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Sample records for astrocyte cell line

  1. Construction and identification of immortalized rat astrocyte cell line expressing enkephalin

    Institute of Scientific and Technical Information of China (English)

    XU Ying; TIAN Yu-ke; TIAN Xue-bi; AN Ke; YANG Hui

    2007-01-01

    Objective: To provide a sound cell source for further ex-vivo gene therapy for chronic pain, we attempt to develop an immortalized rat astrocyte cell line that expresses enkephalin regulated by doxycycline.Methods: Retrovirus infection method was employed to develop an immortalized rat astrocyte cell line that could express enkephalin regulated by doxycycline. The hPPE gene expression level of immoralized astroyte cells (IAC)/hPPE was detected by RT-PCR, indirect immunofiuorescence staining and radioimmunoassay.Results: IAC carrying Tet-on system transfected with preproenkephalin gene could secrete enkephalin that was regulated by doxycycline in a dose-dependent manner and hPPE gene activation could be repeated in on-off-on cycles through administration or removal of doxycycline.Conclusion: An immortalized rat astrocyte cell line that secrete enkephalin under the control of doxycycline is established successfully, which provides a research basis for transgenic cell transplantation for analgesia.

  2. Lauric Acid Stimulates Ketone Body Production in the KT-5 Astrocyte Cell Line.

    Science.gov (United States)

    Nonaka, Yudai; Takagi, Tetsuo; Inai, Makoto; Nishimura, Shuhei; Urashima, Shogo; Honda, Kazumitsu; Aoyama, Toshiaki; Terada, Shin

    2016-08-01

    Coconut oil has recently attracted considerable attention as a potential Alzheimer's disease therapy because it contains large amounts of medium-chain fatty acids (MCFAs) and its consumption is thought to stimulate hepatic ketogenesis, supplying an alternative energy source for brains with impaired glucose metabolism. In this study, we first reevaluated the responses of plasma ketone bodies to oral administration of coconut oil to rats. We found that the coconut oil-induced increase in plasma ketone body concentration was negligible and did not significantly differ from that observed after high-oleic sunflower oil administration. In contrast, the administration of coconut oil substantially increased the plasma free fatty acid concentration and lauric acid content, which is the major MCFA in coconut oil. Next, to elucidate whether lauric acid can activate ketogenesis in astrocytes with the capacity to generate ketone bodies from fatty acids, we treated the KT-5 astrocyte cell line with 50 and 100 μM lauric acid for 4 h. The lauric acid treatments increased the total ketone body concentration in the cell culture supernatant to a greater extent than oleic acid, suggesting that lauric acid can directly and potently activate ketogenesis in KT-5 astrocytes. These results suggest that coconut oil intake may improve brain health by directly activating ketogenesis in astrocytes and thereby by providing fuel to neighboring neurons.

  3. Imipramine activates glial cell line-derived neurotrophic factor via early growth response gene 1 in astrocytes.

    Science.gov (United States)

    Kim, Yeni; Kim, Se Hyun; Kim, Yong Sik; Lee, Young Han; Ha, Kyooseob; Shin, Soon Young

    2011-06-01

    Recent evidence has suggested that deficits in glial plasticity contribute to the pathophysiology of depressive disorders. The present study explored early growth response 1 (EGR-1) transcriptional regulation of imipramine-induced glial cell line-derived neurotrophic factor (GDNF) expression in astrocytes. After we observed the induction of GDNF mRNA expression in rat astrocytes in response to imipramine, deletion mutant studies showed that the proximal region between -493 and -114 of the GDNF promoter, which contains three binding sites for EGR-1, was essential for maximal imipramine-induced activation of GDNF promoter. The dose-dependent upregulation of EGR-1 by imipramine, the activation of GDNF by the over-expression of EGR-1 without imipramine and the reduction in the imipramine-induced GDNF mRNA expression after silencing of endogenous EGR-1 demonstrated that EGR-1 is upregulated by imipramine to activate the GDNF promoter. Furthermore, imipramine-induced GDNF mRNA expression was strongly attenuated in primary astrocytes from Egr-1(-/-) mice, and the immunoreactivity to an anti-GDNF antibody in glial fibrillary acidic protein-positive cells was lower in imipramine-treated astrocytes from Egr-1(-/-) mice than in those from Egr-1(+/-) mice. To determine whether mitogen-activated protein kinases (MAPKs) were associated with imipramine-induced EGR-1 expression, we examined the induction of MAPK phosphorylation in response to imipramine. Pretreatment of rat primary astrocytes with the MAPK kinase inhibitor U0126 or the JNK inhibitor SP600125 strongly inhibited imipramine-stimulated EGR-1 expression. In conclusion, we found that imipramine induction of EGR-1 upregulated GDNF in astrocytes in a dose-dependent manner. This upregulation may occur through the MEK/ERK and JNK MAPK pathways, which suggests a new therapeutic mechanism of action for depressive disorders.

  4. 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.

  5. 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.

  6. Impact of flattening-filter-free radiation on the clonogenic survival of astrocytic cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Steenken, Caroline; Fleckenstein, Jens; Kegel, Stefan; Jahnke, Lennart; Simeonova, Anna; Hartmann, Linda; Kuebler, Jens; Veldwijk, Marlon R.; Wenz, Frederik; Herskind, Carsten; Giordano, Frank Anton [Universitaetsmedizin Mannheim (UMM), Medical Faculty Mannheim, Heidelberg University, Department of Radiation Oncology, Mannheim (Germany)

    2015-07-15

    Flattening-filter-free (FFF) beams are increasingly used in radiotherapy as delivery times can be substantially reduced. However, the relative biologic effectiveness (RBE) of FFF may be increased relative to conventional flattened (FLAT) beams due to differences in energy spectra. Therefore, we investigated the effects of FFF and FLAT beams on the clonogenic survival of astrocytoma cells. Three cell lines (U251, U251-MGMT, and U87) were irradiated with 6-MV and 10-MV X-rays from a linear accelerator in FFF- or FLAT-beam modes at dose rates in the range of 0.5-24 Gy/min. The surviving fraction (SF) as function of dose (2-12 Gy) was determined by the colony formation assay and fitted by the linear-quadratic model. For both beams (FFF or FLAT), the cells were pelleted in conical 15-ml centrifuge tubes and irradiated at 2-cm depth in a 1 x 1-cm{sup 2} area on the central axis of a 30 x 30-cm{sup 2} field. Dosimetry was performed with a 0.3-cm{sup 3} rigid ionization chamber. RBE was determined for FFF versus FLAT irradiation. The RBE of FFF at 7.3-11.3 Gy was 1.027 ± 0.013 and 1.063 ± 0.018 relative to FLAT beams for 6- and 10-MV beams, respectively, and was only significantly higher than 1 for 10 MV. Significantly increased survival rates were seen for lower dose rates (0.5 Gy/min FLAT vs. 5 Gy/min FLAT) at higher doses (11.9 Gy), while no differences were seen at dose rates ≥ 1.4 Gy/min (1.4 Gy/min FFF vs. 14 Gy/min FFF and 2.4 Gy/min FFF vs. 24 Gy/min FFF). FFF beams showed only a slightly increased RBE relative to FLAT beams in this experimental set-up, which is unlikely to result in clinically relevant differences in outcome. (orig.) [German] Die Flattening-Filter-freie (FFF) Bestrahlungstechnik findet zunehmend Verwendung, da sich die Applikationsdauer der einzelnen Fraktionen deutlich verkuerzen laesst. Aufgrund der Unterschiede im Spektrum koennte die relative biologische Wirksamkeit (RBW) von FFF jedoch hoeher sein als bei konventioneller Technik (d.h. bei

  7. PrP{sup C} displays an essential protective role from oxidative stress in an astrocyte cell line derived from PrP{sup C} knockout mice

    Energy Technology Data Exchange (ETDEWEB)

    Bertuchi, Fernanda R. [Center for Natural Sciences and Humanities, Federal University of ABC - UFABC, Avenida dos Estados, 5001, Bloco B, 09210-170, Santo Andre, SP (Brazil); Bourgeon, Dominique M.G.; Landemberger, Michele C.; Martins, Vilma R. [International Center for Research and Education, A.C. Camargo Hospital, Rua Tagua 440, 01505-010 Sao Paulo, SP (Brazil); Cerchiaro, Giselle, E-mail: giselle.cerchiaro@ufabc.edu.br [Center for Natural Sciences and Humanities, Federal University of ABC - UFABC, Avenida dos Estados, 5001, Bloco B, 09210-170, Santo Andre, SP (Brazil)

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer PrP{sup C} in solution acts as a radical scavenger. Black-Right-Pointing-Pointer PrP{sup C} reduces hydrogen peroxide toxicity in astrocytes. Black-Right-Pointing-Pointer Increase in ROS disrupted the cell cycle in the PrP{sup C}-knockout astrocytes. Black-Right-Pointing-Pointer PrP{sup C} prevents the cell death independently of an SOD-like activity. -- Abstract: The PrP{sup C} protein, which is especially present in the cellular membrane of nervous system cells, has been extensively studied for its controversial antioxidant activity. In this study, we elucidated the free radical scavenger activity of purified murine PrP{sup C} in solution and its participation as a cell protector in astrocytes that were subjected to treatment with an oxidant. In vitro and using an EPR spin-trapping technique, we observed that PrP{sup C} decreased the oxidation of the DMPO trap in a Fenton reaction system (Cu{sup 2+}/ascorbate/H{sub 2}O{sub 2}), which was demonstrated by approximately 70% less DMPO/OH{sup {center_dot}}. In cultured PrP{sup C}-knockout astrocytes from mice, the absence of PrP{sup C} caused an increase in intracellular ROS (reactive oxygen species) generation during the first 3 h of H{sub 2}O{sub 2} treatment. This rapid increase in ROS disrupted the cell cycle in the PrP{sup C}-knockout astrocytes, which increased the population of cells in the sub-G1 phase when compared with cultured wild-type astrocytes. We conclude that PrP{sup C} in solution acts as a radical scavenger, and in astrocytes, it is essential for protection from oxidative stress caused by an external chemical agent, which is a likely condition in human neurodegenerative CNS disorders and pathological conditions such as ischemia.

  8. Peptide gH625 enters into neuron and astrocyte cell lines and crosses the blood–brain barrier in rats

    Directory of Open Access Journals (Sweden)

    Valiante S

    2015-03-01

    Full Text Available Salvatore Valiante,1,* Annarita Falanga,2,3,* Luisa Cigliano,1 Giuseppina Iachetta,1 Rosa Anna Busiello,1 Valeria La Marca,1 Massimiliano Galdiero,4 Assunta Lombardi,1 Stefania Galdiero1,2 1Department of Biology, 2Department of Pharmacy, 3DFM Scarl, University of Naples Federico II, 4Department of Experimental Medicine, II University of Naples, Naples, Italy *These authors contributed equally to this paper and are considered joint first authors Abstract: Peptide gH625, derived from glycoprotein H of herpes simplex virus type 1, can enter cells efficiently and deliver a cargo. Nanoparticles armed with gH625 are able to cross an in vitro model of the blood–brain barrier (BBB. In the present study, in vitro experiments were performed to investigate whether gH625 can enter and accumulate in neuron and astrocyte cell lines. The ability of gH625 to cross the BBB in vivo was also evaluated. gH625 was administered in vivo to rats and its presence in the liver and in the brain was detected. Within 3.5 hours of intravenous administration, gH625 can be found beyond the BBB in proximity to cell neurites. gH625 has no toxic effects in vivo, since it does not affect the maximal oxidative capacity of the brain or the mitochondrial respiration rate. Our data suggest that gH625, with its ability to cross the BBB, represents a novel nanocarrier system for drug delivery to the central nervous system. These results open up new possibilities for direct delivery of drugs into patients in the field of theranostics and might address the treatment of several human diseases. Keywords: drug delivery, neurons, astrocytes, blood–brain barrier, peptide

  9. Peptide gH625 enters into neuron and astrocyte cell lines and crosses the blood–brain barrier in rats

    Science.gov (United States)

    Valiante, Salvatore; Falanga, Annarita; Cigliano, Luisa; Iachetta, Giuseppina; Busiello, Rosa Anna; La Marca, Valeria; Galdiero, Massimiliano; Lombardi, Assunta; Galdiero, Stefania

    2015-01-01

    Peptide gH625, derived from glycoprotein H of herpes simplex virus type 1, can enter cells efficiently and deliver a cargo. Nanoparticles armed with gH625 are able to cross an in vitro model of the blood–brain barrier (BBB). In the present study, in vitro experiments were performed to investigate whether gH625 can enter and accumulate in neuron and astrocyte cell lines. The ability of gH625 to cross the BBB in vivo was also evaluated. gH625 was administered in vivo to rats and its presence in the liver and in the brain was detected. Within 3.5 hours of intravenous administration, gH625 can be found beyond the BBB in proximity to cell neurites. gH625 has no toxic effects in vivo, since it does not affect the maximal oxidative capacity of the brain or the mitochondrial respiration rate. Our data suggest that gH625, with its ability to cross the BBB, represents a novel nanocarrier system for drug delivery to the central nervous system. These results open up new possibilities for direct delivery of drugs into patients in the field of theranostics and might address the treatment of several human diseases. PMID:25792823

  10. Peptide gH625 enters into neuron and astrocyte cell lines and crosses the blood-brain barrier in rats.

    Science.gov (United States)

    Valiante, Salvatore; Falanga, Annarita; Cigliano, Luisa; Iachetta, Giuseppina; Busiello, Rosa Anna; La Marca, Valeria; Galdiero, Massimiliano; Lombardi, Assunta; Galdiero, Stefania

    2015-01-01

    Peptide gH625, derived from glycoprotein H of herpes simplex virus type 1, can enter cells efficiently and deliver a cargo. Nanoparticles armed with gH625 are able to cross an in vitro model of the blood-brain barrier (BBB). In the present study, in vitro experiments were performed to investigate whether gH625 can enter and accumulate in neuron and astrocyte cell lines. The ability of gH625 to cross the BBB in vivo was also evaluated. gH625 was administered in vivo to rats and its presence in the liver and in the brain was detected. Within 3.5 hours of intravenous administration, gH625 can be found beyond the BBB in proximity to cell neurites. gH625 has no toxic effects in vivo, since it does not affect the maximal oxidative capacity of the brain or the mitochondrial respiration rate. Our data suggest that gH625, with its ability to cross the BBB, represents a novel nanocarrier system for drug delivery to the central nervous system. These results open up new possibilities for direct delivery of drugs into patients in the field of theranostics and might address the treatment of several human diseases.

  11. 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

  12. 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

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

    Science.gov (United States)

    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.

  14. 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.

  15. 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.

    Science.gov (United States)

    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.

