Fabrizio; Jahroudi, Nadia

    2015-01-01

    Neurodegenerative diseases are characterized by chronic and progressive structural or functional loss of neurons. Limitations related to the animal models of these human diseases have impeded the development of effective drugs. This emphasizes the need to establish disease models using human-derived cells. The discovery of induced pluripotent stem cell (iPSC) technology has provided novel opportunities in disease modeling, drug development, screening, and the potential for “patient-matched” cellular therapies in neurodegenerative diseases. In this study, with the objective of establishing reliable tools to study neurodegenerative diseases, we reprogrammed human umbilical vein endothelial cells (HUVECs) into iPSCs (HiPSCs). Using a novel and direct approach, HiPSCs were differentiated into cells of central nervous system (CNS) lineage, including neuronal, astrocyte and glial cells, with high efficiency. HiPSCs expressed embryonic genes such as nanog, sox2 and Oct-3/4, and formed embryoid bodies that expressed markers of the 3 germ layers. Expression of endothelial-specific genes was not detected in HiPSCs at RNA or protein levels. HiPSC-derived neurons possess similar morphology but significantly longer neurites compared to primary human fetal neurons. These stem cell-derived neurons are susceptible to inflammatory cell-mediated neuronal injury. HiPSC-derived neurons express various amino acids that are important for normal function in the CNS. They have functional receptors for a variety of neurotransmitters such as glutamate and acetylcholine. HiPSC-derived astrocytes respond to ATP and acetylcholine by elevating cytosolic Ca2+ concentrations. In summary, this study presents a novel technique to generate differentiated and functional HiPSC-derived neurons and astrocytes. These cells are appropriate tools for studying the development of the nervous system, the pathophysiology of various neurodegenerative diseases and the development of potential drugs for their

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

    Directory of Open Access Journals (Sweden)

    Anat Elmann

    2013-01-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  9. Glycogen serves as an energy source that maintains astrocyte cell proliferation in the neonatal telencephalon.

    Science.gov (United States)

    Gotoh, Hitoshi; Nomura, Tadashi; Ono, Katsuhiko

    2017-06-01

    Large amounts of energy are required when cells undergo cell proliferation and differentiation for mammalian neuronal development. Early neonatal mice face transient starvation and use stored energy for survival or to support development. Glycogen is a branched polysaccharide that is formed by glucose, and serves as an astrocytic energy store for rapid energy requirements. Although it is present in radial glial cells and astrocytes, the role of glycogen during development remains unclear. In the present study, we demonstrated that glycogen accumulated in glutamate aspartate transporter (GLAST)+ astrocytes in the subventricular zone and rostral migratory stream. Glycogen levels markedly decreased after birth due to the increase of glycogen phosphorylase, an essential enzyme for glycogen metabolism. In primary cultures and in vivo, the inhibition of glycogen phosphorylase decreased the proliferation of astrocytic cells. The number of cells in the G1 phase increased in combination with the up-regulation of cyclin-dependent kinase inhibitors or down-regulation of the phosphorylation of retinoblastoma protein (pRB), a determinant for cell cycle progression. These results suggest that glycogen accumulates in astrocytes located in specific areas during the prenatal stage and is used as an energy source to maintain normal development in the early postnatal stage.

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

    Science.gov (United States)

    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.

  11. Sphingosine 1-phosphate signaling in astrocytes: Implications for progressive multiple sclerosis.

    Science.gov (United States)

    Farez, Mauricio F; Correale, Jorge

    2016-02-15

    Multiple sclerosis is an autoimmune disorder characterized by recurrent attacks against the central nervous system. After many years, certain patients enter a progressive disease phase, characterized by steady clinical deterioration. However, in 10-15% of patients, the disease is progressive from the beginning, and thus diagnosed as Primary Progressive Multiple Sclerosis. Unlike relapsing-remitting forms, progressive MS lacks effective therapy. Astrocytes are a major component of glial cells and are now thought to play a role in disease progression. Sphingosine 1-phophate is a molecule with extensive receptor expression on both immune and glial cells and is also a target of fingolimod, a drug used in relapsing remitting patients that sequesters lymphocytes within lymph nodes. However, because sphingosine 1-phosphate receptors are also expressed in astrocytes, and also because modification of this pathway has shown interesting benefits in animal models of Multiple Sclerosis, this astrocyte pathway has become an interesting target for developing potential new therapeutic approaches for Multiple Sclerosis.

  12. Dopamine Burden Triggers Neurodegeneration via Production and Release of TNF-α from Astrocytes in Minimal Hepatic Encephalopathy.

    Science.gov (United States)

    Ding, Saidan; Wang, Weikan; Wang, Xuebao; Liang, Yong; Liu, Leping; Ye, Yiru; Yang, Jianjing; Gao, Hongchang; Zhuge, Qichuan

    2016-10-01

    Dopamine (DA)-induced learning and memory impairment is well documented in minimal hepatic encephalopathy (MHE), but the contribution of DA to neurodegeneration and the involved underlying mechanisms are not fully understood. In this study, the effect of DA on neuronal apoptosis was initially detected. The results showed that MHE/DA (10 μg)-treated rats displayed neuronal apoptosis. However, we found that DA (10 μM) treatment did not induce evident apoptosis in primary cultured neurons (PCNs) but did produce TNF-α in primary cultured astrocytes (PCAs). Furthermore, co-cultures between PCAs and PCNs exposed to DA exhibited increased astrocytic TNF-α levels and neuronal apoptosis compared with co-cultures exposed to the vehicle, indicating the attribution of the neuronal apoptosis to astrocytic TNF-α. We also demonstrated that DA enhanced TNF-α production from astrocytes by activation of the TLR4/MyD88/NF-κB pathway, and secreted astrocytic TNF-α-potentiated neuronal apoptosis through inactivation of the PI3K/Akt/mTOR pathway. Overall, the findings from this study suggest that DA stimulates substantial production and secretion of astrocytic TNF-α, consequently and indirectly triggering progressive neurodegeneration, resulting in cognitive decline and memory loss in MHE.

  13. Human Resource Architectures for New Teachers in Flemish Primary Education

    Science.gov (United States)

    Vekeman, Eva; Devos, Geert; Valcke, Martin

    2016-01-01

    Since research shows that the quality of a school's teaching force is related to its personnel practices, there is a growing interest in human resource management (HRM) in education. Existing research has generated insights into the differences, constraints and effects of single and isolated HR practices. Yet, little research is available…

  14. Human Resource Architectures for New Teachers in Flemish Primary Education

    Science.gov (United States)

    Vekeman, Eva; Devos, Geert; Valcke, Martin

    2016-01-01

    Since research shows that the quality of a school's teaching force is related to its personnel practices, there is a growing interest in human resource management (HRM) in education. Existing research has generated insights into the differences, constraints and effects of single and isolated HR practices. Yet, little research is available…

  15. Devolution and human resources in primary healthcare in rural Mali.

    Science.gov (United States)

    Lodenstein, Elsbet; Dao, Dramane

    2011-06-08

    Devolution, as other types of decentralization (e.g. deconcentration, delegation, privatization), profoundly changes governance relations in the health system. Devolution is meant to affect performance of the health system by transferring responsibilities and authority to locally elected governments. The key question of this article is: what does devolution mean for human resources for health in Mali?This article assesses the key advantages and dilemmas associated with devolution such as responsiveness to local needs, downward accountability and health worker retention. Challenges of politics and capacities are also addressed in relation to human resources for health at the local level. Examples are derived from experiences in Mali with a capacity development programme and from case studies of other countries. It is not research findings that are presented, but highlights of key issues at stake aimed at inspiring the debate in Mali and elsewhere.A first lesson from the discussion suggests that in the context of human resources for health, decentralization of authority and resources is not the main issue. The challenge is to develop or strengthen accountability of those who decide and act, whether they are local politicians, bureaucrats or community representatives. If decentralization policies do not address public accountability, they will not fundamentally change human resource management, quality and equity of staffing. A second lesson is that successful devolution requires innovations in capacity development of all actors involved and in designing effective incentive measures. A final key conclusion is that the topic of devolution policy and its effects on human resources for health, and vice versa, merit more attention. A better understanding may lead to more appropriate policy designs and better preparation for the actors involved in countries that are embarking on decentralization, as is the case in Mali.

  16. Devolution and human resources in primary healthcare in rural Mali

    Directory of Open Access Journals (Sweden)

    Dao Dramane

    2011-06-01

    Full Text Available Abstract Devolution, as other types of decentralization (e.g. deconcentration, delegation, privatization, profoundly changes governance relations in the health system. Devolution is meant to affect performance of the health system by transferring responsibilities and authority to locally elected governments. The key question of this article is: what does devolution mean for human resources for health in Mali? This article assesses the key advantages and dilemmas associated with devolution such as responsiveness to local needs, downward accountability and health worker retention. Challenges of politics and capacities are also addressed in relation to human resources for health at the local level. Examples are derived from experiences in Mali with a capacity development programme and from case studies of other countries. It is not research findings that are presented, but highlights of key issues at stake aimed at inspiring the debate in Mali and elsewhere. A first lesson from the discussion suggests that in the context of human resources for health, decentralization of authority and resources is not the main issue. The challenge is to develop or strengthen accountability of those who decide and act, whether they are local politicians, bureaucrats or community representatives. If decentralization policies do not address public accountability, they will not fundamentally change human resource management, quality and equity of staffing. A second lesson is that successful devolution requires innovations in capacity development of all actors involved and in designing effective incentive measures. A final key conclusion is that the topic of devolution policy and its effects on human resources for health, and vice versa, merit more attention. A better understanding may lead to more appropriate policy designs and better preparation for the actors involved in countries that are embarking on decentralization, as is the case in Mali.

  17. Altered astrocytic swelling in the cortex of α-syntrophin-negative GFAP/EGFP mice.

    Directory of Open Access Journals (Sweden)

    Miroslava Anderova

    Full Text Available Brain edema accompanying ischemic or traumatic brain injuries, originates from a disruption of ionic/neurotransmitter homeostasis that leads to accumulation of K(+ and glutamate in the extracellular space. Their increased uptake, predominantly provided by astrocytes, is associated with water influx via aquaporin-4 (AQP4. As the removal of perivascular AQP4 via the deletion of α-syntrophin was shown to delay edema formation and K(+ clearance, we aimed to elucidate the impact of α-syntrophin knockout on volume changes in individual astrocytes in situ evoked by pathological stimuli using three dimensional confocal morphometry and changes in the extracellular space volume fraction (α in situ and in vivo in the mouse cortex employing the real-time iontophoretic method. RT-qPCR profiling was used to reveal possible differences in the expression of ion channels/transporters that participate in maintaining ionic/neurotransmitter homeostasis. To visualize individual astrocytes in mice lacking α-syntrophin we crossbred GFAP/EGFP mice, in which the astrocytes are labeled by the enhanced green fluorescent protein under the human glial fibrillary acidic protein promoter, with α-syntrophin knockout mice. Three-dimensional confocal morphometry revealed that α-syntrophin deletion results in significantly smaller astrocyte swelling when induced by severe hypoosmotic stress, oxygen glucose deprivation (OGD or 50 mM K(+. As for the mild stimuli, such as mild hypoosmotic or hyperosmotic stress or 10 mM K(+, α-syntrophin deletion had no effect on astrocyte swelling. Similarly, evaluation of relative α changes showed a significantly smaller decrease in α-syntrophin knockout mice only during severe pathological conditions, but not during mild stimuli. In summary, the deletion of α-syntrophin markedly alters astrocyte swelling during severe hypoosmotic stress, OGD or high K(+.