  16. An experimental study on astrocytes promoting production of neural stem cells derived from mouse embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    ZHOU Yu-feng; FANG Feng; FU Jin-rong; DONG Yong-sui; YE Du-yun; SHU Sai-nan; ZHEN Hong; LI Ge

    2005-01-01

    Background The production of neural stem cells (NSCs) derived from embryonic stem (ES) cells was usually very low according to previous studies, which was a major obstacle for meeting the needs of clinical application. This study aimed at investigating whether astrocytes could promote production of NSCs derived from ES cells in vitro.Methods Mouse ES cells line-D3 was used to differentiate into NSCs with astrocytes as inducing stromal cells by means of three-stage differentiation procedure. Another group without astrocytes served as control. The totipotency of ES cells was identified by observation of cells' morphology and formation of teratoma in severe combined immunodeficiency disease (SCID) mice. The quantity and purity of NSCs derived from ES cells were analyzed using clonogenic assay, immunohistochemical staining and flow cytometry assay. The plasticity of NSCs was detected by differentiating test. Octamer-binding transcription factor 4 (Oct-4) and nestin, the specific marker genes of ES cells and NSCs respectively, were detected continuously using reverse transcription-polymerase chain reaction (RT-PCR) method to monitor the process of cell differentiation. Results The ES cells of D3 line could maintain the ability of differentiating into cellular derivations of all three primary germ layers after continuous passage culture. At the end of two-stage of inducing process, 23.2±3.5 neurospheres per plate formed in astrocyte-induced group and only 0.8±0.3 per plate in the control group (clonogenic assay, P<0.01), and the ratio of nestin positive cells was (50.2±2.8)% in astrocyte-induced group and only (1.4±0.5)% in the control group (flow cytometry, P<0.01). With the induction undergoing, the expression of Oct-4 gradually decreased and then disappeared, while the expression of nestin was increased step by step, and the ratio of nestin positive cells was up to 91.4% by the three-stage differentiation. The nestin positive cells could be further induced into

  17. Evidence for heterogeneity of astrocyte de-differentiation in vitro: astrocytes transform into intermediate precursor cells following induction of ACM from scratch-insulted astrocytes.

    Science.gov (United States)

    Yang, Hao; Qian, Xin-Hong; Cong, Rui; Li, Jing-wen; Yao, Qin; Jiao, Xi-Ying; Ju, Gong; You, Si-Wei

    2010-04-01

    Our previous study definitely demonstrated that the mature astrocytes could undergo a de-differentiation process and further transform into pluripotential neural stem cells (NSCs), which might well arise from the effect of diffusible factors released from scratch-insulted astrocytes. However, these neurospheres passaged from one neurosphere-derived from de-differentiated astrocytes possessed a completely distinct characteristic in the differentiation behavior, namely heterogeneity of differentiation. The heterogeneity in cell differentiation has become a crucial but elusive issue. In this study, we show that purified astrocytes could de-differentiate into intermediate precursor cells (IPCs) with addition of scratch-insulted astrocyte-conditioned medium (ACM) to the culture, which can express NG2 and A2B5, the IPCs markers. Apart from the number of NG2(+) and A2B5(+) cells, the percentage of proliferative cells as labeled with BrdU progressively increased with prolonged culture period ranging from 1 to 10 days. Meanwhile, the protein level of A2B5 in cells also increased significantly. These results revealed that not all astrocytes could de-differentiate fully into NSCs directly when induced by ACM, rather they generated intermediate or more restricted precursor cells that might undergo progressive de-differentiation to generate NSCs.

  18. Direct Signaling from Astrocytes to Neurons in Cultures of Mammalian Brain Cells

    Science.gov (United States)

    Nedergaard, Maiken

    1994-03-01

    Although astrocytes have been considered to be supportive, rather than transmissive, in the adult nervous system, recent studies have challenged this assumption by demonstrating that astrocytes possess functional neurotransmitter receptors. Astrocytes are now shown to directly modulate the free cytosolic calcium, and hence transmission characteristics, of neighboring neurons. When a focal electric field potential was applied to single astrocytes in mixed cultures of rat forebrain astrocytes and neurons, a prompt elevation of calcium occurred in the target cell. This in turn triggered a wave of calcium increase, which propagated from astrocyte to astrocyte. Neurons resting on these astrocytes responded with large increases in their concentration of cytosolic calcium. The gap junction blocker octanol attenuated the neuronal response, which suggests that the astrocytic-neuronal signaling is mediated through intercellular connections rather than synaptically. This neuronal response to local astrocytic stimulation may mediate local intercellular communication within the brain.

  19. 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...

  20. 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.

  1. Ischemic brain cell-derived conditioned medium protects astrocytes against ischemia through GDNF/ERK/NF-kB signaling pathway.

    Science.gov (United States)

    Chu, Lan-Feng; Wang, Wei-Ti; Ghanta, Vithal K; Lin, Chi-Hsin; Chiang, Yung-Yen; Hsueh, Chi-Mei

    2008-11-06

    Conditioned medium (CM) collected from cultures of ischemic microglia, astrocytes, and neurons were protective to astrocytes under the in vitro ischemic condition (deprivation of oxygen, glucose and serum). Molecular and signaling pathway(s) responsible for the CMs protective activity were investigated. Results showed that CMs from the ischemic microglia (MCM), astrocytes (ACM) and neurons (NCM) contained glial cell line-derived neurotrophic factor (GDNF), which protects astrocytes against the in vitro ischemia. Expression of extra cellular signal-regulated kinase (ERK1/2) and nuclear factor-kappa B (NF-kB) by GDNF led to the inhibition of apoptosis of the ischemic astrocytes in a caspase 3-independent manner. However, CMs- and GDNF-mediated protection of the ischemic astrocytes was protein kinase B (Akt) independent. These results provided mechanistic data regarding how GDNF- and CMs-mediated protection of the ischemic astrocytes is taking place. These observations provide information for the use of GDNF and GDNF containing CMs in the control of cerebral ischemia.

  2. Novel cell separation method for molecular analysis of neuron-astrocyte cocultures

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    Andrea eGoudriaan

    2014-01-01

    Full Text Available Over the last decade, the importance of astrocyte-neuron communication in neuronal development and synaptic plasticity has become increasingly clear. Since neuron-astrocyte interactions represent highly dynamic and reciprocal processes, we hypothesized that many astrocyte genes may be regulated as a consequence of their interactions with maturing neurons. In order to identify such neuron-responsive astrocyte genes in vitro, we sought to establish an expedite technique for separation of neurons from co-cultured astrocytes. Our newly established method makes use of cold jet, which exploits different adhesion characteristics of subpopulations of cells (Jirsova et al., 1997, and is rapid, performed under ice-cold conditions and avoids protease-mediated isolation of astrocytes or time-consuming centrifugation, yielding intact astrocyte mRNA with approximately 90% of neuronal RNA removed. Using this purification method, we executed genome-wide profiling in which RNA derived from astrocyte-only cultures was compared with astrocyte RNA derived from differentiating neuron-astrocyte co-cultures. Data analysis determined that many astrocytic mRNAs and biological processes are regulated by neuronal interaction. Our results validate the cold jet as an efficient method to separate astrocytes from neurons in co-culture, and reveals that neurons induce robust gene-expression changes in co-cultured astrocytes.

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

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    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.

  4. 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...

  5. "Cell therapy for stroke: use of local astrocytes"

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    Melek eChouchane

    2012-10-01

    Full Text Available Stroke refers to a variety of conditions caused by the occlusion or hemorrhage of blood vessels supplying the brain, which is one of the main causes of death and the leading cause of disability worldwide. In the last years, cell-based therapies have been proposed as a new approach to ameliorate post stroke deficits. However, the most appropriate type of cell to be used in such therapies, as well as their sources, remains a matter of intense research. A good candidate cell should, in principle, display high plasticity to generate diverse types of neurons and, at the same type, low risk to cause undesired outcomes, such as malignant transformation. Recently, a new approach grounded on the reprogramming of endogenous astrocytes towards neuronal fates emerged as an alternative to restore neurological functions in several central nervous system diseases. In this perspective, we review data about the potential of astrocytes to become functional neurons following expression of neurogenic genes and discuss the potential benefits and risks of reprogramming astrocytes in the glial scar to replace neurons lost after stroke.

  6. GABAA increases calcium in subventricular zone astrocyte-like cells through L- and T-type voltage-gated calcium channels

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    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.

  7. GDNF reverses the inhibitory properties of reactive astrocytes allowing robust axonal regeneration through Schwann cell-seeded guidance channels after spinal cord injury

    OpenAIRE

    Deng, Ling-Xiao; Hu, Jianguo; Liu, Naikui; Wang, Xiaofei; Smith, George M.; Wen, Xuejun; Xu, Xiao-Ming

    2011-01-01

    Reactive astrogliosis has been considered as a major impediment for axonal regeneration after injuries in the mammalian central nervous system (CNS). Here we report that glial cell line-derived neurotrophic factor (GDNF), combined with transplanted Schwann cells (SCs), effectively reversed the inhibitory properties of astrocytes at graft-host interfaces allowing robust axonal regeneration, concomitant with vigorous migration of host astrocytes, into SC-seeded semi-permeable guidance channels ...

  8. 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.

  9. GDNF facilitates differentiation of the adult dentate gyrus-derived neural precursor cells into astrocytes via STAT3

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    Boku, Shuken, E-mail: shuboku@med.hokudai.ac.jp [Department of Psychiatry, Hokkaido University Graduate School of Medicine, Sapporo (Japan); Nakagawa, Shin [Department of Psychiatry, Hokkaido University Graduate School of Medicine, Sapporo (Japan); Takamura, Naoki [Pharmaceutical Laboratories, Dainippon Sumitomo Pharma Co. Ltd., Osaka (Japan); Kato, Akiko [Department of Psychiatry, Hokkaido University Graduate School of Medicine, Sapporo (Japan); Takebayashi, Minoru [Department of Psychiatry, National Hospital Organization Kure Medical Center, Kure (Japan); Hisaoka-Nakashima, Kazue [Department of Pharmacology, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima (Japan); Omiya, Yuki; Inoue, Takeshi; Kusumi, Ichiro [Department of Psychiatry, Hokkaido University Graduate School of Medicine, Sapporo (Japan)

    2013-05-17

    Highlights: •GDNF has no effect on ADP proliferation and apoptosis. •GDNF increases ADP differentiation into astrocyte. •A specific inhibitor of STAT3 decreases the astrogliogenic effect of GDNF. •STAT3 knockdown by lentiviral shRNA vector also decreases the astrogliogenic effect of GDNF. •GDNF increases the phosphorylation of STAT3. -- Abstract: While the pro-neurogenic actions of antidepressants in the adult hippocampal dentate gyrus (DG) are thought to be one of the mechanisms through which antidepressants exert their therapeutic actions, antidepressants do not increase proliferation of neural precursor cells derived from the adult DG. Because previous studies showed that antidepressants increase the expression and secretion of glial cell line-derived neurotrophic factor (GDNF) in C6 glioma cells derived from rat astrocytes and GDNF increases neurogenesis in adult DG in vivo, we investigated the effects of GDNF on the proliferation, differentiation and apoptosis of cultured neural precursor cells derived from the adult DG. Data showed that GDNF facilitated the differentiation of neural precursor cells into astrocytes but had no effect on their proliferation or apoptosis. Moreover, GDNF increased the phosphorylation of STAT3, and both a specific inhibitor of STAT3 and lentiviral shRNA for STAT3 decreased their differentiation into astrocytes. Taken together, our findings suggest that GDNF facilitates astrogliogenesis from neural precursor cells in adult DG through activating STAT3 and that this action might indirectly affect neurogenesis.

  10. Astrocytes and Müller cells changes during retinal degeneration in a transgenic rat model of retinitis pigmentosa.

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    Laura eFernández-Sánchez

    2015-12-01

    Full Text Available Purpose: Retinitis pigmentosa includes a group of progressive retinal degenerative diseases that affect the structure and function of photoreceptors. Secondarily to the loss of photoreceptors, there is a reduction in retinal vascularization, which seems to influence the cellular degenerative process. Retinal macroglial cells, astrocytes and Müller cells provide support for retinal neurons and are fundamental for maintaining normal retinal function. The aim of this study was to investigate the evolution of macroglial changes during retinal degeneration in P23H rats. Methods: Homozygous P23H line-3 rats aged from P18 to 18 months were used to study the evolution of the disease, and SD rats were used as controls. Immunolabeling with antibodies against GFAP, vimentin, and transducin were used to visualize macroglial cells and cone photoreceptors. Results: In P23H rats, increased GFAP labeling in Müller cells was observed as an early indicator of retinal gliosis. At 4 and 12 months of age, the apical processes of Müller cells in P23H rats clustered in firework-like structures, which were associated with ring-like shaped areas of cone degeneration in the outer nuclear layer. These structures were not observed at 16 months of age. The number of astrocytes was higher in P23H rats than in the SD matched controls at 4 and 12 months of age, supporting the idea of astrocyte proliferation. As the disease progressed, astrocytes exhibited a deteriorated morphology and marked hypertrophy. The increase in the complexity of the astrocytic processes correlated with greater connexin 43 expression and higher density of connexin 43 immunoreactive puncta within the ganglion cell layer of P23H versus SD rat retinas. Conclusions: In the P23H rat model of retinitis pigmentosa, the loss of photoreceptors triggers major changes in the number and morphology of glial cells affecting the inner retina.

  11. Nanoscale Properties of Neural Cell Prosthetic and Astrocyte Response

    Science.gov (United States)

    Flowers, D. A.; Ayres, V. M.; Delgado-Rivera, R.; Ahmed, I.; Meiners, S. A.

    2009-03-01

    Preliminary data from in-vivo investigations (rat model) suggest that a nanofiber prosthetic device of fibroblast growth factor-2 (FGF-2)-modified nanofibers can correctly guide regenerating axons across an injury gap with aligned functional recovery. Scanning Probe Recognition Microscopy (SPRM) with auto-tracking of individual nanofibers is used for investigation of the key nanoscale properties of the nanofiber prosthetic device for central nervous system tissue engineering and repair. The key properties under SPRM investigation include nanofiber stiffness and surface roughness, nanofiber curvature, nanofiber mesh density and porosity, and growth factor presentation and distribution. Each of these factors has been demonstrated to have global effects on cell morphology, function, proliferation, morphogenesis, migration, and differentiation. The effect of FGF-2 modification on the key nanoscale properties is investigated. Results from the nanofiber prosthetic properties investigations are correlated with astrocyte response to unmodified and FGF-2 modified scaffolds, using 2D planar substrates as a control.

  12. [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.

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

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    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.

  14. Astrocyte-like glial cells physiologically regulate olfactory processing through the modification of ORN-PN synaptic strength in Drosophila.

    Science.gov (United States)

    Liu, He; Zhou, Bangyu; Yan, Wenjun; Lei, Zhengchang; Zhao, Xiaoliang; Zhang, Ke; Guo, Aike

    2014-09-01

    Astrocyte-like glial cells are abundant in the central nervous system of adult Drosophila and exhibit morphology similar to astrocytes of mammals. Previous evidence has shown that astrocyte-like glial cells are strongly associated with synapses in the antennal lobe (AL), the first relay of the olfactory system, where olfactory receptor neurons (ORNs) transmit information into projection neurons (PNs). However, the function of astrocyte-like glia in the AL remains obscure. In this study, using in vivo calcium imaging, we found that astrocyte-like glial cells exhibited spontaneous microdomain calcium elevations. Using simultaneous manipulation of glial activity and monitoring of neuronal function, we found that the astrocyte-like glial activation, but not ensheathing glial activation, could inhibit odor-evoked responses of PNs. Ensheathing glial cells are another subtype of glia, and are of functional importance in the AL. Electrophysiological experiments indicated that astrocyte-like glial activation decreased the amplitude and slope of excitatory postsynaptic potentials evoked through electrical stimulation of the antennal nerve. These results suggest that astrocyte-like glial cells may regulate olfactory processing through negative regulation of ORN-PN synaptic strength. Beyond the antennal lobe we observed astrocyte-like glial spontaneous calcium activities in the ventromedial protocerebrum, indicating that astrocyte-like glial spontaneous calcium elevations might be general in the adult fly brain. Overall, our study demonstrates a new function for astrocyte-like glial cells in the physiological modulation of olfactory information transmission, possibly through regulating ORN-PN synapse strength.

  15. 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

  16. 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.

  17. Modeling astrocytoma pathogenesis in vitro and in vivo using cortical astrocytes or neural stem cells from conditional, genetically engineered mice.

    Science.gov (United States)

    McNeill, Robert S; Schmid, Ralf S; Bash, Ryan E; Vitucci, Mark; White, Kristen K; Werneke, Andrea M; Constance, Brian H; Huff, Byron; Miller, C Ryan

    2014-08-12

    Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

  18. Heterogeneity of astrocytes: from development to injury - single cell gene expression.

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    Vendula Rusnakova

    Full Text Available Astrocytes perform control and regulatory functions in the central nervous system; heterogeneity among them is still a matter of debate due to limited knowledge of their gene expression profiles and functional diversity. To unravel astrocyte heterogeneity during postnatal development and after focal cerebral ischemia, we employed single-cell gene expression profiling in acutely isolated cortical GFAP/EGFP-positive cells. Using a microfluidic qPCR platform, we profiled 47 genes encoding glial markers and ion channels/transporters/receptors participating in maintaining K(+ and glutamate homeostasis per cell. Self-organizing maps and principal component analyses revealed three subpopulations within 10-50 days of postnatal development (P10-P50. The first subpopulation, mainly immature glia from P10, was characterized by high transcriptional activity of all studied genes, including polydendrocytic markers. The second subpopulation (mostly from P20 was characterized by low gene transcript levels, while the third subpopulation encompassed mature astrocytes (mainly from P30, P50. Within 14 days after ischemia (D3, D7, D14, additional astrocytic subpopulations were identified: resting glia (mostly from P50 and D3, transcriptionally active early reactive glia (mainly from D7 and permanent reactive glia (solely from D14. Following focal cerebral ischemia, reactive astrocytes underwent pronounced changes in the expression of aquaporins, nonspecific cationic and potassium channels, glutamate receptors and reactive astrocyte markers.

  19. 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.

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

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    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  2. 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.

  3. 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.

  4. Non-cell autonomous influence of the astrocyte system xc− on hypoglycaemic neuronal cell death

    Directory of Open Access Journals (Sweden)

    Sandra J Hewett

    2012-02-01

    Full Text Available Despite longstanding evidence that hypoglycaemic neuronal injury is mediated by glutamate excitotoxicity, the cellular and molecular mechanisms involved remain incompletely defined. Here, we demonstrate that the excitotoxic neuronal death that follows GD (glucose deprivation is initiated by glutamate extruded from astrocytes via system xc− – an amino acid transporter that imports l-cystine and exports l-glutamate. Specifically, we find that depriving mixed cortical cell cultures of glucose for up to 8 h injures neurons, but not astrocytes. Neuronal death is prevented by ionotropic glutamate receptor antagonism and is partially sensitive to tetanus toxin. Removal of amino acids during the deprivation period prevents – whereas addition of l-cystine restores – GD-induced neuronal death, implicating the cystine/glutamate antiporter, system xc−. Indeed, drugs known to inhibit system xc− ameliorate GD-induced neuronal death. Further, a dramatic reduction in neuronal death is observed in chimaeric cultures consisting of neurons derived from WT (wild-type mice plated on top of astrocytes derived from sut mice, which harbour a naturally occurring null mutation in the gene (Slc7a11 that encodes the substrate-specific light chain of system xc− (xCT. Finally, enhancement of astrocytic system xc− expression and function via IL-1β (interleukin-1β exposure potentiates hypoglycaemic neuronal death, the process of which is prevented by removal of l-cystine and/or addition of system xc− inhibitors. Thus, under the conditions of GD, our studies demonstrate that astrocytes, via system xc−, have a direct, non-cell autonomous effect on cortical neuron survival.

  5. Non-cell autonomous influence of the astrocyte system xc- on hypoglycaemic neuronal cell death.

    Science.gov (United States)

    Jackman, Nicole A; Melchior, Shannon E; Hewett, James A; Hewett, Sandra J

    2012-02-08

    Despite longstanding evidence that hypoglycaemic neuronal injury is mediated by glutamate excitotoxicity, the cellular and molecular mechanisms involved remain incompletely defined. Here, we demonstrate that the excitotoxic neuronal death that follows GD (glucose deprivation) is initiated by glutamate extruded from astrocytes via system xc---an amino acid transporter that imports L-cystine and exports L-glutamate. Specifically, we find that depriving mixed cortical cell cultures of glucose for up to 8 h injures neurons, but not astrocytes. Neuronal death is prevented by ionotropic glutamate receptor antagonism and is partially sensitive to tetanus toxin. Removal of amino acids during the deprivation period prevents--whereas addition of L-cystine restores--GD-induced neuronal death, implicating the cystine/glutamate antiporter, system xc-. Indeed, drugs known to inhibit system xc- ameliorate GD-induced neuronal death. Further, a dramatic reduction in neuronal death is observed in chimaeric cultures consisting of neurons derived from WT (wild-type) mice plated on top of astrocytes derived from sut mice, which harbour a naturally occurring null mutation in the gene (Slc7a11) that encodes the substrate-specific light chain of system xc- (xCT). Finally, enhancement of astrocytic system xc- expression and function via IL-1β (interleukin-1β) exposure potentiates hypoglycaemic neuronal death, the process of which is prevented by removal of l-cystine and/or addition of system xc- inhibitors. Thus, under the conditions of GD, our studies demonstrate that astrocytes, via system xc-, have a direct, non-cell autonomous effect on cortical neuron survival.

  6. Non-Cell Autonomous Influence of the Astrocyte System xc − on Hypoglycaemic Neuronal Cell Death

    Directory of Open Access Journals (Sweden)

    Nicole A Jackman

    2012-01-01

    Full Text Available Despite longstanding evidence that hypoglycaemic neuronal injury is mediated by glutamate excitotoxicity, the cellular and molecular mechanisms involved remain incompletely defined. Here, we demonstrate that the excitotoxic neuronal death that follows GD (glucose deprivation is initiated by glutamate extruded from astrocytes via system xc −– – an amino acid transporter that imports L-cystine and exports L-glutamate. Specifically, we find that depriving mixed cortical cell cultures of glucose for up to 8 h injures neurons, but not astrocytes. Neuronal death is prevented by ionotropic glutamate receptor antagonism and is partially sensitive to tetanus toxin. Removal of amino acids during the deprivation period prevents – whereas addition of L-cystine restores – GD-induced neuronal death, implicating the cystine/glutamate antiporter, system xc−–. Indeed, drugs known to inhibit system xc −– ameliorate GD-induced neuronal death. Further, a dramatic reduction in neuronal death is observed in chimaeric cultures consisting of neurons derived from WT (wild-type mice plated on top of astrocytes derived from sut mice, which harbour a naturally occurring null mutation in the gene (Slc7a11 that encodes the substrate-specific light chain of system xc −– (xCT. Finally, enhancement of astrocytic system xc −– expression and function via IL-1β (interleukin-1β exposure potentiates hypoglycaemic neuronal death, the process of which is prevented by removal of L-cystine and/or addition of system xc −– inhibitors. Thus, under the conditions of GD, our studies demonstrate that astrocytes, via system xc −–, have a direct, non-cell autonomous effect on cortical neuron survival.

  7. Quantification of Metabolic Rearrangements During Neural Stem Cells Differentiation into Astrocytes by Metabolic Flux Analysis.

    Science.gov (United States)

    Sá, João V; Kleiderman, Susanne; Brito, Catarina; Sonnewald, Ursula; Leist, Marcel; Teixeira, Ana P; Alves, Paula M

    2017-01-01

    Proliferation and differentiation of neural stem cells (NSCs) have a crucial role to ensure neurogenesis and gliogenesis in the mammalian brain throughout life. As there is growing evidence for the significance of metabolism in regulating cell fate, knowledge on the metabolic programs in NSCs and how they evolve during differentiation into somatic cells may provide novel therapeutic approaches to address brain diseases. In this work, we applied a quantitative analysis to assess how the central carbon metabolism evolves upon differentiation of NSCs into astrocytes. Murine embryonic stem cell (mESC)-derived NSCs and astrocytes were incubated with labelled [1-(13)C]glucose and the label incorporation into intracellular metabolites was followed by GC-MS. The obtained (13)C labelling patterns, together with uptake/secretion rates determined from supernatant analysis, were integrated into an isotopic non-stationary metabolic flux analysis ((13)C-MFA) model to estimate intracellular flux maps. Significant metabolic differences between NSCs and astrocytes were identified, with a general downregulation of central carbon metabolism during astrocytic differentiation. While glucose uptake was 1.7-fold higher in NSCs (on a per cell basis), a high lactate-secreting phenotype was common to both cell types. Furthermore, NSCs consumed glutamine from the medium; the highly active reductive carboxylation of alpha-ketoglutarate indicates that this was converted to citrate and used for biosynthetic purposes. In astrocytes, pyruvate entered the TCA cycle mostly through pyruvate carboxylase (81%). This pathway supported glutamine and citrate secretion, recapitulating well described metabolic features of these cells in vivo. Overall, this fluxomics study allowed us to quantify the metabolic rewiring accompanying astrocytic lineage specification from NSCs.

  8. Cell death pathways in astrocytes with a modified model of oxygen-glucose deprivation.

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    Qiaoying Huang

    Full Text Available Traditional oxygen-glucose deprivation (OGD models do not produce sufficiently stable and continuous deprivation to induce cell death in the ischemic core. Therefore, we modified the OGD model to mimic the observed damage in the ischemic core following stroke and utilized this new model to study cell death pathways in astrocytes. The PO2 and pH levels in the astrocyte culture medium were compared between a physical OGD group, a chemical OGD group and a mixed OGD group. The mixed OGD group was able to maintain anaerobic conditions in astrocyte culture medium for 6 h, while the physical and the chemical groups failed to maintain such conditions. Astrocyte viability decreased and LDH release into in the medium increased as a function of exposure to OGD. Compared to the control group, the expression of active caspase-3 in the mixed OGD group increased within 2 h after OGD, but decreased after 2 h of OGD. Additionally, porimin mRNA levels did not significantly increase during the first 2 h of OGD, while bcl-2 mRNA levels decreased at 1 h. However, both porimin and bcl-2 mRNA levels increased after 2 h of OGD; interestingly, they both suddenly decreased at 4 h of OGD. Taken together, these results indicate that apoptosis and oncosis are the two cell death pathways responsible for astrocyte death in the ischemic core. However, the main death pathway varies depending on the OGD period.

  9. 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.

  10. Death and survival of neuronal and astrocytic cells in ischemic brain injury: a role of autophagy

    Institute of Scientific and Technical Information of China (English)

    Min XU; Hui-ling ZHANG

    2011-01-01

    Autophagy is a highly regulated cellular mechanism that leads to degradation of long-lived proteins and dysfunctional organelles. The process has been implicated in a variety of physiological and pathological conditions relevant to neurological diseases. Recent studies show the existence of autophagy in cerebral ischemia, but no consensus has yet been reached regarding the functions of autophagy in this condition. This article highlights the activation of autophagy during cerebral ischemia and/or reperfusion, especially in neurons and astrocytes, as well as the role of autophagy in neuronal or astrocytic cell death and survival. We propose that physiological levels of autophagy, presumably caused by mild to modest hypoxia or ischemia, appear to be protective. However, high levels of autophagy caused by severe hypoxia or ischemia and/or reperfusion may cause self-digestion and eventual neuronal and astrocytic cell death. We also discuss that oxidative and endoplasmic reticulum (ER) stresses in cerebral hypoxia or ischemia and/or reperfusion are potent stimuli of autophagy in neurons and astrocytes. In addition, we review the evidence suggesting a considerable overlap between autophagy on one hand, and apoptosis, necrosis and necroptosis on the other hand, in determining the outcomes and final morphology of damaged neurons and astrocytes.

  11. Reactive Astrocytes: Phenotypic and Functional Characteristics and Astrocytes as Neural Stem Cells

    Science.gov (United States)

    2006-01-01

    proteins, and contained inclusion bodies identical both histologically and antigenically to the Rosenthal fibers of Alexander’s disease (Messing et al...observations. The most notable were from Koelliker, His, and Ramón y Cajal (Koelliker, 1896; His, 1904; Ramón y Cajal , 1999; Gross, 2000). Using...layer and into the molecular layer with the cell body generally located in the granule cell layer. EM evaluation of type 1 cells showed vascular end

  12. Purinergic P2X7 receptors mediate cell death in mouse cerebellar astrocytes in culture.

    Science.gov (United States)

    Salas, Elvira; Carrasquero, Luz María G; Olivos-Oré, Luis A; Bustillo, Diego; Artalejo, Antonio R; Miras-Portugal, Maria Teresa; Delicado, Esmerilda G

    2013-12-01

    The brain distribution and functional role of glial P2X7 receptors are broader and more complex than initially anticipated. We characterized P2X7 receptors from cerebellar astrocytes at the molecular, immunocytochemical, biophysical, and cell physiologic levels. Mouse cerebellar astrocytes in culture express mRNA coding for P2X7 receptors, which is translated into P2X7 receptor protein as proven by Western blot analysis and immunocytochemistry. Fura-2 imaging showed cytosolic calcium responses to ATP and the synthetic analog 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) exhibited two components, namely an initial transient and metabotropic component followed by a sustained one that depended on extracellular calcium. This latter component, which was absent in astrocytes from P2X7 receptor knockout mice (P2X7 KO), was modulated by extracellular Mg(2+), and was sensitive to Brilliant Blue G (BBG) and 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine (A438079) antagonism. BzATP also elicited inwardly directed nondesensitizing whole-cell ionic currents that were reduced by extracellular Mg(2+) and P2X7 antagonists (BBG and calmidazolium). In contrast to that previously reported in rat cerebellar astrocytes, sustained BzATP application induced a gradual increase in membrane permeability to large cations, such as N-methyl-d-glucamine and 4-[3-methyl-2(3H)-benzoxazolylidene)-methyl]-1-[3-(triethylammonio)propyl]diiodide, which ultimately led to the death of mouse astrocytes. Cerebellar astrocyte cell death was prevented by BBG but not by calmidazolium, removal of extracellular calcium, or treatment with the caspase-3 inhibitor, benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone, thus suggesting a necrotic-type mechanism of cell death. Since this cellular response was not observed in astrocytes from P2X7 KO mice, this study suggests that stimulation of P2X7 receptor may convey a cell death signal to cerebellar astrocytes in a species-specific manner.