  18. 几种激素和IL-1β对人脑星形胶质细胞IL-6 、TNF-α分泌的影响%Effect of Some Hormones and IL-1β on the Production of IL-6 and TNF-α in Astrocytes from Human Fetal Brain in Vitro

    Institute of Scientific and Technical Information of China (English)

    李兰英; 孙云; 庞智玲

    2001-01-01

    目的 观察T3等因素对体外培养人胎大脑星形胶质细胞分泌IL-6、TNF-α的调节作用。方法 纯化培养人胎大脑星形胶质细胞,应用酶联免疫分析(ELISA)方法检测培养上清液中IL-6、TNF-α的水平。结果 (1)星形胶质细胞(AC)在体外培养条件下可自发分泌IL-6,而TNF-α则几乎检测不到。(2)LPS(0.1μg/mL)即可诱导AC产生IL-6和TNF-α。(3)IL-1β是IL-6分泌的主要诱导剂,但不诱导TNF-α分泌。(4)氢化可的松可明显抑制AC分泌IL-6、TNF-α。(5)T3在72h可刺激IL-6的分泌。(6)胰岛素对IL-6的分泌没有明显的调节作用。结论 AC可通过分泌细胞因子参与炎症反应等病理过程并维持中枢神经系统的正常发育、内环境的稳定,且受多种因素的调节。在中枢神经系统中T3、胰岛素主要参与调节发育和代谢,可能不直接参与炎症和免疫机制调节。%Objective Studing the effect of some hormons and IL-6 on theproduction of IL-6 and TNF-α in astrocytes from human fetal brain. Methods The purified cultures of astrocytes were prepared from second-trimester human fetal brain. The levels of IL-6 and TNF-α were detected by ELISA before and after stimulation with IL- 1β,LPS , insulin, triiodothyronine(T3)and hydrocortisone. Results (1)Before stimulated the level of IL-6 in medium was (73.60±9.14)pg/mL at 24 h and(68.46±22.60)pg/mL at 72 h respectively and few TNF-α were detected. (2)LPS(0.1 μg/mL) could induce the production of IL- 6 and TNF-α in astrocytes (P <0.001).(3)IL-1β was a strong stimulus for production of IL-6 in a dose-dependent fashion and the level of IL-6 induced by IL-1β(100 U/mL) is 30 fold more than the base level . IL-1β could not stimulate TNF-α secretion . (4) T3 (10-8 mol/L)could increase the level of IL-6 at 72 h(P<0.05) . (5) Hydrocortisone suppress the production of IL-6 and TNF-α before and after stimulation with IL-1β and LPS . (6)Insulin failed to

  19. Stimulus-specific delay activity in human primary visual cortex.

    Science.gov (United States)

    Serences, John T; Ester, Edward F; Vogel, Edward K; Awh, Edward

    2009-02-01

    Working memory (WM) involves maintaining information in an on-line state. One emerging view is that information in WM is maintained via sensory recruitment, such that information is stored via sustained activity in the sensory areas that encode the to-be-remembered information. Using functional magnetic resonance imaging, we observed that key sensory regions such as primary visual cortex (V1) showed little evidence of sustained increases in mean activation during a WM delay period, though such amplitude increases have typically been used to determine whether a region is involved in on-line maintenance. However, a multivoxel pattern analysis of delay-period activity revealed a sustained pattern of activation in V1 that represented only the intentionally stored feature of a multifeature object. Moreover, the pattern of delay activity was qualitatively similar to that observed during the discrimination of sensory stimuli, suggesting that WM representations in V1 are reasonable "copies" of those evoked during pure sensory processing.

  20. Primary study of leptin and human hepatocellular carcinoma in vitro

    Institute of Scientific and Technical Information of China (English)

    Jing Zhou; Wei Lei; Lei Shen; He-Sheng Luo; Zhi-Xiang Shen

    2008-01-01

    AIM: To investigate the expression level and effects of leptin in human hepatocellular carcinoma cells in vitro and to explore the correlation between them.METHODS: Human hepatocellular carcinoma cell line HepG2 was cultured in vitro, and (the expression level)mRNA of leptin and leptin receptors in HepG2 were assessed using reverse transcription polymerase chain reaction (RT-PCR). Effects of different concentrations of leptin (50 ng/mL, 100 ng/mL, 200 ng/mL) on HepG2 were detected with colorimetric assay by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) after incubation periods of 24 h, 48 h,and 72 h. Flow cytometry was performed to assess cell cycle progression of different concentrations of leptin as stated above after each 24 h incubation period.RESULTS: mRNA of leptin and leptin receptors (including short and long isoforms) were expressed in HepG2.The 72 h incubation of leptin at different concentrations (50 ng/mL, 100 ng/mL, 200 ng/mL) promoted proliferation of HepG2 in a concentration- and timedependent manner. The experimental group shows significant statistical differences when compared to the controlled group which contained 0 ng/mL of leptin. As the concentration of leptin increases, significant fewer cells were detected in G0-G1 phase and more cells in S and G2-M phases.CONCLUSION: Leptin and leptin receptor are simultaneously expressed in human hepatocellular carcinoma cell line HepG2. Addition of leptin (O ng/mL200 ng/mL) in 72 h periods indicated there is a concentration- and time-dependent correlation in the stimulation of HepG2 cell proliferation. The effect of proliferation by leptin is due to promotion of DNA synthesis and enhancement of mitotic activity. The relationship between leptin and human hepatocellular carcinoma cells might indicate that adipokine could be associated with the progression of human hepatocellular carcinoma.

  1. Multivoxel fMRI analysis of color tuning in human primary visual cortex

    NARCIS (Netherlands)

    Parkes, Laura M.; Marsman, Jan-Bernard C.; Oxley, David C.; Goulermas, John Y.; Wuerger, Sophie M.

    2009-01-01

    We use multivoxel pattern analysis (MVPA) to study the spatial clustering of color-selective neurons in the human brain. Our main objective was to investigate whether MVPA reveals the spatial arrangements of color-selective neurons in human primary visual cortex (V1). We measured the distributed fMR

  2. Fcgamma receptor-mediated suppression of human immunodeficiency virus type 1 replication in primary human macrophages.

    Science.gov (United States)

    Perez-Bercoff, Danielle; David, Annie; Sudry, Hugues; Barré-Sinoussi, Françoise; Pancino, Gianfranco

    2003-04-01

    Permissiveness of monocytes and macrophages to human immunodeficiency virus (HIV) infection is modulated by various stimuli. In this study we demonstrate that stimulation of primary monocytes and monocyte-derived macrophages (MDM) through the receptors for the Fc portion of immunoglobulin G (IgG) (FcgammaR) inhibits HIV type 1 (HIV-1) replication. Viral p24 production was decreased by 1.5 to 3 log units in MDM infected with both R5 and X4 HIV-1 strains upon stimulation by immobilized IgG but not upon stimulation by soluble IgG or by F(ab')(2) IgG fragments. Although MDM activation by immobilized IgG induced high levels of macrophage-derived chemokine secretion as well as a sustained down-regulation of CD4 and a transient decrease in CCR5 expression, these factors did not appear to play a major role in the suppression of HIV-1 replication. Single-cycle infection of FcgammaR-stimulated MDM with HIV-1 virions pseudotyped with either HIV-1 R5 or vesicular stomatitis virus G envelopes was inhibited, suggesting a postentry restriction of viral replication. PCR analyses of HIV-1 DNA intermediate replication forms suggested that reverse transcription is not affected by stimulation with immobilized human IgG, at least during the first replication cycle. The accumulation of PCR products corresponding to nuclear unintegrated two-long-terminal-repeat circles and the relative decrease of integrated HIV-1 DNA signals suggest an inhibition of proviral integration. Our data, showing that FcgammaR-mediated activation of MDM is a potent mechanism of HIV-1 suppression, raise the possibility that FcgammaR cross-linking by immune complexes may contribute to the control of viral replication in macrophages.

  3. Human synaptic plasticity gene expression profile and dendritic spine density changes in HIV-infected human CNS cells: role in HIV-associated neurocognitive disorders (HAND.

    Directory of Open Access Journals (Sweden)

    Venkata Subba Rao Atluri

    Full Text Available HIV-associated neurocognitive disorders (HAND is characterized by development of cognitive, behavioral and motor abnormalities, and occur in approximately 50% of HIV infected individuals. Our current understanding of HAND emanates mainly from HIV-1 subtype B (clade B, which is prevalent in USA and Western countries. However very little information is available on neuropathogenesis of HIV-1 subtype C (clade C that exists in Sub-Saharan Africa and Asia. Therefore, studies to identify specific neuropathogenic mechanisms associated with HAND are worth pursuing to dissect the mechanisms underlying this modulation and to prevent HAND particularly in clade B infection. In this study, we have investigated 84 key human synaptic plasticity genes differential expression profile in clade B and clade C infected primary human astrocytes by using RT(2 Profile PCR Array human Synaptic Plasticity kit. Among these, 31 and 21 synaptic genes were significantly (≥3 fold down-regulated and 5 genes were significantly (≥3 fold up-regulated in clade B and clade C infected cells, respectively compared to the uninfected control astrocytes. In flow-cytometry analysis, down-regulation of postsynaptic density and dendrite spine morphology regulatory proteins (ARC, NMDAR1 and GRM1 was confirmed in both clade B and C infected primary human astrocytes and SK-N-MC neuroblastoma cells. Further, spine density and dendrite morphology changes by confocal microscopic analysis indicates significantly decreased spine density, loss of spines and decreased dendrite diameter, total dendrite and spine area in clade B infected SK-N-MC neuroblastoma cells compared to uninfected and clade C infected cells. We have also observed that, in clade B infected astrocytes, induction of apoptosis was significantly higher than in the clade C infected astrocytes. In conclusion, this study suggests that down-regulation of synaptic plasticity genes, decreased dendritic spine density and induction of

  4. Rotation is the primary motion of paired human epidermal keratinocytes.

    Science.gov (United States)

    Tate, Sota; Imai, Matome; Matsushita, Natsuki; Nishimura, Emi K; Higashiyama, Shigeki; Nanba, Daisuke

    2015-09-01

    Collective motion of keratinocytes is involved in morphogenesis, homeostasis, and wound healing of the epidermis. Yet how the collective motion of keratinocytes emerges from the behavior of individual cells is still largely unknown. The aim of this study was to find the cellular behavior that links single and collective motion of keratinocytes. We investigated the behavior of two-cell colonies of HaCaT keratinocytes by a combination of time-lapse imaging and image processing. The two-cell colonies of HaCaT cells were formed as a contacted pair of keratinocyte clones. Image analysis and cell culture experiments revealed that the rotational speed of two-cell colonies was positively associated with their proliferative capacity. α6 integrin was required for the rotational motion of two-cell keratinocyte colonies. We also confirmed that two-cell colonies of keratinocytes predominantly exhibited the rotational, but not translational, motion, two modes of motion in a contact pair of rotating objects. The rotational motion is the primary motion of two-cell keratinocyte colonies and its speed is positively associated with their proliferative capacity. This study suggests that the assembly of rotating keratinocytes generates the collective motion of proliferative keratinocytes during morphogenesis and wound healing of the epidermis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  5. Tick-borne encephalitis virus infects rat astrocytes but does not affect their viability.