  13. MK-801 treatment affects glycolysis in oligodendrocytes more than in astrocytes and neuronal cells: insights for schizophrenia

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    Paul C Guest

    2015-05-01

    Full Text Available As a multifactorial disease, the underlying causes of schizophrenia require analysis by multiplex methods such as proteomics to allow identification of whole protein networks. Previous post-mortem proteomic studies on brain tissues from schizophrenia patients have demonstrated changes in activation of glycolytic and energy metabolism pathways. However, it is not known whether these changes occur in neurons or in glial cells. To address this question, we treated neuronal, astrocyte and oligodendrocyte cell lines with the NMDA receptor antagonist MK-801 and measured the levels of six glycolytic enzymes by Western blot analysis. MK-801 acts on the glutamatergic system and has been proposed as a pharmacological means of modeling schizophrenia. Treatment with MK-801 resulted in significant changes in the levels of glycolytic enzymes in all cell types. Most of the differences were found in oligodendrocytes, which had altered levels of hexokinase 1 (HK1, enolase 2 (ENO2, phosphoglycerate kinase (PGK and phosphoglycerate mutase 1 (PGAM1 after acute MK-801 treatment (8 hours, and HK1, ENO2, PGK and triosphosphate isomerase (TPI following long term treatment (72 hours. Addition of the antipsychotic clozapine to the cultures resulted in counter-regulatory effects to the MK-801 treatment by normalizing the levels of ENO2 and PGK in both the acute and long term cultures. In astrocytes, MK-801 affected only aldolase C (ALDOC under both acute conditions and HK1 and ALDOC following long term treatment, and TPI was the only enzyme affected under long term conditions in the neuronal cells. In conclusion, MK-801 affects glycolysis in oligodendrocytes to a larger extent than neuronal cells and this may be modulated by antipsychotic treatment. Although cell culture studies do not necessarily reflect the in vivo pathophysiology and drug effects within the brain, these results suggest that neurons, astrocytes and oligodendrocytes are affected differently in

  14. Storage of cell lines.

    Science.gov (United States)

    Parker, Katharine A

    2011-01-01

    The successful storage of cell lines depends upon many factors, including the condition of the cells to be frozen and the experience of the operator. Attempting to freeze down unhealthy, contaminated or poorly labelled cells can have huge implications for a research laboratory. This chapter outlines the importance of good record keeping, vigilant monitoring, aseptic technique, and high-quality reagents in the successful storage and downstream propagation of cell lines.

  15. 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.

  16. Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

    Energy Technology Data Exchange (ETDEWEB)

    Su, Xin; Guan, Wuqiang; Yu, Yong-Chun; Fu, Yinghui, E-mail: fuyh@fudan.edu.cn

    2014-07-18

    Highlights: • No new neurons and astrocytes are generated in adult mouse cerebellum. • Very few mash1{sup +} or nestin{sup +} stem cells exist, and most of them are quiescent. • Cell proliferation rate is diversified among cerebellar regions and decreases over time. - Abstract: Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU{sup +} cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU{sup +} cells, very few are mash1{sup +} or nestin{sup +} stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1{sup +} microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition.

  17. 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.

  18. Astrocytes Upregulate Survival Genes in Tumor Cells and Induce Protection from Chemotherapy

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    Sun-Jin Kim

    2011-03-01

    Full Text Available In the United States, more than 40% of cancer patients develop brain metastasis. The median survival for untreated patients is 1 to 2 months, which may be extended to 6 months with conventional radiotherapy and chemotherapy. The growth and survival of metastasis depend on the interaction of tumor cells with host factors in the organ microenvironment. Brain metastases are surrounded and infiltrated by activated astrocytes and are highly resistant to chemotherapy. We report here that coculture of human breast cancer cells or lung cancer cells with murine astrocytes (but not murine fibroblasts led to the up-regulation of survival genes, including GSTA5, BCL2L1, and TWIST1, in the tumor cells. The degree of up-regulation directly correlated with increased resistance to all tested chemotherapeutic agents. We further show that the up-regulation of the survival genes and consequent resistance are dependent on the direct contact between the astrocytes and tumor cells through gap junctions and are therefore transient. Knocking down these genes with specific small interfering RNA rendered the tumor cells sensitive to chemotherapeutic agents. These data clearly demonstrate that host cells in the microenvironment influence the biologic behavior of tumor cells and reinforce the contention that the organ microenvironment must be taken into consideration during the design of therapy.

  19. 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.

  20. 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.

  1. Hippocalcin Is Required for Astrocytic Differentiation through Activation of Stat3 in Hippocampal Neural Precursor Cells.

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    Min-Jeong Kang

    2016-10-01

    Full Text Available Hippocalcin (Hpca is a neuronal calcium sensor protein expressed in the mammalian brain. However, its function in neural stem/precursor cells has not yet been studied. Here, we clarify the function of Hpca in astrocytic differentiation in hippocampal neural precursor cells (HNPCs. When we overexpressed Hpca in HNPCs in the presence or absence of bFGF, expression levels of nerve-growth factors such as neurotrophin-3 (NT-3, neurotrophin-4/5 (NT-4/5 and brain-derived neurotrophic factor (BDNF, together with the proneural basic helix loop helix (bHLH transcription factors neuroD and neurogenin 1 (ngn1, increased significantly. In addition, there was an increase in the number of cells expressing glial fibrillary acidic protein (GFAP, an astrocyte marker, and in dendrite outgrowth, indicating astrocytic differentiation of the HNPCs. Downregulation of Hpca by transfection with Hpca siRNA reduced expression of NT-3, NT-4/5, BDNF, neuroD and ngn1 as well as levels of GFAP protein. Furthermore, overexpression of Hpca increased the phosphorylation of STAT3 (Ser727, and this effect was abolished by treatment with a STAT3 inhibitor (S3I-201, suggesting that STAT3 (Ser727 activation is involved in Hpca-mediated astrocytic differentiation. As expected, treatment with Stat3 siRNA or STAT3 inhibitor caused a complete inhibition of astrogliogenesis induced by Hpca overexpression. Taken together, this is the first report to show that Hpca, acting through Stat3, has an important role in the expression of neurotrophins and proneural bHLH transcription factors, and that it is an essential regulator of astrocytic differentiation and dendrite outgrowth in HNPCs.

  2. Oligodendroglioma cells shed microvesicles which contain TRAIL as well as molecular chaperones and induce cell death in astrocytes.

    Science.gov (United States)

    Lo Cicero, Alessandra; Schiera, Gabriella; Proia, Patrizia; Saladino, Patrizia; Savettieri, Giovanni; Di Liegro, Carlo Maria; Di Liegro, Italia

    2011-12-01

    Microvesicles (MVs) shed from G26/24 oligodendroglioma cells were previously reported to cause a reproducible, dose-dependent, inhibitory effect on neurite outgrowth, and eventually neuronal apoptosis, when added to primary cultures of rat cortical neurons. These effects were reduced but not abolished by functional monoclonal antibodies against Fas-L. In order to investigate whether MVs contain other factors able to induce cell death, we tested them for TRAIL and found clear evidence of its presence in the vesicles. This finding suggests the possibility that Fas-L and TRAIL cooperate in inducing brain cell death. Aimed at understanding the route through which the vesicles deliver their messages to the target cells, we labeled oligodendroglioma cells with radioactive methionine and then added the labeled vesicles shed from tumor cells to unlabeled astrocytes in culture. Here we report that labeled proteins were delivered to the test cells. In order to investigate whether astrocytes, like neurons, are sensitive to oligodendroglioma-derived vesicles, MVs were prepared from media conditioned by G26/24 oligodendroglioma cells and added to primary cultures of rat cortical astrocytes. These cells were clearly more resistant than neurons to microvesicle-induced damage: a high dose (40 µg) of shed MVs induced cell death in only about 40% of astrocytes. Finally, we demonstrated that Hsp70 is specifically enriched in MVs which also contain, even if at lower level, the Hsc70 constitutive chaperone.

  3. Use of RNAi silencing to target preconditioned glial cell line-derived neurotrophic factor in neuronal apoptosis

    Institute of Scientific and Technical Information of China (English)

    Hongliang Guo; Zhongxin Xu; Xinhua Li; Jing Mang; Ying Xing; Jinting He; Guihua Xu; Shijun Yan; Lifeng Liu; Chunli Mei

    2011-01-01

    Several studies have suggested that exogenous glial cell line-derived neurotrophic factor may protect neurons from cerebral ischemic injury. However, the mechanisms underlying the neuroprotective effects of endogenous glial cell line-derived neurotrophic factor remain unclear. The present experiments sought to elucidate the influence of various conditioned media on neuronal apoptosis, using a normal culture medium for astrocytes, an astrocyte medium highly expressing glial cell line-derived neurotrophic factor, and an astrocyte medium in which glial cell line-derived neurotrophic factor expression was silenced using RNAi technology. The results confirmed that the use of RNAi silencing to target pretreated glial cell line-derived neurotrophic factor expression promoted neuronal apoptosis. In addition, oxygen and glucose deprivation preconditioning was found to upregulate glial cell line-derived neurotrophic factor expression, and significantly reduce neuronal apoptosis.

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

    OpenAIRE

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

    2016-01-01

    Testosterone is a hormone that has been shown to confer neuroprotection from different insults affecting the central nervous system (CNS). Testosterone induces this protection by different mechanisms that include the activation of anti-apoptotic pathways that are directly implicated in neuronal survival. However, little attention has been devoted to its actions on glial cells. In the present study, we have assessed whether testosterone exerts protection in a human astrocyte cell model, the T9...

  5. 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

  6. The speed of swelling kinetics modulates cell volume regulation and calcium signaling in astrocytes: A different point of view on the role of aquaporins.

    Science.gov (United States)

    Mola, Maria Grazia; Sparaneo, Angelo; Gargano, Concetta Domenica; Spray, David C; Svelto, Maria; Frigeri, Antonio; Scemes, Eliana; Nicchia, Grazia Paola

    2016-01-01

    Regulatory volume decrease (RVD) is a process by which cells restore their original volume in response to swelling. In this study, we have focused on the role played by two different Aquaporins (AQPs), Aquaporin-4 (AQP4), and Aquaporin-1 (AQP1), in triggering RVD and in mediating calcium signaling in astrocytes under hypotonic stimulus. Using biophysical techniques to measure water flux through the plasma membrane of wild-type (WT) and AQP4 knockout (KO) astrocytes and of an astrocyte cell line (DI TNC1) transfected with AQP4 or AQP1, we here show that AQP-mediated fast swelling kinetics play a key role in triggering and accelerating RVD. Using calcium imaging, we show that AQP-mediated fast swelling kinetics also significantly increases the amplitude of calcium transients inhibited by Gadolinium and Ruthenium Red, two inhibitors of the transient receptor potential vanilloid 4 (TRPV4) channels, and prevented by removing extracellular calcium. Finally, inhibition of TRPV4 or removal of extracellular calcium does not affect RVD. All together our study provides evidence that (1) AQP influenced swelling kinetics is the main trigger for RVD and in mediating calcium signaling after hypotonic stimulus together with TRPV4, and (2) calcium influx from the extracellular space and/or TRPV4 are not essential for RVD to occur in astrocytes.

  7. Extracellular Microvesicles from Astrocytes Contain Functional Glutamate Transporters: Regulation by Protein Kinase C and Cell Activation

    Directory of Open Access Journals (Sweden)

    Romain-Daniel eGosselin

    2013-12-01

    Full Text Available Glutamate transport through astrocytic excitatory amino-acid transporters (EAAT-1 and EAAT-2 is paramount for neural homeostasis. EAAT-1 has been reported in secreted extracellular microvesicles (eMV, such as exosomes and because the Protein Kinase C (PKC family controls the sub-cellular distribution of EAATs, we have explored whether PKCs drive EAATs into eMV. Using rat primary astrocytes, confocal immunofluorescence and ultracentrifugation on sucrose gradient we here report that PKC activation by phorbol myristate acetate (PMA reorganizes EAAT-1 distribution and reduces functional [3H]-aspartate reuptake. Western-blots show that EAAT-1 is present in eMV from astrocyte conditioned medium, together with NaK ATPase and glutamine synthetase all being further increased after PMA treatment. However, nanoparticle tracking analysis reveals that PKC activation did not change particle concentration. Functional analysis indicates that eMV have the capacity to reuptake [3H]-aspartate. In vivo, we demonstrate that spinal astrocytic reaction induced by peripheral nerve lesion (spared nerve injury, SNI is associated with a phosphorylation of PKC δ together with a shift of EAAT distribution ipsilaterally. Ex vivo, spinal explants from SNI rats release eMV with an increased content of NaK ATPase, EAAT-1 and EAAT-2. These data indicate PKC and cell activation as important regulators of EAAT-1 incorporation in eMV, and raise the possibility that microvesicular EAAT-1 may exert extracellular functions. Beyond a putative role in neuropathic pain, this phenomenon may be important for understanding neural homeostasis and a wide range of neurological diseases associated with astrocytic reaction as well as non-neurological diseases linked to eMV release.

  8. Endothelial Cells and Astrocytes: A Concerto en Duo in Ischemic Pathophysiology

    Directory of Open Access Journals (Sweden)

    Vincent Berezowski

    2012-01-01

    Full Text Available The neurovascular/gliovascular unit has recently gained increased attention in cerebral ischemic research, especially regarding the cellular and molecular changes that occur in astrocytes and endothelial cells. In this paper we summarize the recent knowledge of these changes in association with edema formation, interactions with the basal lamina, and blood-brain barrier dysfunctions. We also review the involvement of astrocytes and endothelial cells with recombinant tissue plasminogen activator, which is the only FDA-approved thrombolytic drug after stroke. However, it has a narrow therapeutic time window and serious clinical side effects. Lastly, we provide alternative therapeutic targets for future ischemia drug developments such as peroxisome proliferator- activated receptors and inhibitors of the c-Jun N-terminal kinase pathway. Targeting the neurovascular unit to protect the blood-brain barrier instead of a classical neuron-centric approach in the development of neuroprotective drugs may result in improved clinical outcomes after stroke.

  9. 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.

  10. 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.