    Directory of Open Access Journals (Sweden)

    Maja Potokar

    Full Text Available Tick-borne encephalitis virus (TBEV causes one of the most dangerous human neuroinfections in Europe and Asia. To infect neurons it must cross the blood-brain-barrier (BBB, and presumably also cells adjacent to the BBB, such as astrocytes, the most abundant glial cell type. However, the knowledge about the viral infection of glial cells is fragmental. Here we studied whether TBEV infects rat astrocytes. Rats belong to an animal group serving as a TBEV amplifying host. We employed high resolution quantitative fluorescence microscopy to investigate cell entry and cytoplasmic mobility of TBEV particles along with the effect on the cell cytoskeleton and cell survival. We report that infection of astrocytes with TBEV increases with time of exposure to TBEV and that with post-infection time TBEV particles gained higher mobility. After several days of infection actin cytoskeleton was affected, but cell survival was unchanged, indicating that rat astrocytes resist TBEV-mediated cell death, as reported for other mammalian cells. Therefore, astrocytes may present an important pool of dormant TBEV infections and a new target for therapeutic intervention.

  6. Tick-Borne Encephalitis Virus Infects Rat Astrocytes but Does Not Affect Their Viability

    Science.gov (United States)

    Potokar, Maja; Korva, Miša; Jorgačevski, Jernej; Avšič-Županc, Tatjana; Zorec, Robert

    2014-01-01

    Tick-borne encephalitis virus (TBEV) causes one of the most dangerous human neuroinfections in Europe and Asia. To infect neurons it must cross the blood-brain-barrier (BBB), and presumably also cells adjacent to the BBB, such as astrocytes, the most abundant glial cell type. However, the knowledge about the viral infection of glial cells is fragmental. Here we studied whether TBEV infects rat astrocytes. Rats belong to an animal group serving as a TBEV amplifying host. We employed high resolution quantitative fluorescence microscopy to investigate cell entry and cytoplasmic mobility of TBEV particles along with the effect on the cell cytoskeleton and cell survival. We report that infection of astrocytes with TBEV increases with time of exposure to TBEV and that with post-infection time TBEV particles gained higher mobility. After several days of infection actin cytoskeleton was affected, but cell survival was unchanged, indicating that rat astrocytes resist TBEV-mediated cell death, as reported for other mammalian cells. Therefore, astrocytes may present an important pool of dormant TBEV infections and a new target for therapeutic intervention. PMID:24465969

  7. Influence of PrP 106 - 126 on expression of laminin and fibronectin in astrocyte

    Institute of Scientific and Technical Information of China (English)

    LI YuRong; GUAN LeLuo; YANG JianMin; ZHOU XiangMei; YIN XiaoMin; ZHAO DeMing

    2008-01-01

    Astrogliosis is a hallmark of prion disease, but the metabolic alterations of astrocytes remain poorly documented. A synthetic pepUde corresponding to amino acid 106-126 of the human prion protein (PrP) has been shown to be toxic to neurons. In this study, the effects of PrP 106-126 on astrocytes were investigated in vitro. The proliferation of astrocytes was significantly (P < 0.05) increased when grown in media conditioned with PrP 106-126 (80 μmol/L) from microglia. The expression of laminin (LN) and fibronectin (FN) was examined at both mRNA and protein levels. The results showed that ex-posure of astrocytes to PrP 106-126 enhanced the expression of LN and FN. The increase of FN in astrocyte cultures required cytokines previously released by activated microglia. This study reveals the expression ofLN and FN affected by PrP106-126.

  8. Sodium signaling and astrocyte energy metabolism.

    Science.gov (United States)

    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.

  9. Sodium signaling and astrocyte energy metabolism

    KAUST Repository

    Chatton, Jean-Yves

    2016-03-31

    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. © 2016 Wiley Periodicals, Inc.

  10. Gene expression analysis of primary normal human hepatocytes infected with human hepatitis B virus

    Institute of Scientific and Technical Information of China (English)

    Hyun Mi Ryu; Sung Gyoo Park; Sung Su Yea; Won Hee Jang; Young-Il Yang; Guhung Jung

    2006-01-01

    AIM: To find the relationship between hepatitis B virus (HBV) and hepatocytes during the initial state of infection by cDNA microarray.METHODS: Primary normal human hepatocytes (PNHHs)were isolated and infected with HBV. From the PNHHs,RNA was isolated and inverted into complement DNA (cDNA) with Cy3- or Cy5- labeled dUTP for microarray analysis. The labeled cDNA was hybridized with microarray chip, including 4224 cDNAs. From the image of the microarray, expression profiles were produced and some of them were confirmed by RT-PCR, immunoblot analysis, and NF-κB luciferase reporter assay.RESULTS: From the cDNA microarray, we obtained 98differentially regulated genes. Of the 98 genes, 53 were up regulated and 45 down regulated. Interestingly, in the up regulated genes, we found the TNF signaling pathway-related genes: LT-α, TRAF2, and NIK. By using RT-PCR, we confirmed the up-regulation of these genes in HepG2, Huh7, and Chang liver cells, which were transfected with pHBV1.2x, a plasmid encoding all HBV messages. Moreover, these three genes participated in HBVmediated NF-κB activation.CONCLUSION: During the initial state of HBV infection,hepatocytes facilitate the activation of NF-κB through up regulation of LT-α, TRAF2, and NIK.

  11. Primary function analysis of human mental retardation related gene CRBN.

    Science.gov (United States)

    Xin, Wang; Xiaohua, Ni; Peilin, Chen; Xin, Chen; Yaqiong, Sun; Qihan, Wu

    2008-06-01

    The mutation of human cereblon gene (CRBN) is revealed to be related with mild mental retardation. Since the molecular characteristics of CRBN have not been well presented, we investigated the general properties of CRBN. We analyzed its gene structure and protein homologues. The CRBN protein might belong to a family of adenosine triphosphate (ATP)-dependent Lon protease. We also found that CRBN was widely expressed in different tissues, and the expression level in testis is significantly higher than other tissues. This may suggested it could play some important roles in several other tissues besides brain. Transient transfection experiment in AD 293 cell lines suggested that both CRBN and CRBN mutant (nucleotide position 1,274(C > T)) are located in the whole cells. This may suggest new functions of CRBN in cell nucleolus besides its mitochondria protease activity in cytoplasm.

  12. Hypoxia inducible factor-2α regulates the development of retinal astrocytic network by maintaining adequate supply of astrocyte progenitors.

    Directory of Open Access Journals (Sweden)

    Li-Juan Duan

    Full Text Available Here we investigate the role of hypoxia inducible factor (HIF-2α in coordinating the development of retinal astrocytic and vascular networks. Three Cre mouse lines were used to disrupt floxed Hif-2α, including Rosa26(CreERT2, Tie2(Cre, and GFAP(Cre. Global Hif-2α disruption by Rosa26(CreERT2 led to reduced astrocytic and vascular development in neonatal retinas, whereas endothelial disruption by Tie2(Cre had no apparent effects. Hif-2α deletion in astrocyte progenitors by GFAP(Cre significantly interfered with the development of astrocytic networks, which failed to reach the retinal periphery and were incapable of supporting vascular development. Perplexingly, the abundance of strongly GFAP(+ mature astrocytes transiently increased at P0 before they began to lag behind the normal controls by P3. Pax2(+ and PDGFRα(+ astrocytic progenitors and immature astrocytes were dramatically diminished at all stages examined. Despite decreased number of astrocyte progenitors, their proliferation index or apoptosis was not altered. The above data can be reconciled by proposing that HIF-2α is required for maintaining the supply of astrocyte progenitors by slowing down their differentiation into non-proliferative mature astrocytes. HIF-2α deficiency in astrocyte progenitors may accelerate their differentiation into astrocytes, a change which greatly interferes with the replenishment of astrocyte progenitors due to insufficient time for proliferation. Rapidly declining progenitor supply may lead to premature cessation of astrocyte development. Given that HIF-2α protein undergoes oxygen dependent degradation, an interesting possibility is that retinal blood vessels may regulate astrocyte differentiation through their oxygen delivery function. While our findings support the consensus that retinal astrocytic template guides vascular development, they also raise the possibility that astrocytic and vascular networks may mutually regulate each other

  13. Astrocytic expression of the Alzheimer's disease beta-secretase (BACE1) is stimulus-dependent

    DEFF Research Database (Denmark)

    Hartlage-Rübsamen, Maike; Zeitschel, Ulrike; Apelt, Jenny

    2003-01-01

    The beta-site APP-cleaving enzyme (BACE1) is a prerequisite for the generation of beta-amyloid peptides, which give rise to cerebrovascular and parenchymal beta-amyloid deposits in the brain of Alzheimer's disease patients. BACE1 is neuronally expressed in the brains of humans and experimental...... paradigms studied. In contrast, BACE1 expression by reactive astrocytes was evident in chronic but not in acute models of gliosis. Additionally, we observed BACE1-immunoreactive astrocytes in proximity to beta-amyloid plaques in the brains of aged Tg2576 mice and Alzheimer's disease patients....

  14. Food Safety - The Primary Objective of Human Society Existence

    Directory of Open Access Journals (Sweden)

    Tabita Cornelia Adamov

    2010-10-01

    Full Text Available Food has played, plays and will play a decisive role in the existence and development of human society. The level of food quality, causes physical, social and moral society health. In this sense food security is an essential goal of economic and social development, being an essential component of security of life and national security. Evolution of the Common Agricultural Policy (CAP, in terms of food security, was due not only changes in agriculture, but also came in response to the demands of society in general. Amoung these is growing concerns about hygiene and food safety and animal welfare. European consumers want safe and wholesome food and the EU wants to ensure that all its citizens consume food with high quality standards. Food safety policy has undergone an extensive refurbishment. The objective of this reform was to ensure that EU legislation on food safety is as complete as possible, and consumers benefit as much information about potential risks and measures to be taken to minimize them. The goal of a modern economy, is the correlation of quantitative and qualitative food production with consumer demand. It thus requires knowledge of the physiological needs of consumers, leading to demand for agricultural products.

  15. Glutathione-Dependent Detoxification Processes in Astrocytes

    DEFF Research Database (Denmark)

    Dringen, Ralf; Brandmann, Maria; Hohnholt, Michaela C

    2015-01-01

    component in many of the astrocytic detoxification processes is the tripeptide glutathione (GSH) which serves as electron donor in the GSH peroxidase-catalyzed reduction of peroxides. In addition, GSH is substrate in the detoxification of xenobiotics and endogenous compounds by GSH-S-transferases which......Astrocytes have a pivotal role in brain as partners of neurons in homeostatic and metabolic processes. Astrocytes also protect other types of brain cells against the toxicity of reactive oxygen species and are considered as first line of defence against the toxic potential of xenobiotics. A key...... generate GSH conjugates that are efficiently exported from the cells by multidrug resistance proteins. Moreover, GSH reacts with the reactive endogenous carbonyls methylglyoxal and formaldehyde to intermediates which are substrates of detoxifying enzymes. In this article we will review the current...

  16. Neuroinflammatory TNFα Impairs Memory via Astrocyte Signaling.

    Science.gov (United States)

    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.

  17. Metastatic behaviour of primary human tumours in a zebrafish xenotransplantation model

    Directory of Open Access Journals (Sweden)

    Heidecke Claus-Dieter

    2009-04-01

    Full Text Available Abstract Background Aberrant regulation of cell migration drives progression of many diseases, including cancer cell invasion and metastasis formation. Analysis of tumour invasion and metastasis in living organisms to date is cumbersome and involves difficult and time consuming investigative techniques. For primary human tumours we establish here a simple, fast, sensitive and cost-effective in vivo model to analyse tumour invasion and metastatic behaviour. Methods We fluorescently labelled small explants from gastrointestinal human tumours and investigated their metastatic behaviour after transplantation into zebrafish embryos and larvae. The transparency of the zebrafish embryos allows to follow invasion, migration and micrometastasis formation in real-time. High resolution imaging was achieved through laser scanning confocal microscopy of live zebrafish. Results In the transparent zebrafish embryos invasion, circulation of tumour cells in blood vessels, migration and micrometastasis formation can be followed in real-time. Xenografts of primary human tumours showed invasiveness and micrometastasis formation within 24 hours after transplantation, which was absent when non-tumour tissue was implanted. Furthermore, primary human tumour cells, when organotopically implanted in the zebrafish liver, demonstrated invasiveness and metastatic behaviour, whereas primary control cells remained in the liver. Pancreatic tumour cells showed no metastatic behaviour when injected into cloche mutant embryos, which lack a functional vasculature. Conclusion Our results show that the zebrafish is a useful in vivo animal model for rapid analysis of invasion and metastatic behaviour of primary human tumour specimen.

  18. Intervention timing and effect of PJ34 on astrocytes during oxygen-glucose deprivation/reperfusion and cell death pathways.

    Science.gov (United States)

    Cai, Chuan; Zhang, Rui; Huang, Qiao-Ying; Cao, Xu; Zou, Liang-Yu; Chu, Xiao-Fan

    2015-06-01

    Poly (ADP-ribose) polymerase-1 (PARP-1) plays as a double edged sword in cerebral ischemia-reperfusion, hinging on its effect on the intracellular energy storage and injury severity, and the prognosis has relationship with intervention timing. During ischemia injury, apoptosis and oncosis are the two main cell death pathway sin the ischemic core. The participation of astrocytes in ischemia-reperfusion induced cell death has triggered more and more attention. Here, we examined the protective effects and intervention timing of the PARP-1 inhibitor PJ34, by using a mixed oxygen-glucose deprivation/reperfusion (OGDR) model of primary rat astrocytes in vitro, which could mimic the ischemia-reperfusion damage in the "ischemic core". Meanwhile, cell death pathways of various PJ34 treated astrocytes were also investigated. Our results showed that PJ34 incubation (10 μmol/L) did not affect release of lactate dehydrogenase (LDH) from astrocytes and cell viability or survival 1 h after OGDR. Interestingly, after 3 or 5 h OGDR, PJ34 significantly reduced LDH release and percentage of PI-positive cells and increased cell viability, and simultaneously increased the caspase-dependent apoptotic rate. The intervention timing study demonstrated that an earlier and longer PJ34 intervention during reperfusion was associated with more apparent protective effects. In conclusion, earlier and longer PJ34 intervention provides remarkable protective effects for astrocytes in the "ischaemic core" mainly by reducing oncosis of the astrocytes, especially following serious OGDR damage.

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

    Science.gov (United States)

    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 manganese or oxidants and NO induced activation, oxidation/nitration of NKCC1 play an important role in the astrocyte swelling. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses.

    Science.gov (United States)

    Shigetomi, Eiji; Bushong, Eric A; Haustein, Martin D; Tong, Xiaoping; Jackson-Weaver, Olan; Kracun, Sebastian; Xu, Ji; Sofroniew, Michael V; Ellisman, Mark H; Khakh, Baljit S

    2013-05-01

    Intracellular Ca(2+) transients are considered a primary signal by which astrocytes interact with neurons and blood vessels. With existing commonly used methods, Ca(2+) has been studied only within astrocyte somata and thick branches, leaving the distal fine branchlets and endfeet that are most proximate to neuronal synapses and blood vessels largely unexplored. Here, using cytosolic and membrane-tethered forms of genetically encoded Ca(2+) indicators (GECIs; cyto-GCaMP3 and Lck-GCaMP3), we report well-characterized approaches that overcome these limitations. We used in vivo microinjections of adeno-associated viruses to express GECIs in astrocytes and studied Ca(2+) signals in acute hippocampal slices in vitro from adult mice (aged ∼P80) two weeks after infection. Our data reveal a sparkling panorama of unexpectedly numerous, frequent, equivalently scaled, and highly localized Ca(2+) microdomains within entire astrocyte territories in situ within acute hippocampal slices, consistent with the distribution of perisynaptic branchlets described using electron microscopy. Signals from endfeet were revealed with particular clarity. The tools and experimental approaches we describe in detail allow for the systematic study of Ca(2+) signals within entire astrocytes, including within fine perisynaptic branchlets and vessel-associated endfeet, permitting rigorous evaluation of how astrocytes contribute to brain function.

  1. Overexpression of serum response factor in astrocytes improves neuronal plasticity in a model of early alcohol exposure.

    Science.gov (United States)

    Paul, A P; Medina, A E

    2012-09-27

    Neuronal plasticity deficits underlie many of the cognitive problems seen in fetal alcohol spectrum disorders (FASD). We have developed a ferret model showing that early alcohol exposure leads to a persistent disruption in ocular dominance (OD) plasticity. Recently, we showed that this deficit could be reversed by overexpression of serum response factor (SRF) in the primary visual cortex during the period of monocular deprivation (MD). Surprisingly, this restoration was observed throughout the extent of visual cortex and most of the cells transfected by the virus were positive for the astrocytic marker GFAP rather than the neuronal marker NeuN. Here we test whether overexpression of SRF exclusively in astrocytes is sufficient to restore OD plasticity in alcohol-exposed ferrets. To accomplish that, first we exposed cultured astrocytes to Sindbis viruses carrying either a constitutively active form of SRF (SRF+), a dominant negative (SRF-) or control Green Fluorescent Protein (GFP). After 24h, these astrocytes were implanted in the visual cortex of alcohol-exposed animals or saline controls one day before MD. Optical imaging of intrinsic signals showed that alcohol-exposed animals that were implanted with astrocytes expressing SRF, but not SRF- or GFP, showed robust restoration of OD plasticity in all visual cortex. These findings suggest that overexpression of SRF exclusively in astrocytes can improve neuronal plasticity in FASD. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

  2. Orientation anisotropies in human primary visual cortex depend on contrast.

    Science.gov (United States)

    Maloney, Ryan T; Clifford, Colin W G

    2015-10-01

    Orientation processing in visual cortex appears matched to the environment, such that larger neural populations are tuned to cardinal (horizontal/vertical) than oblique orientations. This may be manifested perceptually as a cardinal bias: poorer sensitivity to oblique compared to cardinal orientations (the "oblique effect"). However, a growing body of psychophysical data reveals the opposite pattern of anisotropy: a bias towards the oblique over the cardinal orientations (the "horizontal effect"), something matched by recent functional magnetic resonance imaging (fMRI) studies that have found an increased response to the oblique over the cardinal orientations in early visual cortex. This may reveal the operation of an efficient coding strategy optimised to the diet of orientations encountered during natural viewing. From consideration of coding efficiency, it might be expected that the anisotropies would change as the quality/strength of the oriented stimulus changes. In two experiments, fMRI response modulations were measured in retinotopically-defined human early visual cortex as a function of the contrast and orientation of sinusoidal gratings. Both experiments revealed a marked change in the V1 response from a cardinal (vertical) bias at low contrast to an oblique bias at high contrast. In Experiment 2, this was also apparent in areas V2 and V3. On average, there was no systematic "radial bias" (a preference for orientations aligned with the visual field meridian) in V1, although it was present in some individual subjects. The change in orientation anisotropies with contrast is consistent with an adaptive stimulus coding strategy in cortex that shifts according to the strength of the sensory inputs.

  3. Maturation and demise of human primary monocytes by carbon nanotubes

    Energy Technology Data Exchange (ETDEWEB)

    De Nicola, Milena, E-mail: milena.de.nicola@uniroma2.it [University of Rome ' Tor Vergata' , Department of Biology (Italy); Mirabile Gattia, Daniele, E-mail: daniele.mirabile@enea.it [UTTMAT, ENEA-C.R. Casaccia (Italy); Traversa, Enrico, E-mail: Enrico.Traversa@kaust.edu.sa [King Abdullah University of Science and Technology (KAUST), Division of Physical Science and Engineering (Saudi Arabia); Ghibelli, Lina, E-mail: ghibelli@uniroma2.it [University of Rome ' Tor Vergata' , Department of Biology (Italy)

    2013-06-15

    The possibility of exploiting carbon nanotubes (CNT) in biomedical practices requires thorough analysis of the chemical or bulk effects they may exert on the immune system, the complex network that recognizes and eliminates foreign particles. In particular, the phagocytosing ability of cells belonging to the monocyte/macrophage lineage may render these immune cells an ideal toxicological target of pristine CNT, which may form aggregates of size exceeding monocyte/macrophage phagocytosing plasticity. To shed light on this issue, we analyzed the effects that pristine multi-walled CNT (MWCNT) without metal or biological impurities exert on survival and activation of freshly explanted human peripheral blood monocytes, analyzing in parallel the non-phagocytosing lymphocytes, and using graphite as control carbon material. MWCNT (diameter 10-50 nm, length up to 10 {mu}m) exert two different toxic effects on mononuclear leukocytes: a minor apoptogenic effect (on lymphocytes > monocytes), and a major, apoptosis-independent effect that exclusively and deeply affect monocyte homeostasis. Analysis of monocyte number, adhesion, redox equilibrium, and the differentiation markers CD14 and CD11b reveals that MWCNT cause the selective disappearance of phagocytosis-competent monocytes by mechanisms related to the presence of large nanoparticle aggregates, suggesting phenomena of bulk toxicity possibly consisting of frustrated phagocytosis. At the same time, MWCNT stimulate adhesion of the phagocytosis-incompetent monocytes, and their differentiation toward a peculiar maturation asset. These observations point out novel mechanisms of CNT toxicity, renewing concerns that they may impair the innate immune system deranging the inflammatory responses.