  11. Preclinical Studies of Induced Pluripotent Stem Cell-Derived Astrocyte Transplantation in ALS

    Science.gov (United States)

    2013-10-01

    to neuralize the cultures. This was considered day 1 of differentiation. At day 3, retinoic acid (RA, 1 μM, Sigma-Aldrich) and ascorbic acid (0.4...whether these iPSC-derived cells may in fact harbor ALS-specific abnormalities that may lack benefit or, potentially exacerbate disease. By comparing...mature properties in vivo which may be essential for their potential therapeutic benefits in patients. In evaluation of the hiPSC-derived astrocyte

  12. Generation of reactive astrocytes from NG2 cells is regulated by sonic hedgehog.

    Science.gov (United States)

    Honsa, Pavel; Valny, Martin; Kriska, Jan; Matuskova, Hana; Harantova, Lenka; Kirdajova, Denisa; Valihrach, Lukas; Androvic, Peter; Kubista, Mikael; Anderova, Miroslava

    2016-09-01

    NG2 cells, a fourth glial cell type in the adult mammalian central nervous system, produce oligodendrocytes in the healthy nervous tissue, and display wide differentiation potential under pathological conditions, where they could give rise to reactive astrocytes. The factors that control the differentiation of NG2 cells after focal cerebral ischemia (FCI) are largely unknown. Here, we used transgenic Cspg4-cre/Esr1/ROSA26Sortm14(CAG-tdTomato) mice, in which tamoxifen administration triggers the expression of red fluorescent protein (tomato) specifically in NG2 cells and cells derived therefrom. Differentiation potential (in vitro and in vivo) of tomato-positive NG2 cells from control or postischemic brains was determined using the immunohistochemistry, single cell RT-qPCR and patch-clamp method. The ischemic injury was induced by middle cerebral artery occlusion, a model of FCI. Using genetic fate-mapping method, we identified sonic hedgehog (Shh) as an important factor that influences differentiation of NG2 cells into astrocytes in vitro. We also manipulated Shh signaling in the adult mouse brain after FCI. Shh signaling activation significantly increased the number of astrocytes derived from NG2 cells in the glial scar around the ischemic lesion, while Shh signaling inhibition caused the opposite effect. Since Shh signaling modifications did not change the proliferation rate of NG2 cells, we can conclude that Shh has a direct influence on the differentiation of NG2 cells and therefore, on the formation and composition of a glial scar, which consequently affects the degree of the brain damage. GLIA 2016;64:1518-1531.

  13. 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.

  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. The development of blood-retinal barrier during the interaction of astrocytes with vascular wall cells

    Institute of Scientific and Technical Information of China (English)

    Huanling Yao; Tianshi Wang; Jiexin Deng; Ding Liu; Xiaofei Li; Jinbo Deng

    2014-01-01

    Astrocytes are intimately involved in the formation and development of retinal vessels. Astrocyte dysfunction is a major cause of blood-retinal barrier injury and other retinal vascular diseases. In this study, the development of the retinal vascular system and the formation of the blood-ret-inal barrier in mice were investigated using immunolfuorescence staining, gelatin-ink perfusion, and transmission electron microscopy. The results showed that the retinal vascular system of mice develops from the optic disc after birth, and radiates out gradually to cover the entire retina, taking the papilla optica as the center. First, the superifcial vasculature is formed on the inner retinal layer;then, the vasculature extends into the inner and outer edges of the retinal inner nuclear layer, forming the deep vasculature that is parallel to the superifcial vasculature. The blood-retinal barrier is mainly composed of endothelium, basal lamina and the end-feet of astrocytes, which become mature during mouse development. Initially, the naive endothelial cells were immature with few organelles and many microvilli. The basal lamina was uniform in thickness, and the glial end-feet surrounded the outer basal lamina incompletely. In the end, the blood-retinal barrier matures with smooth endothelia connected through tight junctions, rela-tively thin and even basal lamina, and relatively thin glial cell end-feet. These ifndings indicate that the development of the vasculature in the retina follows the rules of“center to periphery”and“superifcial layer to deep layers”. Its development and maturation are spatially and tempo-rally consistent with the functional performance of retinal neurons and photosensitivity. The blood-retinal barrier gradually becomes mature via the process of interactions between astro-cytes and blood vessel cells.

  16. Cell-type-specific expression and regulation of a c-fos-NGF fusion gene in neurons and astrocytes of transgenic mice.

    Science.gov (United States)

    Onténiente, B; Horellou, P; Neveu, I; Makeh, I; Suzuki, F; Bourdet, C; Grimber, G; Colin, P; Brachet, P; Mallet, J

    1994-02-01

    A mouse line transgenic for nerve growth factor (NGF) was developed using the mouse prepro-NGF cDNA inserted within a plasmid containing the proximal region (-10 to -550 bp) of the c-fos promoter and the transcription termination and polyadenylation signals of the rabbit beta-globin gene. No significant modification of gross behavior or central nervous system anatomy was detected in adult animals as assessed by immunohistochemistry and in situ hybridization for NGF and choline acetyltransferase. The expression of the transgene and the possible regulation of its expression by agents acting on the promoter were investigated in vitro. Despite the presence of an additional pool of NGF mRNA specific to the transgene, basal levels of NGF in the supernatant of transgenic astrocytes were similar to normal ones. On the other hand, transgenic neurons spontaneously synthesized and released levels of NGF two to three times higher than normal neurons, while mRNA levels were barely detectable by conventional Northern blotting. The tissue-specificity of NGF expression was respected, with higher levels in hippocampal than neocortical neurons. Increases of NGF mRNA by agents acting on the promoter could be observed in normal and transgenic astrocytes only after inhibition of the protein synthesis by cycloheximide, suggesting a similar rapid turnover of normal and transgenic transcripts. Cyclic AMP agonists specifically increased the secretion of NGF protein by transgenic astrocytes and neurons, while activators of the protein kinase C had a similar effect on transgenic and normal cells. Differences between amounts of NGF secreted by neurons and astrocytes with regards to their respective content in mRNA suggest that transgenic transcripts are subject to normal cell- and tissue-specific post-transcriptional regulations. Agents acting on the c-fos promoter through the protein kinase C or cyclic AMP routes differentially increased the secretion of NGF by transgenic astrocytes or

  17. 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.

  18. Effect of diclofenac and antidepressants on the inflammatory response in astrocyte cell culture.

    Science.gov (United States)

    Al-Amin, Md Mamun; Uddin, Mir Muhammad Nasir; Rahman, Md Mahbubur; Reza, Hasan Mahmud; Rana, Md Sohel

    2013-12-01

    Central nervous system (CNS) has a completely separate immune system that communicates with the neurons by small molecules called cytokines. Cytokines are involved in many crucial processes in neuron including cell metabolism and neurotransmitter synthesis. It has been reported that cytokine imbalance is involved in the progression of many CNS diseases such as neuropsychiatric disorders (depression, schizophrenia, autism, and bipolar disorder) and neurodegenerative disorders (Parkinson's and Alzheimer's disease). Here, the effects of diclofenac, different antidepressants (sertraline, venlafaxine, and fluvoxamine), and vitamin B₆ (pyridoxine) on IL-10 and tumor necrosis factor-α (TNF-α) change with and without immune challenges with lipopolysaccharide (LPS) were investigated in in vitro culture of astrocytes from 2-day-old Swiss-Albino mice. Diclofenac and Sertraline significantly (p 0.05) TNF-α in the astrocyte culture. Antidepressant (sertraline) showed positive effects (increased IL-10 and reduced TNF-α level) possibly through the suppression of Th1 lymphocytes and monocytes and stimulation of Th2 lymphocytes and monocytes/macrophages. NSAID (diclofenac) showed positive immune regulation effect possibly through the inhibition of cyclo-oxygenase enzyme. Based on these findings, it may conclude that, diclofenac and antidepressants (sertraline) may positively contribute in the cytokine production in astrocyte cell culture.

  19. Hypoxic regulation of Ca2+ signalling in astrocytes and endothelial cells.

    Science.gov (United States)

    Peers, C; Kang, P; Boyle, J P; Porter, K E; Pearson, H A; Smith, I F; Kemp, P J

    2006-01-01

    Acute hypoxia is well known to modulate plasmalemmal ion channels in specific tissue types, thereby modulating [Ca2+]i. Alternative mechanisms by which acute hypoxia could modulate [Ca2+]i are less well explored, particularly in non-excitable cells. Here, we describe experiments employing microfluorimetric recordings from Fura-2-loaded rat cortical astrocytes and human saphenous vein endothelial cells designed to explore any effects of hypoxia (pO2 20-30 mmHg) on [Ca2+]i. In both cell types, hypoxia evoked small rises of [Ca2+]i in the majority of cells during perfusion with a Ca(2+)-free solution, indicating hypoxia can release Ca2+ from an intracellular pool. Capacitative Ca2+ entry was observed when Ca2+ was subsequently restored to the extracellular solution. These effects were abolished by pre-treatment of cells with thapsigargin or prior application of inositol 1,4,5-trisphosphate (IP3)-generating agonists. Antioxidants fully prevented this effect of hypoxia in both cell types. Mitochondrial uncoupling significantly enhanced the effects of hypoxia in astrocytes, yet markedly suppressed the effects of hypoxia in endothelial cells. Our findings indicate that hypoxia can modulate [Ca2+]i in non-excitable cells; most importantly, it can evoke Ca2+ release from intracellular stores via a mechanism which involves reactive oxygen species. The involvement of mitochondria in this effect appears to be tissue specific.

  20. 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...

  1. 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.

  2. Immune Privilege as an Intrinsic CNS Property: Astrocytes Protect the CNS against T-Cell-Mediated Neuroinflammation

    Directory of Open Access Journals (Sweden)

    Ulrike Gimsa

    2013-01-01

    Full Text Available Astrocytes have many functions in the central nervous system (CNS. They support differentiation and homeostasis of neurons and influence synaptic activity. They are responsible for formation of the blood-brain barrier (BBB and make up the glia limitans. Here, we review their contribution to neuroimmune interactions and in particular to those induced by the invasion of activated T cells. We discuss the mechanisms by which astrocytes regulate pro- and anti-inflammatory aspects of T-cell responses within the CNS. Depending on the microenvironment, they may become potent antigen-presenting cells for T cells and they may contribute to inflammatory processes. They are also able to abrogate or reprogram T-cell responses by inducing apoptosis or secreting inhibitory mediators. We consider apparently contradictory functions of astrocytes in health and disease, particularly in their interaction with lymphocytes, which may either aggravate or suppress neuroinflammation.

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  5. A hit and run approach to inducible direct reprogramming of astrocytes to neural stem cells

    Directory of Open Access Journals (Sweden)

    Maria ePoulou

    2016-04-01

    Full Text Available Temporal and spatial control of gene expression can be achieved using an inducible system as a fundamental tool for regulated transcription in basic, applied and eventually in clinical research. We describe a novel hit and run inducible direct reprogramming approach. In a single step, two days post-transfection, transiently transfected Sox2FLAG under the Leu3p-αIPM inducible control (iSox2 triggers the activation of endogenous Sox2, redirecting primary astrocytes into abundant distinct nestin-positive radial glia cells. This technique introduces a unique novel tool for safe, rapid and efficient reprogramming amendable to regenerative medicine.

  6. 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....

  7. Acceleration of astrocytic differentiation in neural stem cells surviving X-irradiation.

    Science.gov (United States)

    Ozeki, Ayumi; Suzuki, Keiji; Suzuki, Masatoshi; Ozawa, Hiroki; Yamashita, Shunichi

    2012-03-28

    Neural stem cells (NSCs) are highly susceptible to DNA double-strand breaks; however, little is known about the effects of radiation in cells surviving radiation. Although the nestin-positive NSCs predominantly became glial fibrillary acidic protein (GFAP)-positive in differentiation-permissive medium, little or no cells were GFAP positive in proliferation-permissive medium. We found that more than half of the cells surviving X-rays became GFAP positive in proliferation-permissive medium. Moreover, localized irradiation stimulated differentiation of cells outside the irradiated area. These results indicate for the first time that ionizing radiation is able to stimulate astrocyte-specific differentiation of surviving NSCs, whose process is mediated both by the direct activation of nuclear factor-κB and by the indirect bystander effect induced by X-irradiation.

  8. A human astrocytoma cell line is highly susceptible to infection with Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    Juan Camilo Vargas-Zambrano

    2013-04-01

    Full Text Available Astrocytes play a vital role in neuronal protection, homeostasis, vascular interchange and the local immune response. Some viruses and parasites can cross the blood-brain barrier and infect glia. Trypanosoma cruzi, the aetiological agent of Chagas disease, can seriously compromise the central nervous system, mainly in immune-suppressed individuals, but also during the acute phase of the infection. In this report, the infective capacity of T. cruzi in a human astrocyte tumour-derived cell line was studied. Astrocytes exposed to trypomastigotes (1:10 ratio produced intracellular amastigotes and new trypomastigotes emerged by day 4 post-infection (p.i.. At day 6 p.i., 93% of the cells were infected. Using flow cytometry, changes were observed in both the expression of major histocompatibility complex class I and II molecules and the chemokine secretion pattern of astrocytes exposed to the parasite. Blocking the low-density lipoprotein receptor on astrocytes did not reduce parasite intracellular infection. Thus, T. cruzi can infect astrocytes and modulate the immune response during central nervous system infection.

  9. CLO : The cell line ontology

    NARCIS (Netherlands)

    Sarntivijai, Sirarat; Lin, Yu; Xiang, Zuoshuang; Meehan, Terrence F.; Diehl, Alexander D.; Vempati, Uma D.; Schuerer, Stephan C.; Pang, Chao; Malone, James; Parkinson, Helen; Liu, Yue; Takatsuki, Terue; Saijo, Kaoru; Masuya, Hiroshi; Nakamura, Yukio; Brush, Matthew H.; Haendel, Melissa A.; Zheng, Jie; Stoeckert, Christian J.; Peters, Bjoern; Mungall, Christopher J.; Carey, Thomas E.; States, David J.; Athey, Brian D.; He, Yongqun

    2014-01-01

    Background: Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO

  10. Phenotypic transition of microglia into astrocyte-like cells associated with disease onset in a model of inherited ALS

    Directory of Open Access Journals (Sweden)

    Emiliano eTrias

    2013-12-01

    Full Text Available Microglia and reactive astrocytes accumulate in the spinal cord of rats expressing the ALS-linked SOD1G93A mutation. We previously reported that the rapid progression of paralysis in ALS rats is associated with the appearance of proliferative astrocyte-like cells thatsurroundmotor neurons. These cells, designated as Aberrant Astrocytes (AbA cells because of their atypical astrocytic phenotype, exhibit high toxicity to motor neurons. However, the cellular origin of AbA cells remains unknown. Because AbA cells are labeled with the proliferation marker Ki67, we analyzed the phenotypic makers of proliferating glial cells that surround motor neurons by immunohistochemistry. The number of Ki67+AbA cells sharply increased in symptomatic rats, displaying large cell bodies with processes embracing motor neurons. Most were co-labeled with astrocytic marker GFAP concurrently with the microglial markers Iba1 and CD163. Cultures of spinal cord prepared from symptomatic SOD1G93A rats yielded large numbers of microglia expressing Iba1, CD11b and CD68. Cells sortedfor CD11b expression by flow cytometry transformed into AbA cells within two weeks. During these two weeks, the expression of microglial markers largely disappeared, while GFAP and S100β expression increased. The phenotypic transition to AbA cells was stimulated by forskolin. These findings provide evidence for a subpopulation of proliferating microglial cells in SOD1G93A rats that undergo a phenotypic transition into AbA cells after onset of paralysis that may promote the fulminant disease progression. These cells could be a therapeutic target for slowing paralysis progression in ALS.

  11. Microglia-derived interleukin-6 and leukaemia inhibitory factor promote astrocytic differentiation of neural stem/progenitor cells.

    Science.gov (United States)

    Nakanishi, Masaya; Niidome, Tetsuhiro; Matsuda, Satoru; Akaike, Akinori; Kihara, Takeshi; Sugimoto, Hachiro

    2007-02-01

    Neural stem/progenitor cells (NSPCs) proliferate and differentiate depending on their intrinsic properties and local environment. It has been recognized that astrocytes promote neurogenic differentiation of NSPCs, suggesting the importance of cell-cell interactions between glial cells and NSPCs. Recent studies have demonstrated that microglia, one type of glial cells, play an important role in neurogenesis. However, little is known about how activated microglia control the proliferation and differentiation of NSPCs. In this study, we investigated the possibility that microglia-derived soluble factors regulate the behaviour of NSPCs. To this end, NSPCs and microglial cultures were obtained from rat embryonic day 16 subventricular zone (SVZ) and rat postnatal 1 day cortex, respectively, and the conditioned medium from microglia was prepared. Microglial-conditioned medium had no significant effect on the proliferation of NSPCs. In contrast, it increased the percentage of cells positive for a marker of astrocytes, glial fibrillary acidic protein (GFAP) during differentiation. The induction of astrocytic differentiation by microglial-conditioned medium was reduced by the inhibition of the Janus kinase/signal transducer and activation of transcription (JAK/STAT) and mitogen-activated protein kinase (MAPK) pathways. Furthermore, microglia-derived interleukin (IL)-6 and leukaemia inhibitory factor (LIF) were identified as essential molecules for this astrocytic differentiation using neutralizing antibodies and recombinant cytokines. Our results suggest that microglia as well as astrocytes contribute to the integrity of the local environment of NSPCs, and at least IL-6 and LIF released by activated microglia promote astrocytic differentiation of NSPCs via the activation of the JAK/STAT and MAPK pathways.

  12. 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.

  13. Impaired APP activity and altered Tau splicing in embryonic stem cell-derived astrocytes obtained from an APPsw transgenic minipig

    DEFF Research Database (Denmark)

    Hall, Vanessa Jane; Lindblad, Maiken Marie; Jakobsen, Jannik E.;

    2015-01-01

    onset mechanisms related to cell renewal and function in familial AD astrocytes. These outcomes also highlight that radial glia could be a potentially useful population of cells for drug discovery, and that altered APP expression and altered tau phosphorylation can be detected in an in vitro model...

  14. 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.

  15. Manipulation of Schwann cell migration across the astrocyte boundary by polysialyltransferase-loaded superparamagnetic nanoparticles under magnetic field

    Directory of Open Access Journals (Sweden)

    Xia B

    2016-12-01

    Full Text Available Bing Xia,* Liangliang Huang,* Lei Zhu, Zhongyang Liu, Teng Ma, Shu Zhu, Jinghui Huang, Zhuojing Luo Department of Orthopaedics, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China *These authors contributed equally to this work Abstract: Schwann cell (SC transplantation is an attractive strategy for spinal cord injury (SCI. However, the efficacy of SC transplantation has been limited by the poor migratory ability of SCs in the astrocyte-rich central nervous system (CNS environment and the inability to intermingle with the host astrocyte. In this study, we first magnetofected SCs by polysialyltransferase-functionalized superparamagnetic iron oxide nanoparticles (PST/SPIONs to induce overexpression of polysialylation of neural cell adhesion molecule (PSA-NCAM to enhance SC migration ability, before manipulating the direction of SC migration with the assistance of an applied magnetic field (MF. It was found that magnetofection with PST/SPIONs significantly upregulated the expression of PSA-NCAM in SCs, which significantly enhanced the migration ability of SCs, but without preferential direction in the absence of MF. The number and averaged maximum distance of SCs with PST/SPIONs migrating into the astrocyte domain were significantly enhanced by an applied MF. In a 300 µm row along the astrocyte boundary, the number of SCs with PST/SPIONs migrating into the astrocyte domain under an MF was 2.95 and 6.71 times higher than that in the absence of MF and the intact control SCs, respectively. More interestingly, a confrontation assay demonstrated that SCs with PST/SPIONs were in close contact with astrocytes and no longer formed boundaries in the presence of MF. In conclusion, SCs with PST/SPIONs showed enhanced preferential migration along the axis of a magnetic force, which might be beneficial for the formation of Büngner bands in the CNS. These findings raise the possibilities of enhancing the

  16. Astrocytes and Microglia as Non-cell Autonomous Players in the Pathogenesis of ALS

    Science.gov (United States)

    Hyeon, Seung Jae; Im, Hyeonjoo; Ryu, Hyun; Kim, Yunha

    2016-01-01

    Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder that leads to a progressive muscle wasting and paralysis. The pathological phenotypes are featured by severe motor neuron death and glial activation in the lumbar spinal cord. Proposed ALS pathogenic mechanisms include glutamate cytotoxicity, inflammatory pathway, oxidative stress, and protein aggregation. However, the exact mechanisms of ALS pathogenesis are not fully understood yet. Recently, a growing body of evidence provides a novel insight on the importance of glial cells in relation to the motor neuronal damage via the non-cell autonomous pathway. Accordingly, the aim of the current paper is to overview the role of astrocytes and microglia in the pathogenesis of ALS and to better understand the disease mechanism of ALS. PMID:27790057

  17. 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.

  18. Light and electron microscopic localization of GABAA-receptors on cultured cerebellar granule cells and astrocytes using immunohistochemical techniques

    DEFF Research Database (Denmark)

    Hansen, G H; Hösli, E; Belhage, B;

    1991-01-01

    GABAA-receptors were localized in explant cultures of rat cerebellum and in dissociated primary cultures of rat cerebellar granule cells and rat cerebellar astrocytes using the monoclonal antibody bd-17 directed against the beta-subunit of the GABAA/benzodiazepine/chloride channel complex. At the...

  19. Multilineage potential of stable human mesenchymal stem cell line derived from fetal marrow.

    Directory of Open Access Journals (Sweden)

    Atsushi Nagai

    Full Text Available Human bone marrow contains two major cell types, hematopoietic stem cells (HSCs and mesenchymal stem cells (MSCs. MSCs possess self-renewal capacity and pluripotency defined by their ability to differentiate into osteoblasts, chondrocytes, adipocytes and muscle cells. MSCs are also known to differentiate into neurons and glial cells in vitro, and in vivo following transplantation into the brain of animal models of neurological disorders including ischemia and intracerebral hemorrhage (ICH stroke. In order to obtain sufficient number and homogeneous population of human MSCs, we have clonally isolated permanent and stable human MSC lines by transfecting primary cell cultures of fetal human bone marrow MSCs with a retroviral vector encoding v-myc gene. One of the cell lines, HM3.B10 (B10, was found to differentiate into neural cell types including neural stem cells, neurons, astrocytes and oligodendrocytes in vitro as shown by expression of genetic markers for neural stem cells (nestin and Musashi1, neurons (neurofilament protein, synapsin and MAP2, astrocytes (glial fibrillary acidic protein, GFAP and oligodendrocytes (myelin basic protein, MBP as determined by RT-PCR assay. In addition, B10 cells were found to differentiate into neural cell types as shown by immunocytochical demonstration of nestin (for neural stem cells, neurofilament protein and beta-tubulin III (neurons GFAP (astrocytes, and galactocerebroside (oligodendrocytes. Following brain transplantation in mouse ICH stroke model, B10 human MSCs integrate into host brain, survive, differentiate into neurons and astrocytes and induce behavioral improvement in the ICH animals. B10 human MSC cell line is not only a useful tool for the studies of organogenesis and specifically for the neurogenesis, but also provides a valuable source of cells for cell therapy studies in animal models of stroke and other neurological disorders.

  20. Mesoporous silica nanoparticles enhance MTT formazan exocytosis in HeLa cells and astrocytes.

    Science.gov (United States)

    Fisichella, Matthieu; Dabboue, Hinda; Bhattacharyya, Sanjib; Saboungi, Marie-Louise; Salvetat, Jean-Paul; Hevor, Tobias; Guerin, Martine

    2009-06-01

    We report on the observation that mesoporous silica nanoparticles (MSNs), after being endocytosed, interfere with the MTT test in HeLa cells and astrocytes by accelerating the exocytosis of formazan crystals. The stimulation of MTT formazan exocytosis is probably related to perturbation of intracellular vesicle trafficking by MSN uptake as revealed by experiments in presence of chloroquine and genistein. Similar effect has been previously observed with a number of chemicals, especially with neurotoxic beta amyloid peptides, but not with nanoparticles. We showed also that MTT reduction test gives an overestimation of the cytotoxicity of mesoporous silica nanoparticles compared to other tests such as LDH activity, WST-1 test and flow cytometry. These findings show that MTT assay should not be used for the study of MSN toxicity, and that perturbation of intracellular trafficking has to be taken into account in evaluating biocompatibility of MSNs.

  1. Determining the Oxidation States of Manganese in NT2 Cells and Cultured Astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Gunter,K.; Aschner, M.; Miller, L.; Eliseev, R.; Salter, J.; Andersen, K.; Gunter, T.

    2006-01-01

    Excessive brain manganese (Mn) can produce a syndrome called 'manganism', which correlates with loss of striatal dopamine and cell death in the striatum and globus pallidus. The prevalent hypothesis for the cause of this syndrome has been oxidation of cell components by the strong oxidizing agent, Mn{sup 3+}, either formed by oxidation of intracellular Mn{sup 2+} or transported into the cell as Mn{sup 3+}. We have recently used X-ray absorption near edge structure spectroscopy (XANES) to determine the oxidation states of manganese complexes in brain and liver mitochondria and in nerve growth factor (NGF)-induced and non-induced PC12 cells. No evidence was found for stabilization or accumulation of Mn{sup 3+} complexes because of oxidation of Mn{sup 2+} by reactive oxygen species in these tissues. Here we extend these studies of manganese oxidation state to cells of brain origin, human neuroteratocarcinoma (NT2) cells and primary cultures of rat astrocytes. Again we find no evidence for stabilization or accumulation of any Mn{sup 3+} complex derived from oxidation of Mn{sup 2+} under a range of conditions.

  2. Culturing of PC12 Cells, Neuronal Cells, Astrocytes Cultures and Brain Slices in an Open Microfluidic System

    DEFF Research Database (Denmark)

    Al Atraktchi, Fatima Al-Zahraa; Bakmand, Tanya; Rømer Sørensen, Ane

    cells, neuronal cells, astrocytes cultures and brain slices. The microfluidic system provides efficient nutrient delivery, waste removal, access to oxygen, fine control over the neurochemical environment and access to modern microscopy. Additionally, the setup consists of an in vitro culturing......The brain is the center of the nervous system, where serious neurodegenerative diseases such as Parkinson’s, Alzheimer’s and Huntington’s are products of functional loss in the neural cells (1). Typical techniques used to investigate these diseases lack precise control of the cellular surroundings...... and electrochemical sensor system that enables real time detection of metabolites, e.g. dopamine from cell cultures and brain slices. In summary we present results on culturing of brain slices and cells in the microfluidic system as well as on the incorporation of an electrochemical sensor system for characterization...

  3. Immunochemical expression of proliferative cell nuclear antigen in aging cultured astrocytes

    Directory of Open Access Journals (Sweden)

    M. C. Vanzani

    2003-08-01

    Full Text Available Cell differentiation degree and mitotic activity were sequentially assessed by immunoperoxidase labeling of glial fibrillary acidic protein (GFAP and proliferative cell nuclear antigen (PCNA, respectively, in rat brain cultured astrocytes maintained up to 60 days in vitro (DIV of first subculture, or weekly passaged until their 12th subculture. Cell count was performed through a 0.01 mm2 section reticule and morphometric analysis with a stereological grid. The number of double immunoreactive cells peaked by 2 DIV to achieve its lowest value at 60 DIV. At 24 hs of cell seeding of successive passages, such values peaked by the 6th subculture to gradually decrease thereafter. Increasing cell hypertrophy was found during the long-term first subculture but not after passaging. At the end of the observation period, doubly immunolabeled astrocytes were still recorded, thus evidencing retention of proliferative potential despite aging.El grado de diferenciación celular y la actividad mitótica fueron secuencialmente determinados mediante marcación por inmunoperoxidasa de la proteína gliofibrilar ácida (GFAP y del antígeno nuclear de proliferación celular (PCNA, respectivamente, en cultivos astrocitarios obtenidos de encéfalo de rata y mantenidos hasta 60 días in vitro (DIV de su primer subcultivo, o mediante pasajes semanales hasta el 12do subcultivo. El conteo celular se realizó mediante una retícula de 0.01-mm2 de sección y el análisis morfométrico con una grilla estereológica. El número de células doblemente inmunorreactivas alcanzó valores máximos a los 2 DIV para descender a los menores a los 60 DIV. A las 24 hs de sembrado celular de los sucesivos pasajes, esos valores ascendieron hacia el 6to subcultivo para luego declinar. En cuanto a la hipertrofia celular, se observó en todo el curso del primer subcultivo, pero no durante los posteriores pasajes. Al final del período de observación, todavía se continuaban detectando

  4. Neural Stem Cell Transplantation Induces Stroke Recovery by Upregulating Glutamate Transporter GLT-1 in Astrocytes

    Science.gov (United States)

    Russo, Gianluca Luigi; Peruzzotti-Jametti, Luca; Rossi, Silvia; Sandrone, Stefano; Butti, Erica; De Ceglia, Roberta; Bergamaschi, Andrea; Motta, Caterina; Gallizioli, Mattia; Studer, Valeria; Colombo, Emanuela; Farina, Cinthia; Comi, Giancarlo; Politi, Letterio Salvatore; Muzio, Luca; Villani, Claudia; Invernizzi, Roberto William; Hermann, Dirk Matthias; Centonze, Diego