  4. Matrigel improves functional properties of primary human salivary gland cells.

    Science.gov (United States)

    Maria, Ola M; Zeitouni, Anthony; Gologan, Olga; Tran, Simon D

    2011-05-01

    Currently, there is no effective treatment available to patients with irreversible loss of functional salivary acini caused by Sjogren's syndrome or after radiotherapy for head and neck cancer. A tissue-engineered artificial salivary gland would help these patients. The graft cells for this device must establish tight junctions in addition to being of fluid-secretory nature. This study analyzed a graft source from human salivary glands (huSG) cultured on Matrigel. Cells were obtained from parotid and submandibular glands, expanded in vitro, and then plated on either Matrigel-coated (2 mg/mL) or uncoated culture dish. Immunohistochemistry, transmission electron microscopy, quantitative real-time-polymerase chain reaction, Western blot, and transepithelial electrical resistance were employed. On Matrigel, huSG cells adopted an acinar phenotype by forming three-dimensional acinar-like units (within 24 h of plating) as well as a monolayer of cells. On uncoated surfaces (plastic), huSG cells only formed monolayers of ductal cells. Both types of culture conditions allowed huSG cells to express tight junction proteins (claudin-1, -2, -3, -4; occludin; JAM-A; and ZO-1) and adequate transepithelial electrical resistance. Importantly, 99% of huSG cells on Matrigel expressed α-amylase and the water channel protein Aquaporin-5, as compared to cells on plastic. Transmission electron microscopy confirmed an acinar phenotype with many secretory granules. Matrigel increased the secretion of α-amylase two to five folds into the media, downregulated certain salivary genes, and regulated the translation of acinar proteins. This three-dimensional in vitro serum-free cell culture method allows the organization and differentiation of huSG cells into salivary cells with an acinar phenotype.

  5. Maturation and demise of human primary monocytes by carbon nanotubes

    KAUST Repository

    De Nicola, Milena D.

    2013-05-17

    The possibility of exploiting carbon nanotubes (CNT) in biomedical practices requires thorough analysis of the chemical or bulk effects they may exert on the immune system, the complex network that recognizes and eliminates foreign particles. In particular, the phagocytosing ability of cells belonging to the monocyte/macrophage lineage may render these immune cells an ideal toxicological target of pristine CNT, which may form aggregates of size exceeding monocyte/macrophage phagocytosing plasticity. To shed light on this issue, we analyzed the effects that pristine multi-walled CNT (MWCNT) without metal or biological impurities exert on survival and activation of freshly explanted human peripheral blood monocytes, analyzing in parallel the non-phagocytosing lymphocytes, and using graphite as control carbon material. MWCNT (diameter 10-50 nm, length up to 10 μm) exert two different toxic effects on mononuclear leukocytes: a minor apoptogenic effect (on lymphocytes > monocytes), and a major, apoptosis-independent effect that exclusively and deeply affect monocyte homeostasis. Analysis of monocyte number, adhesion, redox equilibrium, and the differentiation markers CD14 and CD11b reveals that MWCNT cause the selective disappearance of phagocytosis-competent monocytes by mechanisms related to the presence of large nanoparticle aggregates, suggesting phenomena of bulk toxicity possibly consisting of frustrated phagocytosis. At the same time, MWCNT stimulate adhesion of the phagocytosis-incompetent monocytes, and their differentiation toward a peculiar maturation asset. These observations point out novel mechanisms of CNT toxicity, renewing concerns that they may impair the innate immune system deranging the inflammatory responses. © 2013 Springer Science+Business Media Dordrecht.

  6. From stem cell to astrocyte: Decoding the regulation of GFAP

    NARCIS (Netherlands)

    R. Kanski

    2014-01-01

    The research presented in this thesis focuses on glial fibrillary acidic protein (GFAP), the main intermediate filament (IF) in astrocytes and astrocyte subpopulations such as neural stem cells (NSCs). In neurodegenerative diseases or upon brain damage, astrocytes respond to an injury with an upregu

  7. Astrocyte-Derived CCL2 is Associated with M1 Activation and Recruitment of Cultured Microglial Cells

    Directory of Open Access Journals (Sweden)

    Mingfeng He

    2016-02-01

    Full Text Available Background/Aims: Microglia are an essential player in central nervous system inflammation. Recent studies have demonstrated that the astrocytic chemokine, CCL2, is associated with microglial activation in vivo. However, CCL2-induced microglial activation has not yet been studied in vitro. The purpose of the current study was to understand the role of astrocyte-derived CCL2 in microglial activation and to elucidate the underlying mechanism(s. Methods: Primary astrocytes were pre-treated with CCL2 siRNA and stimulated with TNF-α. The culture medium (CM was collected and added to cultures of microglia, which were incubated with and without CCR2 inhibitor. Microglial cells were analyzed by quantitative RT-PCR to determine whether they polarized to the M1 or M2 state. Microglial migratory ability was assessed by transwell migration assay. Results: TNF-α stimulated the release of CCL2 from astrocytes, even if the culture media containing TNF-α was replaced with fresh media after 3 h. CM from TNF-α-stimulated astrocytes successfully induced microglial activation, which was ascertained by increased activation of M1 and enhanced migration ability. In contrast, CM from astrocytes pretreated with CCL2 siRNA showed no effect on microglial activation, compared to controls. Additionally, microglia pre-treated with RS102895, a CCR2 inhibitor, were resistant to activation by CM from TNF-α-stimulated astrocytes. Conclusion: This study demonstrates that the CCL2/CCR2 pathway of astrocyte-induced microglial activation is associated with M1 polarization and enhanced migration ability, indicating that this pathway could be a useful target to ameliorate inflammation in the central nervous system.

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

    Science.gov (United States)

    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. Copyright © 2014 Wiley Periodicals, Inc.

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

    Science.gov (United States)

    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.

  10. Hepcidin attenuates amyloid beta-induced inflammatory and pro-oxidant responses in astrocytes and microglia.

    Science.gov (United States)

    Urrutia, Pamela J; Hirsch, Etienne C; González-Billault, Christian; Núñez, Marco T

    2017-07-01

    Alzheimer's disease (AD) is characterized by extracellular senile plaques, intracellular neurofibrillary tangles, and neuronal death. Aggregated amyloid-β (Aβ) induces inflammation and oxidative stress, which have pivotal roles in the pathogenesis of AD. Hepcidin is a key regulator of systemic iron homeostasis. Recently, an anti-inflammatory response to hepcidin was reported in macrophages. Under the hypothesis that hepcidin mediates anti-inflammatory response in the brain, in this study, we evaluated the putative anti-inflammatory role of hepcidin on Aβ-activated astrocytes and microglia. Primary culture of astrocytes and microglia were treated with Aβ, with or without hepcidin, and cytokine levels were then evaluated. In addition, the toxicity of Aβ-treated astrocyte- or microglia-conditioned media was tested on neurons, evaluating cellular death and oxidative stress generation. Finally, mice were injected in the right lateral ventricle with Aβ, with or without hepcidin, and hippocampus glial activation and oxidative stress were evaluated. Pre-treatment with hepcidin reduced the expression and secretion of TNF-α and IL-6 in astrocytes and microglia treated with Aβ. Hepcidin also reduced neurotoxicity and oxidative damage triggered by conditioned media obtained from astrocytes and microglia treated with Aβ. Stereotaxic intracerebral injection of hepcidin reduced glial activation and oxidative damage triggered by Aβ injection in mice. Overall, these results are consistent with the hypothesis that in astrocytes and microglia hepcidin down-regulates the inflammatory and pro-oxidant processes induced by Aβ, thus protecting neighboring neurons. This is a newly described property of hepcidin in the central nervous system, which may be relevant for the development of strategies to prevent the neurodegenerative process associated with AD. © 2017 International Society for Neurochemistry.

  11. GABAA increases calcium in subventricular zone astrocyte-like cells through L- and T-type voltage-gated calcium channels

    Directory of Open Access Journals (Sweden)

    Stephanie Z Young

    2010-04-01

    Full Text Available In the adult neurogenic subventricular zone (SVZ, the behavior of astrocyte-like cells and some of their functions depend on changes in intracellular Ca2+ levels and tonic GABAA receptor activation. However, it is unknown whether, and if so how, GABAA receptor activity regulates intracellular Ca2+ dynamics in SVZ astrocytes. To monitor Ca2+ activity selectively in astrocyte-like cells, we used two lines of transgenic mice expressing either GFP fused to a Gq-coupled receptor or DsRed under the human glial fibrillary acidic protein (hGFAP promoter. GABAA receptor activation induced Ca2+ increases in 40-50% of SVZ astrocytes. GABAA-induced Ca2+ increases were prevented with nifedipine and mibefradil, blockers of L- and T-type voltage-gated calcium channels (VGCC. The L-type Ca2+ channel activator BayK 8644 increased the percentage of GABAA-responding astrocyte-like cells to 75%, suggesting that the majority of SVZ astrocytes express functional VGCCs. SVZ astrocytes also displayed spontaneous Ca2+ activity, the frequency of which was regulated by tonic GABAA receptor activation. These data support a role for ambient GABA in tonically regulating intracellular Ca2+ dynamics through GABAA receptors and VGCC in a subpopulation of astrocyte-like cells in the postnatal SVZ.

  12. IFN-γ-induced increase in the mobility of MHC class II compartments in astrocytes depends on intermediate filaments

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    Vardjan Nina

    2012-06-01

    Full Text Available Abstract Background In immune-mediated diseases of the central nervous system, astrocytes exposed to interferon-γ (IFN-γ can express major histocompatibility complex (MHC class II molecules and antigens on their surface. MHC class II molecules are thought to be delivered to the cell surface by membrane-bound vesicles. However, the characteristics and dynamics of this vesicular traffic are unclear, particularly in reactive astrocytes, which overexpress intermediate filament (IF proteins that may affect trafficking. The aim of this study was to determine the mobility of MHC class II vesicles in wild-type (WT astrocytes and in astrocytes devoid of IFs. Methods The identity of MHC class II compartments in WT and IF-deficient astrocytes 48 h after IFN-γ activation was determined immunocytochemically by using confocal microscopy. Time-lapse confocal imaging and Alexa Fluor546-dextran labeling of late endosomes/lysosomes in IFN-γ treated cells was used to characterize the motion of MHC class II vesicles. The mobility of vesicles was analyzed using ParticleTR software. Results Confocal imaging of primary cultures of WT and IF-deficient astrocytes revealed IFN-γ induced MHC class II expression in late endosomes/lysosomes, which were specifically labeled with Alexa Fluor546-conjugated dextran. Live imaging revealed faster movement of dextran-positive vesicles in IFN-γ-treated than in untreated astrocytes. Vesicle mobility was lower in IFN-γ-treated IF-deficient astrocytes than in WT astrocytes. Thus, the IFN-γ-induced increase in the mobility of MHC class II compartments is IF-dependent. Conclusions Since reactivity of astrocytes is a hallmark of many CNS pathologies, it is likely that the up-regulation of IFs under such conditions allows a faster and therefore a more efficient delivery of MHC class II molecules to the cell surface. In vivo, such regulatory mechanisms may enable antigen-presenting reactive astrocytes to respond rapidly and in a

  13. Quantitative proteomics of extracellular vesicles derived from human primary and metastatic colorectal cancer cells

    OpenAIRE

    Gho, Yong Song; Choi, Dong-Sic; Choi, Do-Young; Hong, Bok Sil; Jang, Su Chul; Kim, Dae-Kyum; Lee, Jaewook; Kim, Yoon-Keun; Kim, Kwang Pyo

    2012-01-01

    Cancer cells actively release extracellular vesicles (EVs), including exosomes and microvesicles, into surrounding tissues. These EVs play pleiotropic roles in cancer progression and metastasis, including invasion, angiogenesis, and immune modulation. However, the proteomic differences between primary and metastatic cancer cell-derived EVs remain unclear. Here, we conducted comparative proteomic analysis between EVs derived from human primary colorectal cancer cells (SW480) and their metastat...

  14. Adherence of uropathogenic Escherichia coli to human primary epithelial cells of renal pelvis

    Institute of Scientific and Technical Information of China (English)

    CHAO GU; JIN YING CHEN; MIN HOU; JING DONG HE; JI WU CHANG

    2006-01-01

    Human primary epithelial cells of renal pelvis was established to investigate the adherence of uropathogenic Escherichia coli (UPEC) to this cell line, in which the primary cell culture was performed by using cultivation of the normal epithelium of renal pelvis in keratinocyte serum free medium (K-SFM)with epidermal growth factor (EGF) and bovine pituitary extract (BPE). Both UPEC132 obtained from urine specimen of patients with pyelonephritis and the pilus-free representative strain E. coli K-12p678-54 were used to study the adherence of these strains on human primary epithelial cells of renal pelvis.The UPEC adherence was performed with observation on the morphological changes of the adhered cells,while the adhesion rates and indices were calculated in different times of experiment. In addition, the virulence genes hly and cnf1 of UPEC132 were detected by multiplex PCR assay. In this study, the human primary epithelial cells of renal pelvis was found to exhibit the character of the transitional epithelial cells. Compared with the control group, the adhesion rates and indices began to increase from 15 min of the experiment time and reached its peak in 120 min. The adhesion rate and index of UPEC132 to human primary epithelial cells of renal pelvis were 74.4% and 34.0 respectively. Many microscopic changes in the primary cells adhered with UPEC132 could be detected, such as rounding or irregularity in shape,unevenness in staining and the cytoplasmic and nuclear changes. It suggests that human primary epithelial cells of renal pelvis can be used for the experiment on UPEC adhesion, thus providing a basis for the further study on the pathogenesis of UPEC.

  15. Substrate regulation of ascorbate transport activity in astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, J.X.; Jaworski, E.M.; Kulaga, A.; Dixon, S.J. (Univ. of Western Ontario, London (Canada))

    1990-10-01

    Astrocytes possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na(+)-dependent L-ascorbate transporter located in the plasma membrane. The present experiments examined the effects of deprivation and supplementation of extracellular L-ascorbate on the activity of this transport system. Initial rates of L-ascorbate uptake were measured by incubating primary cultures of rat astrocytes with L-(14C)ascorbate for 1 min at 37 degrees C. We observed that the apparent maximal rate of uptake (Vmax) increased rapidly (less than 1 h) when cultured cells were deprived of L-ascorbate. In contrast, there was no change in the apparent affinity of the transport system for L-(14C)ascorbate. The increase in Vmax was reversed by addition of L-ascorbate, but not D-isoascorbate, to the medium. The effects of external ascorbate on ascorbate transport activity were specific in that preincubation of cultures with L-ascorbate did not affect uptake of 2-deoxy-D-(3H(G))glucose. We conclude that the astroglial ascorbate transport system is modulated by changes in substrate availability. Regulation of transport activity may play a role in intracellular ascorbate homeostasis by compensating for regional differences and temporal fluctuations in external ascorbate levels.

  16. Human enamel veneer restoration: an alternative technique to restore anterior primary teeth.

    Science.gov (United States)

    Oliveira, Luciana Butini; Tamay, Tereza Keiko; Oliveira, Marta Dutra Machado; Rodrigues, Célia Martins Delgado; Wanderley, Marcia Turolla

    2006-01-01

    Restoration of severely decayed primary teeth is a clinical challenge in Pediatric Dentistry. Among the restorative treatment options, the use of prefabricated crowns and resin composite restorations, either by means of direct or indirect techniques is mentioned in the literature. The purpose of this article is to describe the rehabilitation of primary anterior teeth in a 5-year-old patient. Dental treatment consisted on an anterior space maintainer prosthesis made with natural primary teeth, plus human dental enamel veneer (facet) restorations. The advantages of this technique are better esthetics and the natural enamel has physiologic wear and offers superficial smoothness and cervical adaptation compatible with those of the surrounding teeth.

  17. Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis

    Energy Technology Data Exchange (ETDEWEB)

    Woolbright, Benjamin L.; Dorko, Kenneth [Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Antoine, Daniel J.; Clarke, Joanna I. [MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool (United Kingdom); Gholami, Parviz [Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS (United States); Li, Feng [Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Kumer, Sean C.; Schmitt, Timothy M.; Forster, Jameson [Department of Surgery, University of Kansas Medical Center, Kansas City, KS (United States); Fan, Fang [Department of Pathology, University of Kansas Medical Center, Kansas City, KS (United States); Jenkins, Rosalind E.; Park, B. Kevin [MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool (United Kingdom); Hagenbuch, Bruno [Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Olyaee, Mojtaba [Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS (United States); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States)

    2015-03-15

    Accumulation of bile acids is a major mediator of cholestatic liver injury. Recent studies indicate bile acid composition between humans and rodents is dramatically different, as humans have a higher percent of glycine conjugated bile acids and increased chenodeoxycholate content, which increases the hydrophobicity index of bile acids. This increase may lead to direct toxicity that kills hepatocytes, and promotes inflammation. To address this issue, this study assessed how pathophysiological concentrations of bile acids measured in cholestatic patients affected primary human hepatocytes. Individual bile acid levels were determined in serum and bile by UPLC/QTOFMS in patients with extrahepatic cholestasis with, or without, concurrent increases in serum transaminases. Bile acid levels increased in serum of patients with liver injury, while biliary levels decreased, implicating infarction of the biliary tracts. To assess bile acid-induced toxicity in man, primary human hepatocytes were treated with relevant concentrations, derived from patient data, of the model bile acid glycochenodeoxycholic acid (GCDC). Treatment with GCDC resulted in necrosis with no increase in apoptotic parameters. This was recapitulated by treatment with biliary bile acid concentrations, but not serum concentrations. Marked elevations in serum full-length cytokeratin-18, high mobility group box 1 protein (HMGB1), and acetylated HMGB1 confirmed inflammatory necrosis in injured patients; only modest elevations in caspase-cleaved cytokeratin-18 were observed. These data suggest human hepatocytes are more resistant to human-relevant bile acids than rodent hepatocytes, and die through necrosis when exposed to bile acids. These mechanisms of cholestasis in humans are fundamentally different to mechanisms observed in rodent models. - Highlights: • Cholestatic liver injury is due to cytoplasmic bile acid accumulation in hepatocytes. • Primary human hepatocytes are resistant to BA-induced injury

  18. The catalytic and the RNA subunits of human telomerase are required to immortalize equid primary fibroblasts.

    Science.gov (United States)

    Vidale, Pamela; Magnani, Elisa; Nergadze, Solomon G; Santagostino, Marco; Cristofari, Gael; Smirnova, Alexandra; Mondello, Chiara; Giulotto, Elena

    2012-10-01

    Many human primary somatic cells can be immortalized by inducing telomerase activity through the exogenous expression of the human telomerase catalytic subunit (hTERT). This approach has been extended to the immortalization of cell lines from several mammals. Here, we show that hTERT expression is not sufficient to immortalize primary fibroblasts from three equid species, namely donkey, Burchelli's zebra and Grevy's zebra. In vitro analysis of a reconstituted telomerase composed by hTERT and an equid RNA component of telomerase (TERC) revealed a low activity of this enzyme compared to human telomerase, suggesting a low compatibility of equid and human telomerase subunits. This conclusion was also strengthened by comparison of human and equid TERC sequences, which revealed nucleotide differences in key regions for TERC and TERT interaction. We then succeeded in immortalizing equid fibroblasts by expressing hTERT and hTERC concomitantly. Expression of both human telomerase subunits led to telomerase activity and telomere elongation, indicating that human telomerase is compatible with the other equid telomerase subunits and proteins involved in telomere metabolism. The immortalization procedure described herein could be extended to primary cells from other mammals. The availability of immortal cells from endangered species could be particularly useful for obtaining new information on the organization and function of their genomes, which is relevant for their preservation.

  19. Mechanical loading and the synthesis of 1,25(OH)2D in primary human osteoblasts.

    Science.gov (United States)

    van der Meijden, K; Bakker, A D; van Essen, H W; Heijboer, A C; Schulten, E A J M; Lips, P; Bravenboer, N

    2016-02-01

    The metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D) is synthesized from its precursor 25-hydroxyvitamin D (25(OH)D) by human osteoblasts leading to stimulation of osteoblast differentiation in an autocrine or paracrine way. Osteoblast differentiation is also stimulated by mechanical loading through activation of various responses in bone cells such as nitric oxide signaling. Whether mechanical loading affects osteoblast differentiation through an enhanced synthesis of 1,25(OH)2D by human osteoblasts is still unknown. We hypothesized that mechanical loading stimulates the synthesis of 1,25(OH)2D from 25(OH)D in primary human osteoblasts. Since the responsiveness of bone to mechanical stimuli can be altered by various endocrine factors, we also investigated whether 1,25(OH)2D or 25(OH)D affect the response of primary human osteoblasts to mechanical loading. Primary human osteoblasts were pre-incubated in medium with/without 25(OH)D3 (400 nM) or 1,25(OH)2D3 (100 nM) for 24h and subjected to mechanical loading by pulsatile fluid flow (PFF). The response of osteoblasts to PFF was quantified by measuring nitric oxide, and by PCR analysis. The effect of PFF on the synthesis of 1,25(OH)2D3 was determined by subjecting osteoblasts to PFF followed by 24h post-incubation in medium with/without 25(OH)D3 (400 nM). We showed that 1,25(OH)2D3 reduced the PFF-induced NO response in primary human osteoblasts. 25(OH)D3 did not significantly alter the NO response of primary human osteoblasts to PFF, but 25(OH)D3 increased osteocalcin and RANKL mRNA levels, similar to 1,25(OH)2D3. PFF did not increase 1,25(OH)2D3 amounts in our model, even though PFF did increase CYP27B1 mRNA levels and reduced VDR mRNA levels. CYP24 mRNA levels were not affected by PFF, but were strongly increased by both 25(OH)D3 and 1,25(OH)2D3. In conclusion, 1,25(OH)2D3 may affect the response of primary human osteoblasts to mechanical stimuli, at least with respect to NO production. Mechanical stimuli may affect

  20. Activated astrocytes enhance the dopaminergic differentiation of stem cells and promote brain repair through bFGF.

    Science.gov (United States)

    Yang, Fan; Liu, Yunhui; Tu, Jie; Wan, Jun; Zhang, Jie; Wu, Bifeng; Chen, Shanping; Zhou, Jiawei; Mu, Yangling; Wang, Liping

    2014-12-17

    Astrocytes provide neuroprotective effects against degeneration of dopaminergic (DA) neurons and play a fundamental role in DA differentiation of neural stem cells. Here we show that light illumination of astrocytes expressing engineered channelrhodopsin variant (ChETA) can remarkably enhance the release of basic fibroblast growth factor (bFGF) and significantly promote the DA differentiation of human embryonic stem cells (hESCs) in vitro. Light activation of transplanted astrocytes in the substantia nigra (SN) also upregulates bFGF levels in vivo and promotes the regenerative effects of co-transplanted stem cells. Importantly, upregulation of bFGF levels, by specific light activation of endogenous astrocytes in the SN, enhances the DA differentiation of transplanted stem cells and promotes brain repair in a mouse model of Parkinson's disease (PD). Our study indicates that astrocyte-derived bFGF is required for regulation of DA differentiation of the stem cells and may provide a strategy targeting astrocytes for treatment of PD.