    2016-01-01

    Ischemic stroke is the leading cause of disability, but effective therapies are currently widely lacking. Recovery from stroke is very much dependent on the possibility to develop treatments able to both halt the neurodegenerative process as well as to foster adaptive tissue plasticity. Here we show that ischemic mice treated with neural precursor cell (NPC) transplantation had on neurophysiological analysis, early after treatment, reduced presynaptic release of glutamate within the ipsilesional corticospinal tract (CST), and an enhanced NMDA-mediated excitatory transmission in the contralesional CST. Concurrently, NPC-treated mice displayed a reduced CST degeneration, increased axonal rewiring, and augmented dendritic arborization, resulting in long-term functional amelioration persisting up to 60 d after ischemia. The enhanced functional and structural plasticity relied on the capacity of transplanted NPCs to localize in the peri-ischemic and ischemic area, to promote the upregulation of the glial glutamate transporter 1 (GLT-1) on astrocytes and to reduce peri-ischemic extracellular glutamate. The upregulation of GLT-1 induced by transplanted NPCs was found to rely on the secretion of VEGF by NPCs. Blocking VEGF during the first week after stroke reduced GLT-1 upregulation as well as long-term behavioral recovery in NPC-treated mice. Our results show that NPC transplantation, by modulating the excitatory–inhibitory balance and stroke microenvironment, is a promising therapy to ameliorate disability, to promote tissue recovery and plasticity processes after stroke. SIGNIFICANCE STATEMENT Tissue damage and loss of function occurring after stroke can be constrained by fostering plasticity processes of the brain. Over the past years, stem cell transplantation for repair of the CNS has received increasing interest, although underlying mechanism remain elusive. We here show that neural stem/precursor cell transplantation after ischemic stroke is able to foster

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

    Science.gov (United States)

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

    2016-01-01

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

  6. Neural Crest Cells Contribute an Astrocyte-like Glial Population to the Spleen

    Science.gov (United States)

    Barlow-Anacker, Amanda J.; Fu, Ming; Erickson, Christopher S.; Bertocchini, Federica; Gosain, Ankush

    2017-01-01

    Neural crest cells (NCC) are multi-potent cells of ectodermal origin that colonize diverse organs, including the gastrointestinal tract to form the enteric nervous system (ENS) and hematopoietic organs (bone marrow, thymus) where they participate in lymphocyte trafficking. Recent studies have implicated the spleen as an anatomic site for integration of inflammatory signals from the intestine with efferent neural inputs. We have previously observed alterations in splenic lymphocyte subsets in animals with defective migration of NCC that model Hirschsprung’s disease, leading us to hypothesize that there may be a direct cellular contribution of NCC to the spleen. Here, we demonstrate that NCC colonize the spleen during embryogenesis and persist into adulthood. Splenic NCC display markers indicating a glial lineage and are arranged anatomically adjacent to blood vessels, pericytes and nerves, suggesting an astrocyte-like phenotype. Finally, we identify similar neural-crest derived cells in both the avian and non-human primate spleen, showing evolutionary conservation of these cells. PMID:28349968

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  9. 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...

  10. Orexin-A promotes cell migration in cultured rat astrocytes via Ca2+-dependent PKCα and ERK1/2 signals.

    Directory of Open Access Journals (Sweden)

    Qing Shu

    Full Text Available Orexin-A is an important neuropeptide involved in the regulation of feeding, arousal, energy consuming, and reward seeking in the body. The effects of orexin-A have widely studied in neurons but not in astrocytes. Here, we report that OX1R and OX2R are expressed in cultured rat astrocytes. Orexin-A stimulated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2, and then induced the migration of astrocytes via its receptor OX1R but not OX2R. Orexin-A-induced ERK1/2 phosphorylation and astrocytes migration are Ca2+-dependent, since they could be inhibited by either chelating the extracellular Ca2+ or blocking the pathway of store-operated calcium entry (SOCE. Furthermore, both non-selective protein kinase C (PKC inhibitor and PKCα selective inhibitor, but not PKCδ inhibitor, prevented the increase in ERK1/2 phosphorylation and the migration of astrocytes, indicating that the Ca2+-dependent PKCα acts as the downstream of the OX1R activation and mediates the orexin-A-induced increase in ERK1/2 phosphorylation and cell migration. In conclusion, these results suggest that orexin-A can stimulate ERK1/2 phosphorylation and then facilitate the migration of astrocytes via PLC-PKCα signal pathway, providing new knowledge about the functions of the OX1R in astrocytes.

  11. Thyroid cell lines in research on goitrogenesis.

    Science.gov (United States)

    Gerber, H; Peter, H J; Asmis, L; Studer, H

    1991-12-01

    Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes.

  12. Impaired APP activity and altered Tau splicing in embryonic stem cell-derived astrocytes obtained from an APPsw transgenic minipig

    Directory of Open Access Journals (Sweden)

    Vanessa J. Hall

    2015-10-01

    Full Text Available Animal models of familial juvenile onset of Alzheimer's disease (AD often fail to produce diverse pathological features of the disease by modification of single gene mutations that are responsible for the disease. They can hence be poor models for testing and development of novel drugs. Here, we analyze in vitro-produced stem cells and their derivatives from a large mammalian model of the disease created by overexpression of a single mutant human gene (APPsw. We produced hemizygous and homozygous radial glial-like cells following culture and differentiation of embryonic stem cells (ESCs isolated from embryos obtained from mated hemizygous minipigs. These cells were confirmed to co-express varying neural markers, including NES, GFAP and BLBP, typical of type one radial glial cells (RGs from the subgranular zone. These cells had altered expression of CCND1 and NOTCH1 and decreased expression of several ribosomal RNA genes. We found that these cells were able to differentiate into astrocytes upon directed differentiation. The astrocytes produced had decreased α- and β-secretase activity, increased γ-secretase activity and altered splicing of tau. This indicates novel aspects of early onset mechanisms related to cell renewal and function in familial AD astrocytes. These outcomes also highlight that radial glia could be a potentially useful population of cells for drug discovery, and that altered APP expression and altered tau phosphorylation can be detected in an in vitro model of the disease. Finally, it might be possible to use large mammal models to model familial AD by insertion of only a single mutation.

  13. Reactive astrocytes promote the metastatic growth of breast cancer stem-like cells by activating Notch signalling in brain.

    Science.gov (United States)

    Xing, Fei; Kobayashi, Aya; Okuda, Hiroshi; Watabe, Misako; Pai, Sudha K; Pandey, Puspa R; Hirota, Shigeru; Wilber, Andrew; Mo, Yin-Yuan; Moore, Brian E; Liu, Wen; Fukuda, Koji; Iiizumi, Megumi; Sharma, Sambad; Liu, Yin; Wu, Kerui; Peralta, Elizabeth; Watabe, Kounosuke

    2013-03-01

    Brain metastasis of breast cancer profoundly affects the cognitive and sensory functions as well as morbidity of patients, and the 1 year survival rate among these patients remains less than 20%. However, the pathological mechanism of brain metastasis is as yet poorly understood. In this report, we found that metastatic breast tumour cells in the brain highly expressed IL-1β which then 'activated' surrounding astrocytes. This activation significantly augmented the expression of JAG1 in the astrocytes, and the direct interaction of the reactivated astrocytes and cancer stem-like cells (CSCs) significantly stimulated Notch signalling in CSCs. We also found that the activated Notch signalling in CSCs up-regulated HES5 followed by promoting self-renewal of CSCs. Furthermore, we have shown that the blood-brain barrier permeable Notch inhibitor, Compound E, can significantly suppress the brain metastasis in vivo. These results represent a novel paradigm for the understanding of how metastatic breast CSCs re-establish their niche for their self-renewal in a totally different microenvironment, which opens a new avenue to identify a novel and specific target for the brain metastatic disease.

  14. Vitro Culture and Immunohistochemical Identification of Astrocytes of Infantile Optic Nerve

    Institute of Scientific and Technical Information of China (English)

    Jianliang Zheng; Yuqing Lan; Jie Zhang; Yan Guo; Yan Luo

    2000-01-01

    Objective: To culture astrocytes of optic nerve and to establish the cell lines for further study of healing process after optic nerve trauma.Methods: Optic nerve astrocytes of infantile and adults with sudden death were cultured by tissue inoculation or tissue digestion with 0. 25% Trypsin and 0. 06% EDTA. The second and fourth passage cells were stained with HE and anti- GFAP, S-100 protein, Vimentin, and CD34 antibodies.Results: The trypsinized astrocytes of infantile optic nerve reached confluence in 7 days,but the astrocytes of adults weren't successfully cultured. The cultured cells were in polygonal shape with processes; the cytoplasm was abundant and pink; the cells had light-blue nuclei. These cells were positive in GFAP, S-100 protein and vimentin staining, and negative in CD34 staining.Conclusions: The results showed that astrocytes of infantile optic nerve can be successfully cultured and trypsinization is a better method than tissue inoculation. The culture of infantile astrocytes is easier than that of adult astrocytes. Immunohistochemistry were used to determine the source and type of those cultured cells.

  15. A co-culture model of the hippocampal neurogenic niche reveals differential effects of astrocytes, endothelial cells and pericytes on proliferation and differentiation of adult murine precursor cells

    Directory of Open Access Journals (Sweden)

    Fanny Ehret

    2015-11-01

    Full Text Available The niche concept of stem cell biology proposes a functional unit between the precursor cells and their local microenvironment, to which several cell types might contribute by cell–cell contacts, extracellular matrix, and humoral factors. We here established three co-culture models (with cell types separated by membrane for both adherent monolayers and neurospheres to address the potential influence of different niche cell types in the neurogenic zone of the adult hippocampus of mice. Astrocytes and endothelial cells enhanced precursor cell proliferation and neurosphere formation. Endothelial factors also led to a prolonged increase in proliferation after growth factor withdrawal, which otherwise induces differentiation. All niche cell types enhanced cell survival in monolayer cultures, endothelial cells also stimulated neuronal differentiation. A parallel trend elicited by astrocytes did not reach conventional statistical significance. Pericytes had variable effects here. We did not observe changes in differentiation in neurosphere co-cultures. In summary, our data indicate that in precursor cell culture protocols survival could be improved by adding as yet unknown factors physiologically contributed by astrocytes and endothelial cells. Our findings also underscore the complexity of the niche and the differential impact of factors from the different sources on distinct aspects of neuronal development. With the help of the models presented here, identification of these factors and their specific biological activity can now be initiated.

  16. Ultrastructural evidence for differentiation in a human glioblastoma cell line treated with inhibitors of eicosanoid metabolism

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    Wilson, D.E.; Anderson, K.M. (Rush Presbyterian St. Luke' s Medical Center, Chicago, IL (United States)); Seed, T.M. (Argonne National Lab., IL (United States))

    1990-01-01

    Human glioblastoma cells incubated in the presence of inhibitors of eicosanoid biosynthesis show decreased cellular proliferation without cytotoxicity. The authors studied the ultrastructural morphology of a human glioblastoma cell line cultured with nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, or 5,8,11,14-eicosatetraynoic acid, a cyclooxygenase and lipoxygenase inhibitor. When glioblastoma cells were treated for 3 days with antiproliferative concentrations of either agent, they shared many morphological characteristics, including evidence for increased astrocytic differentiation with only limited signs of toxicity. The inhibited glioma cells demonstrated an increase in the number and length of astrocytic processes containing greater numbers of glial filaments, and the NDGA-treated cells also demonstrated extensive lateral pseudopod formation along the processes. The glioblastoma cell shape also become more elongated, losing the usual nuclear lobularity and nuclear inclusions, especially in NDGA-treated cells. Many cytoplasmic organelles packed the cytosol of the inhibited glioma cells, including prominent Golgi apparatus, dilated smooth endoplasmic reticulum evolving into dilated vesicles, cytoplasmic vacuoles, and numerous concentric laminations. There was limited evidence for toxicity, however, as the mitochondria were more pleomorphic with some mitochondrial distension and disruption of the cristae along with an increase in cytoplasmic vacuolization. The authors conclude that the inhibitors of eicosanoid biosynthesis. NDGA and 5,8,11,14-eicosatetraynoic acid, not only suppress glioblastoma cell proliferation, but also include increased astrocytic differentiation.

  17. 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.

  18. Regional Susceptibility to Domoic Acid in Primary Astrocyte Cells Cultured from the Brain Stem and Hippocampus

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    Olga M. Pulido

    2008-02-01

    Full Text Available Domoic acid is a marine biotoxin associated with harmful algal blooms and is the causative agent of amnesic shellfish poisoning in marine animals and humans. It is also an excitatory amino acid analog to glutamate and kainic acid which acts through glutamate receptors eliciting a very rapid and potent neurotoxic response. The hippocampus, among other brain regions, has been identified as a specific target site having high sensitivity to DOM toxicity. Histopathology evidence indicates that in addition to neurons, the astrocytes were also injured. Electron microscopy data reported in this study further supports the light microscopy findings. Furthermore, the effect of DOM was confirmed by culturing primary astrocytes from the hippocampus and the brain stem and subsequently exposing them to domoic acid. The RNA was extracted and used for biomarker analysis. The biomarker analysis was done for the early response genes including c-fos, c-jun, c-myc, Hsp-72; specific marker for the astrocytes- GFAP and the glutamate receptors including GluR 2, NMDAR 1, NMDAR 2A and B. Although, the astrocyte-GFAP and c-fos were not affected, c-jun and GluR 2 were down-regulated. The microarray analysis revealed that the chemokines / cytokines, tyrosine kinases (Trk, and apoptotic genes were altered. The chemokines that were up-regulated included - IL1-a, IL-1B, IL-6, the small inducible cytokine, interferon protein IP-10, CXC chemokine LIX, and IGF binding proteins. The Bax, Bcl-2, Trk A and Trk B were all downregulated. Interestingly, only the hippocampal astrocytes were affected. Our findings suggest that astrocytes may present a possible target for pharmacological interventions for the prevention and treatment of amnesic shellfish poisoning and for other brain pathologies involving excitotoxicity

  19. Increased toll-like receptor 4 in cerebral endothelial cells contributes to the astrocyte swelling and brain edema in acute hepatic encephalopathy.

    Science.gov (United States)

    Jayakumar, Arumugam R; Tong, Xiao Y; Curtis, Kevin M; Ruiz-Cordero, Roberto; Abreu, Maria T; Norenberg, Michael D

    2014-03-01

    Astrocyte swelling and the subsequent increase in intracranial pressure and brain herniation are major clinical consequences in patients with acute hepatic encephalopathy. We recently reported that conditioned media from brain endothelial cells (ECs) exposed to ammonia, a mixture of cytokines (CKs) or lipopolysaccharide (LPS), when added to astrocytes caused cell swelling. In this study, we investigated the possibility that ammonia and inflammatory agents activate the toll-like receptor 4 (TLR4) in ECs, resulting in the release of factors that ultimately cause astrocyte swelling. We found a significant increase in TLR4 protein expression when ECs were exposed to ammonia, CKs or LPS alone, while exposure of ECs to a combination of these agents potentiate such effects. In addition, astrocytes exposed to conditioned media from TLR4-silenced ECs that were treated with ammonia, CKs or LPS, resulted in a significant reduction in astrocyte swelling. TLR4 protein up-regulation was also detected in rat brain ECs after treatment with the liver toxin thioacetamide, and that thioacetamide-treated TLR4 knock-out mice exhibited a reduction in brain edema. These studies strongly suggest that ECs significantly contribute to the astrocyte swelling/brain edema in acute hepatic encephalopathy, likely as a consequence of increased TLR4 protein expression by blood-borne noxious agents.