  1. Exposure to inflammatory cytokines IL-1β and TNFα induces compromise and death of astrocytes; implications for chronic neuroinflammation.

    Directory of Open Access Journals (Sweden)

    Christa van Kralingen

    Full Text Available BACKGROUND: Astrocytes have critical roles in the human CNS in health and disease. They provide trophic support to neurons and are innate-immune cells with keys roles during states-of-inflammation. In addition, they have integral functions associated with maintaining the integrity of the blood-brain barrier. METHODS: We have used cytometric bead arrays and xCELLigence technology to monitor the to monitor the inflammatory response profiles and astrocyte compromise in real-time under various inflammatory conditions. Responses were compared to a variety of inflammatory cytokines known to be released in the CNS during neuroinflammation. Astrocyte compromise measured by xCELLigence was confirmed using ATP measurements, cleaved caspase 3 expression, assessment of nuclear morphology and cell death. RESULTS: Inflammatory activation (IL-1β or TNFα of astrocytes results in the transient production of key inflammatory mediators including IL-6, cell surface adhesion molecules, and various leukocyte chemoattractants. Following this phase, the NT2-astrocytes progressively become compromised, which is indicated by a loss of adhesion, appearance of apoptotic nuclei and reduction in ATP levels, followed by DEATH. The earliest signs of astrocyte compromise were observed between 24-48 h post cytokine treatment. However, significant cell loss was not observed until at least 72 h, where there was also an increase in the expression of cleaved-caspase 3. By 96 hours approximately 50% of the astrocytes were dead, with many of the remaining showing signs of compromise too. Numerous other inflammatory factors were tested, however these effects were only observed with IL-1β or TNFα treatment. CONCLUSIONS: Here we reveal direct sensitivity to mediators of the inflammatory milieu. We highlight the power of xCELLigence technology for revealing the early progressive compromise of the astrocytes, which occurs 24-48 hours prior to substantive cell loss. Death induced by IL

  2. The origin of Rosenthal fibers and their contributions to astrocyte pathology in Alexander disease.

    Science.gov (United States)

    Sosunov, Alexander A; McKhann, Guy M; Goldman, James E

    2017-03-31

    Rosenthal fibers (RFs) are cytoplasmic, proteinaceous aggregates. They are the pathognomonic feature of the astrocyte pathology in Alexander Disease (AxD), a neurodegenerative disorder caused by heterozygous mutations in the GFAP gene, encoding glial fibrillary acidic protein (GFAP). Although RFs have been known for many years their origin and significance remain elusive issues. We have used mouse models of AxD based on the overexpression of human GFAP (transgenic, TG) and a point mutation in mouse GFAP (knock-in, KI) to examine the formation of RFs and to find astrocyte changes that correlate with the appearance of RFs. We found RFs of various sizes and shapes. The smallest ones appear as granular depositions on intermediate filaments. These contain GFAP and the small heat shock protein, alphaB-crystallin. Their aggregation appears to give rise to large RFs. The appearance of new RFs and the growth of previously formed RFs occur over time. We determined that DAPI is a reliable marker of RFs and in parallel with Fluoro-Jade B (FJB) staining defined a high variability in the appearance of RFs, even in neighboring astrocytes. Although many astrocytes in AxD with increased levels of GFAP and with or without RFs change their phenotype, only some cells with large numbers of RFs show a profound reconstruction of cellular processes, with a loss of fine distal processes and the appearance of large, lobulated nuclei, likely due to arrested mitosis. We conclude that 1) RFs appear to originate as small, osmiophilic masses containing both GFAP and alphaB-crystallin deposited on bundles of intermediate filaments. 2) RFs continue to form within AxD astrocytes over time. 3) DAPI is a reliable marker for RFs and can be used with immunolabeling. 4) RFs appear to interfere with the successful completion of astrocyte mitosis and cell division.

  3. Lrp4 in astrocytes modulates glutamatergic transmission.

    Science.gov (United States)

    Sun, Xiang-Dong; Li, Lei; Liu, Fang; Huang, Zhi-Hui; Bean, Jonathan C; Jiao, Hui-Feng; Barik, Arnab; Kim, Seon-Myung; Wu, Haitao; Shen, Chengyong; Tian, Yun; Lin, Thiri W; Bates, Ryan; Sathyamurthy, Anupama; Chen, Yong-Jun; Yin, Dong-Min; Xiong, Lei; Lin, Hui-Ping; Hu, Jin-Xia; Li, Bao-Ming; Gao, Tian-Ming; Xiong, Wen-Cheng; Mei, Lin

    2016-08-01

    Neurotransmission requires precise control of neurotransmitter release from axon terminals. This process is regulated by glial cells; however, the underlying mechanisms are not fully understood. We found that glutamate release in the brain was impaired in mice lacking low-density lipoprotein receptor-related protein 4 (Lrp4), a protein that is critical for neuromuscular junction formation. Electrophysiological studies revealed compromised release probability in astrocyte-specific Lrp4 knockout mice. Lrp4 mutant astrocytes suppressed glutamatergic transmission by enhancing the release of ATP, whose level was elevated in the hippocampus of Lrp4 mutant mice. Consequently, the mutant mice were impaired in locomotor activity and spatial memory and were resistant to seizure induction. These impairments could be ameliorated by blocking the adenosine A1 receptor. The results reveal a critical role for Lrp4, in response to agrin, in modulating astrocytic ATP release and synaptic transmission. Our findings provide insight into the interaction between neurons and astrocytes for synaptic homeostasis and/or plasticity.

  4. New roles for astrocytes: the nightlife of an 'astrocyte'. La vida loca!

    Science.gov (United States)

    Horner, Philip J; Palmer, Theo D

    2003-11-01

    Like a newly popular nightspot, the biology of adult stem cells has emerged from obscurity to become one of the most lively new disciplines of the decade. The neurosciences have not escaped this trendy pastime and, from amid the noise and excitement, the astrocyte emerges as a beguiling companion to the adult neural stem cell. A once receding partner to neurons and oligodendrocytes, the astrocyte even takes on an alter ego of the stem cell itself (S. Goldman, this issue of TINS). Putting ego aside, the 'astrocyte' is also (and perhaps more importantly) an integral component of neural progenitor hotspots, where the craziness or 'la vida loca' of the nightlife might not be so wild when compared with our traditional understanding of the astrocyte. Here, astrocytes contribute to the instructive confluence of location, atmosphere and cellular neighbors that define the daily 'vida local' or everyday local life of an adult stem cell. This review discusses astrocytes as influential components in the local stem cell niche.

  5. The computational power of astrocyte mediated synaptic plasticity

    Directory of Open Access Journals (Sweden)

    Rogier eMin

    2012-11-01

    Full Text Available Research in the last two decades has made clear that astrocytes play a crucial role in the brain beyond their functions in energy metabolism and homeostasis. Many studies have shown that astrocytes can dynamically modulate neuronal excitability and synaptic plasticity, and might participate in higher brain functions like learning and memory. With the plethora of astrocyte-mediated signaling processes described in the literature today, the current challenge is to identify which of these processes happen under what physiological condition, and how this shapes information processing and, ultimately, behavior. To answer these questions will require a combination of advanced physiological, genetical and behavioral experiments. Additionally, mathematical modeling will prove crucial for testing predictions on the possible functions of astrocytes in neuronal networks, and to generate novel ideas as to how astrocytes can contribute to the complexity of the brain. Here, we aim to provide an outline of how astrocytes can interact with neurons. We do this by reviewing recent experimental literature on astrocyte-neuron interactions, discussing the dynamic effects of astrocytes on neuronal excitability and short- and long-term synaptic plasticity. Finally, we will outline the potential computational functions that astrocyte-neuron interactions can serve in the brain. We will discuss how astrocytes could govern metaplasticity in the brain, how they might organize the clustering of synaptic inputs, and how they could function as memory elements for neuronal activity. We conclude that astrocytes can enhance the computational power of neuronal networks in previously unexpected ways.

  6. The computational power of astrocyte mediated synaptic plasticity.

    Science.gov (United States)

    Min, Rogier; Santello, Mirko; Nevian, Thomas

    2012-01-01

    Research in the last two decades has made clear that astrocytes play a crucial role in the brain beyond their functions in energy metabolism and homeostasis. Many studies have shown that astrocytes can dynamically modulate neuronal excitability and synaptic plasticity, and might participate in higher brain functions like learning and memory. With the plethora of astrocyte mediated signaling processes described in the literature today, the current challenge is to identify, which of these processes happen under what physiological condition, and how this shapes information processing and, ultimately, behavior. To answer these questions will require a combination of advanced physiological, genetical, and behavioral experiments. Additionally, mathematical modeling will prove crucial for testing predictions on the possible functions of astrocytes in neuronal networks, and to generate novel ideas as to how astrocytes can contribute to the complexity of the brain. Here, we aim to provide an outline of how astrocytes can interact with neurons. We do this by reviewing recent experimental literature on astrocyte-neuron interactions, discussing the dynamic effects of astrocytes on neuronal excitability and short- and long-term synaptic plasticity. Finally, we will outline the potential computational functions that astrocyte-neuron interactions can serve in the brain. We will discuss how astrocytes could govern metaplasticity in the brain, how they might organize the clustering of synaptic inputs, and how they could function as memory elements for neuronal activity. We conclude that astrocytes can enhance the computational power of neuronal networks in previously unexpected ways.

  7. Spatial organization of astrocytes in ferret visual cortex

    Science.gov (United States)

    López‐Hidalgo, Mónica; Hoover, Walter B.

    2016-01-01

    ABSTRACT Astrocytes form an intricate partnership with neural circuits to influence numerous cellular and synaptic processes. One prominent organizational feature of astrocytes is the “tiling” of the brain with non‐overlapping territories. There are some documented species and brain region–specific astrocyte specializations, but the extent of astrocyte diversity and circuit specificity are still unknown. We quantitatively defined the rules that govern the spatial arrangement of astrocyte somata and territory overlap in ferret visual cortex using a combination of in vivo two‐photon imaging, morphological reconstruction, immunostaining, and model simulations. We found that ferret astrocytes share, on average, half of their territory with other astrocytes. However, a specific class of astrocytes, abundant in thalamo‐recipient cortical layers (“kissing” astrocytes), overlap markedly less. Together, these results demonstrate novel features of astrocyte organization indicating that different classes of astrocytes are arranged in a circuit‐specific manner and that tiling does not apply universally across brain regions and species. J. Comp. Neurol. 524:3561–3576, 2016. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc. PMID:27072916

  8. The computational power of astrocyte mediated synaptic plasticity

    Science.gov (United States)

    Min, Rogier; Santello, Mirko; Nevian, Thomas

    2012-01-01

    Research in the last two decades has made clear that astrocytes play a crucial role in the brain beyond their functions in energy metabolism and homeostasis. Many studies have shown that astrocytes can dynamically modulate neuronal excitability and synaptic plasticity, and might participate in higher brain functions like learning and memory. With the plethora of astrocyte mediated signaling processes described in the literature today, the current challenge is to identify, which of these processes happen under what physiological condition, and how this shapes information processing and, ultimately, behavior. To answer these questions will require a combination of advanced physiological, genetical, and behavioral experiments. Additionally, mathematical modeling will prove crucial for testing predictions on the possible functions of astrocytes in neuronal networks, and to generate novel ideas as to how astrocytes can contribute to the complexity of the brain. Here, we aim to provide an outline of how astrocytes can interact with neurons. We do this by reviewing recent experimental literature on astrocyte-neuron interactions, discussing the dynamic effects of astrocytes on neuronal excitability and short- and long-term synaptic plasticity. Finally, we will outline the potential computational functions that astrocyte-neuron interactions can serve in the brain. We will discuss how astrocytes could govern metaplasticity in the brain, how they might organize the clustering of synaptic inputs, and how they could function as memory elements for neuronal activity. We conclude that astrocytes can enhance the computational power of neuronal networks in previously unexpected ways. PMID:23125832

  9. Mash1 efifciently reprograms rat astrocytes into neurons

    Institute of Scientific and Technical Information of China (English)

    Daofang Ding; Leqin Xu; Hao Xu; Xiaofeng Li; Qianqian Liang; Yongjian Zhao; Yongjun Wang

    2014-01-01

    To date, it remains poorly understood whether astrocytes can be easily reprogrammed into neurons. Mash1 and Brn2 have been previously shown to cooperate to reprogram fibroblasts into neurons. In this study, we examined astrocytes from 2-month-old Sprague-Dawley rats, and found that Brn2 was expressed, but Mash1 was not detectable. Thus, we hypothesized that Mash1 alone could be used to reprogram astrocytes into neurons. We transfected a recombinant MSCV-MASH1 plasmid into astrocytes for 72 hours, and saw that all cells expressed Mash1. One week later, we observed the changes in morphology of astrocytes, which showed typical neuro-nal characteristics. Moreover,β-tubulin expression levels were signiifcantly higher in astrocytes expressing Mash1 than in control cells. These results indicate that Mash1 alone can reprogram astrocytes into neurons.

  10. Endocannabinoids potentiate synaptic transmission through stimulation of astrocytes.

    Science.gov (United States)

    Navarrete, Marta; Araque, Alfonso

    2010-10-06

    Endocannabinoids and their receptor CB1 play key roles in brain function. Astrocytes express CB1Rs that are activated by endocannabinoids released by neurons. However, the consequences of the endocannabinoid-mediated neuron-astrocyte signaling on synaptic transmission are unknown. We show that endocannabinoids released by hippocampal pyramidal neurons increase the probability of transmitter release at CA3-CA1 synapses. This synaptic potentiation is due to CB1R-induced Ca(2+) elevations in astrocytes, which stimulate the release of glutamate that activates presynaptic metabotropic glutamate receptors. While endocannabinoids induce synaptic depression in the stimulated neuron by direct activation of presynaptic CB1Rs, they indirectly lead to synaptic potentiation in relatively more distant neurons by activation of CB1Rs in astrocytes. Hence, astrocyte calcium signal evoked by endogenous stimuli (neuron-released endocannabinoids) modulates synaptic transmission. Therefore, astrocytes respond to endocannabinoids that then potentiate synaptic transmission, indicating that astrocytes are actively involved in brain physiology.

  11. Generation of iPSC lines from primary human chorionic villi cells

    Directory of Open Access Journals (Sweden)

    Björn Lichtner

    2015-11-01

    Full Text Available Primary human chorionic villi (CV cells were used to generate the iPSC line by retroviral transduction of the four Yamanaka-factors OCT4, SOX2, KLF4 and c-MYC. Pluripotency was confirmed both in vivo and in vitro. The transcriptomes of the CV-derived iPSC lines and the human embryonic stem cell lines—H1 and H9 have a Pearson correlation of 0.929 and 0.943 respectively.

  12. Reconstituting development of pancreatic intraepithelial neoplasia from primary human pancreas duct cells

    Science.gov (United States)

    Lee, Jonghyeob; Snyder, Emily R.; Liu, Yinghua; Gu, Xueying; Wang, Jing; Flowers, Brittany M.; Kim, Yoo Jung; Park, Sangbin; Szot, Gregory L.; Hruban, Ralph H.; Longacre, Teri A.; Kim, Seung K.

    2017-01-01

    Development of systems that reconstitute hallmark features of human pancreatic intraepithelial neoplasia (PanINs), the precursor to pancreatic ductal adenocarcinoma, could generate new strategies for early diagnosis and intervention. However, human cell-based PanIN models with defined mutations are unavailable. Here, we report that genetic modification of primary human pancreatic cells leads to development of lesions resembling native human PanINs. Primary human pancreas duct cells harbouring oncogenic KRAS and induced mutations in CDKN2A, SMAD4 and TP53 expand in vitro as epithelial spheres. After pancreatic transplantation, mutant clones form lesions histologically similar to native PanINs, including prominent stromal responses. Gene expression profiling reveals molecular similarities of mutant clones with native PanINs, and identifies potential PanIN biomarker candidates including Neuromedin U, a circulating peptide hormone. Prospective reconstitution of human PanIN development from primary cells provides experimental opportunities to investigate pancreas cancer development, progression and early-stage detection. PMID:28272465

  13. Valproic acid mediates the synaptic excitatory/inhibitory balance through astrocytes--a preliminary study.

    Science.gov (United States)

    Wang, Chao-Chuan; Chen, Po See; Hsu, Chien-Wen; Wu, Shou-Jung; Lin, Chieh-Ting; Gean, Po Wu

    2012-04-27

    Valproic acid (VPA) is one of the most widely used anticonvulsant and mood-stabilizing agents for the treatment of epilepsy and bipolar disorder. However, the underlying therapeutic mechanisms of the treatment of each disease remain unclear. Recently, the anti-epileptic effect of VPA has been found to lead to modulation of the synaptic excitatory/inhibitory balance. In addition, the therapeutic action of VPA has been linked to its effect on astrocytes by regulating gene expression at the molecular level, perhaps through an epigenetic mechanism as a histone deacetylase (HDAC) inhibitor. To provide insight into the mechanisms underlying the actions of VPA, this study investigated whether the synaptic excitatory/inhibitory (E/I) balance could be mediated by VPA through astrocytes. First, using the primary rat neuronal, astroglial, and neuro-glial mixed culture systems, we demonstrated that VPA treatment could regulate the mRNA levels of two post-synaptic cell adhesion molecules(neuroligin-1 and neuregulin-1) and two extracellular matrices (neuronal pentraxin-1and thrombospondin-3) in primary rat astrocyte cultures in a time- and concentration-dependent manner. Moreover, the up-regulation effect of VPA was noted in astrocytes, but not in neurons. In addition, these regulatory effects could be mimicked by sodium butyrate, a HDAC inhibitor, but not by lithium or two other glycogen synthase kinase-3 beta inhibitors. With the known role of these four proteins in regulating the synaptic E/I balance, we further demonstrated that VPA increased excitatory post-synaptic protein (postsynaptic density 95) and inhibitory post-synaptic protein (Gephyrin) in cortical neuro-glial mixed cultures. Our results suggested that VPA might affect the synaptic excitatory/inhibitory balance through its effect on astrocytes. This work provides the basis for future evaluation of the role of astroglial cell adhesion molecules and the extracellular matrix on the control of excitatory and

  14. [Vasculogenic mimicry in human primary gallbladder carcinoma and clinical significance thereof].

    Science.gov (United States)

    Fan, Yue-zu; Sun, Wei; Zhang, Wen-zhong; Ge, Chun-yan

    2007-01-16

    To investigate if there is vasculogenic mimicry (VM) in human primary gallbladder carcinoma and clinical significance thereof. Seventy-four specimens of primary gallbladder carcinoma obtained from operation underwent HE staining and double staining of CD(31) and PAS to observe the existence of VM. The correlation of pathological examination and clinical outcomes was analyzed. VM was seen in 10 of the 74 (13.5%) specimens. VM was not correlated with age, sex, location, diameter, differentiation degree, Nevin stage, and invasion depth of tumor, and existence of lymph node metastasis; but was associated with histological type (chi(2) = 10.241, P = 0.017), hepatic metastasis (chi(2) = 11.904, P = 0.01), and poor overall survival (chi(2) = 5.771, P = 0.016). Cox analysis showed that existence of VM, invasion depth, lymph node metastasis, hepatic metastasis, and operational method were independent risk factors of the prognosis of primary gallbladder carcinoma. VM exists in human primary gallbladder carcinoma. Those cases of human primary gallbladder carcinoma with VM have a poorer prognosis.

  15. Application of modified enzyme digestion method in rapid primary culture of human glioma cells

    Directory of Open Access Journals (Sweden)

    Wei XIANG

    2016-06-01

    Full Text Available Objective  To explore the applied value of modified enzyme digestion method in primary culture of human glioma cells. Methods  A traditional enzyme digestion method was modified based on literatures and our work experience. The glioma cells from 32 glioma patients with different grades were primarily cultured by the modified enzyme digestion method. The morphological features of these cells were observed under an inverted phase contrast microscope. The primary cells were purified by differential adhesion during passage. The primary cells were identified by immunofluorescence technique, and the growth curves were drawn by cell proliferation assays (CCK-8 method for investigating the proliferation of the cells cultured in vitro. Results  The primary human glioma cells were successfully cultured and transferred by the new method, with a success rate of 87.5%. The cells cultured successfully in vitro showed good adherent growth, stable morphologies, thus can be passaged. Fluoroimmunoassay showed positive expression of glial fibrillary acidic protein, which confirms the cultured cells were glioma cells. Cell proliferation assays revealed active cell proliferation in vitro, the higher the tumor grade, the higher the proliferative capacity. Conclusion  The modified enzyme digestion method is simpler and more efficient for primary culture of human glioma cells, and the success rate is also higher, thus being able to provide a good guarantee for fundamental research of glioma. DOI: 10.11855/j.issn.0577-7402.2016.06.06

  16. Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation.

    Science.gov (United States)

    Magaldi, Thomas G; Almstead, Laura L; Bellone, Stefania; Prevatt, Edward G; Santin, Alessandro D; DiMaio, Daniel

    2012-01-05