  20. HIV-1 Entry and Trans-Infection of Astrocytes Involves CD81 Vesicles

    Science.gov (United States)

    Gray, Lachlan R.; Turville, Stuart G.; HItchen, Tina L.; Cheng, Wan-Jung; Ellett, Anne M.; Salimi, Hamid; Roche, Michael J.; Wesselingh, Steve L.; Gorry, Paul R.; Churchill, Melissa J.

    2014-01-01

    Astrocytes are extensively infected with HIV-1 in vivo and play a significant role in the development of HIV-1-associated neurocognitive disorders. Despite their extensive infection, little is known about how astrocytes become infected, since they lack cell surface CD4 expression. In the present study, we investigated the fate of HIV-1 upon infection of astrocytes. Astrocytes were found to bind and harbor virus followed by biphasic decay, with HIV-1 detectable out to 72 hours. HIV-1 was observed to associate with CD81-lined vesicle structures. shRNA silencing of CD81 resulted in less cell-associated virus but no loss of co-localization between HIV-1 and CD81. Astrocytes supported trans-infection of HIV-1 to T-cells without de novo virus production, and the virus-containing compartment required 37°C to form, and was trypsin-resistant. The CD81 compartment observed herein, has been shown in other cell types to be a relatively protective compartment. Within astrocytes, this compartment may be actively involved in virus entry and/or spread. The ability of astrocytes to transfer virus, without de novo viral synthesis suggests they are capable of sequestering and protecting virus and thus, they could potentially facilitate viral dissemination in the CNS. PMID:24587404

  1. Effects of folic acid on in vitro astrocytic differentiation of neural stem cells from neonatal rats

    Institute of Scientific and Technical Information of China (English)

    Xumei Zhang; Guowei Huang; Zhihong Tian; Guanglei Wang; Dalin Ren

    2009-01-01

    ary acidic protein/BrdU-positive ceils (P<0.05), and significantly decreased Ngn1 protein expression (P<0.05).CONCLUSION: Folic acid promotes astrocytic differentiation of NSCs, which might be related to downregulation of Ngnl protein expression.

  2. Activation of the intrinsic cell death pathway, increased apoptosis and modulation of astrocytes in the cerebellum of diabetic rats.

    Science.gov (United States)

    Lechuga-Sancho, Alfonso M; Arroba, Ana I; Frago, Laura M; Pañeda, Covadonga; García-Cáceres, Cristina; Delgado Rubín de Célix, Arancha; Argente, Jesús; Chowen, Julie A

    2006-08-01

    Poorly controlled diabetes mellitus results in structural and functional changes in many brain regions. We demonstrate that in streptozotocin-induced diabetic rats cell death is increased and proliferation decreased in the cerebellum, indicating overall cell loss. Levels of both the proform and cleaved forms of caspases 3, 6 and 9 are increased, with no change in caspases 7, 8 or 12. Colocalization of glial fibrillary acidic protein (GFAP) and cleaved caspase 3 and GFAP in TUNEL-positive cells increased in diabetic rats. Changes in GFAP levels paralleled modifications in proliferating cell nuclear antigen (PCNA), increasing at 1 week of diabetes and decreasing thereafter, and proliferating GFAP-positive cells were decreased in the cerebellum of diabetic rats. These results suggest that astrocytes are dramatically affected in the cerebellum, including an increase in cell death and a decrease in proliferation, and this could play a role in the structural and functional changes in this brain area in diabetes.

  3. 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.

  4. Ornithine and Homocitrulline Impair Mitochondrial Function, Decrease Antioxidant Defenses and Induce Cell Death in Menadione-Stressed Rat Cortical Astrocytes: Potential Mechanisms of Neurological Dysfunction in HHH Syndrome.

    Science.gov (United States)

    Zanatta, Ângela; Rodrigues, Marília Danyelle Nunes; Amaral, Alexandre Umpierrez; Souza, Débora Guerini; Quincozes-Santos, André; Wajner, Moacir

    2016-09-01

    Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is caused by deficiency of ornithine translocase leading to predominant tissue accumulation and high urinary excretion of ornithine (Orn), homocitrulline (Hcit) and ammonia. Although affected patients commonly present neurological dysfunction manifested by cognitive deficit, spastic paraplegia, pyramidal and extrapyramidal signs, stroke-like episodes, hypotonia and ataxia, its pathogenesis is still poorly known. Although astrocytes are necessary for neuronal protection. Therefore, in the present study we investigated the effects of Orn and Hcit on cell viability (propidium iodide incorporation), mitochondrial function (thiazolyl blue tetrazolium bromide-MTT-reduction and mitochondrial membrane potential-ΔΨm), antioxidant defenses (GSH) and pro-inflammatory response (NFkB, IL-1β, IL-6 and TNF-α) in unstimulated and menadione-stressed cortical astrocytes that were previously shown to be susceptible to damage by neurotoxins. We first observed that Orn decreased MTT reduction, whereas both amino acids decreased GSH levels, without altering cell viability and the pro-inflammatory factors in unstimulated astrocytes. Furthermore, Orn and Hcit decreased cell viability and ΔΨm in menadione-treated astrocytes. The present data indicate that the major compounds accumulating in HHH syndrome impair mitochondrial function and reduce cell viability and the antioxidant defenses in cultured astrocytes especially when stressed by menadione. It is presumed that these mechanisms may be involved in the neuropathology of this disease.

  5. A “Hit and Run” Approach to Inducible Direct Reprogramming of Astrocytes to Neural Stem Cells

    Science.gov (United States)

    Poulou, Maria; Mandalos, Nikolaos P.; Karnavas, Theodoros; Saridaki, Marannia; McKay, Ronald D. G.; Remboutsika, Eumorphia

    2016-01-01

    Temporal and spatial control of gene expression can be achieved using an inducible system as a fundamental tool for regulated transcription in basic, applied and eventually in clinical research. We describe a novel “hit and run” inducible direct reprogramming approach. In a single step, 2 days post-transfection, transiently transfected Sox2FLAG under the Leu3p-αIPM inducible control (iSox2) triggers the activation of endogenous Sox2, redirecting primary astrocytes into abundant distinct nestin-positive radial glia cells. This technique introduces a unique novel tool for safe, rapid and efficient reprogramming amendable to regenerative medicine. PMID:27148066

  6. The role of astrocytes in Multiple Sclerosis progression

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    Jorge eCorreale

    2015-08-01

    Full Text Available Multiple sclerosis is an inflammatory disorder causing central nervous system demyelination and axonal injury. Although its etiology remains elusive, several lines of evidence support the concept that autoimmunity plays a major role in disease pathogenesis.The course ofMS is highly variable; nevertheless, the majority of patients initially present a relapsing-remitting clinical course. After 10-15 years of disease, this pattern becomes progressive in up to 50% of untreated patients, during which time clinical symptoms slowly cause constant deterioration over a period of many years. In about 15% of MS patients however, disease progression is relentless from disease onset. Published evidence supports the concept that progressive multiple sclerosis reflects a poorly understood mechanism of insidious axonal degeneration and neuronal loss. Recently, the type of microglial cell and of astrocyte activation and proliferation observed has suggested contribution of resident central nervous system cells may play a critical role in disease progression. Astrocytes could contribute to this process through several mechanisms: a as part of the innate immune system, b as a source of cytotoxic factors, c inhibiting re-myelination and axonal regeneration by forming a glial scar, and d contributing to axonal mitochondrial dysfunction. Furthermore, regulatory mechanisms mediated by astrocytes can be affected by aging. Notably, astrocytes might also limit the detrimental effects of pro-inflammatory factors, while providing support and protection for oligodendrocytes and neurons. Because of the dichotomy observed in astrocytic effects, the design of therapeutic strategies targeting astrocytes becomes a challenging endeavor. Better knowledge of molecular and functional properties of astrocytes therefore, should promote understanding of their specific role in multiple sclerosis pathophysiology, and consequently lead to development of novel and more successful

  7. Id4 and FABP7 are preferentially expressed in cells with astrocytic features in oligodendrogliomas and oligoastrocytomas

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    Nicholas M Kelly

    2005-07-01

    Full Text Available Abstract Background Oligodendroglioma (ODG and oligoastrocytoma (OAC are diffusely infiltrating primary brain tumors whose pathogenesis remains unclear. We previously identified a group of genes whose expression was inversely correlated with survival in a cohort of patients with glioblastoma (GBM, and some of these genes are also reportedly expressed in ODG and OAC. We examined the expression patterns and localization of these survival-associated genes in ODG and OAC in order to analyze their possible roles in the oncogenesis of these two tumor types. Methods We used UniGene libraries derived from GBM and ODG specimens to examine the expression levels of the transcripts for each of the 50 GBM survival-associated genes. We used immunohistochemistry and cDNA microarrays to examine expression of selected survival-associated genes and Id4, a gene believed to control the timing of oligodendrocyte development. The expression of FABP7 and Id4 and the survival of patients with ODG and OAC were also analyzed. Results Transcripts of most survival-associated genes as well as Id4 were present in both GBM and ODG tumors, whereas protein expression of Id4 and one of the survival-associated genes, brain-type fatty acid-binding protein (FABP7, was present in cells with astrocytic features, including reactive and neoplastic astrocytes, but not in neoplastic oligodendrocytes. Id4 was co-expressed with FABP7 in microgemistocytes in ODG and in neoplastic astrocytes in OAC. Id4 and FABP7 expression, however, did not correlate with the clinical outcome of patients with ODG or OAC tumors. Conclusion Expression of Id4 and some of our previously identified GBM survival-associated genes is present in developing or mature oligodendrocytes. However, protein expression of Id4 and FABP7 in GBM, ODG, and OAC suggests that this group of functionally important genes might demonstrate two patterns of expression in these glioma subtypes: one group is universally expressed in

  8. Connexin43 recruits PTEN and Csk to inhibit c-Src activity in glioma cells and astrocytes

    Science.gov (United States)

    González-Sánchez, Ana; Jaraíz-Rodríguez, Myriam; Domínguez-Prieto, Marta; Herrero-González, Sandra; Medina, José M.; Tabernero, Arantxa

    2016-01-01

    Connexin43 (Cx43), the major protein forming gap junctions in astrocytes, is reduced in high-grade gliomas, where its ectopic expression exerts important effects, including the inhibition of the proto-oncogene tyrosine-protein kinase Src (c-Src). In this work we aimed to investigate the mechanism responsible for this effect. The inhibition of c-Src requires phosphorylation at tyrosine 527 mediated by C-terminal Src kinase (Csk) and dephosphorylation at tyrosine 416 mediated by phosphatases, such as phosphatase and tensin homolog (PTEN). Our results showed that the antiproliferative effect of Cx43 is reduced when Csk and PTEN are silenced in glioma cells, suggesting the involvement of both enzymes. Confocal microscopy and immunoprecipitation assays confirmed that Cx43, in addition to c-Src, binds to PTEN and Csk in glioma cells transfected with Cx43 and in astrocytes. Pull-down assays showed that region 266–283 in Cx43 is sufficient to recruit c-Src, PTEN and Csk and to inhibit the oncogenic activity of c-Src. As a result of c-Src inhibition, PTEN was increased with subsequent inactivation of Akt and reduction of proliferation of human glioblastoma stem cells. We conclude that the recruitment of Csk and PTEN to the region between residues 266 and 283 within the C-terminus of Cx43 leads to c-Src inhibition. PMID:27391443

  9. Triptolide upregulates NGF synthesis in rat astrocyte cultures.

    Science.gov (United States)

    Xue, Bing; Jiao, Jian; Zhang, Lei; Li, Kai-Rong; Gong, Yun-Tao; Xie, Jun-Xia; Wang, Xiao-Min

    2007-07-01

    Triptolide (T10), an extract from the traditional Chinese herb, Tripterygium wilfordii Hook F (TWHF), has been shown to attenuate the rotational behavior induced by D: -amphetamine and prevent the loss of dopaminergic neurons in the substantia nigra in rat models of Parkinson's disease. To examine if the neuroprotective effect is mediated by its stimulation of production of neurotrophic factors from astrocytes, we investigated the effect of T10 on synthesis and release of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in rat astrocyte cultures. T10 did not affect the synthesis and release of either BDNF or GDNF. However, it significantly increased NGF mRNA expression. It also increased both intracellular NGF and NGF level in culture medium. These results indicate that the neuroprotective effect of T10 might be mediated, at least in part, via a stimulation of the production and release of NGF in astrocytes.

  10. Preclinical Studies of Induced Pluripotent Stem Cell-Derived Astrocyte Transplantation in ALS

    Science.gov (United States)

    2014-12-01

    a second trial testing transplantation of hESC-derived retinal pigment epithelium for macular degeneration has reported no safety concerns involving...ALS Association Investigator Meeting. Investigating Astrocyte-Mediated Degeneration of Upper Motor Neurons in ALS. Philadelphia, PA, USA 10/24/14...evident that aDepartment of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; bDepartments of Rehabilitation and

  11. 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.

  12. 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.

  13. Light and electron microscopic localization of GABAA-receptors on cultured cerebellar granule cells and astrocytes using immunohistochemical techniques

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Hösli, E; Belhage, B;

    1991-01-01

    . At the light microscope level specific staining of GABAA-receptors was localized in various types of neurones in explant cultures of rat cerebellum using the indirect peroxidase-antiperoxidase (PAP) technique, whereas no specific staining was found in astrocytes. At the electron microscope level labeling...... of GABAA-receptors was observed in the plasma membrane of both the cell bodies and processes in dissociated primary cultures of cerebellar granule cells using an indirect preembedding immunogold staining technique which in contrast to the classical PAP technique allows quantitative estimations...... to be performed. Quantification of the labeling intensity revealed a higher concentration of GABAA-receptors per microns plasma membrane in the cell bodies than in the processes. In discrete areas an extremely high density of the GABAA-receptors was observed. No specific labeling of GABAA-receptors was observed...

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

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    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.

  15. Molluscan cells in culture: primary cell cultures and cell lines.

    Science.gov (United States)

    Yoshino, T P; Bickham, U; Bayne, C J

    2013-06-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome.

  16. Sphingosine 1 Phosphate at the Blood Brain Barrier: Can the Modulation of S1P Receptor 1 Influence the Response of Endothelial Cells and Astrocytes to Inflammatory Stimuli?

    Directory of Open Access Journals (Sweden)

    Simona F Spampinato

    Full Text Available The ability of the Blood Brain Barrier (BBB to maintain proper barrier functions, keeping an optimal environment for central nervous system (CNS activity and regulating leukocytes' access, can be affected in CNS diseases. Endothelial cells and astrocytes are the principal BBB cellular constituents and their interaction is essential to maintain its function. Both endothelial cells and astrocytes express the receptors for the bioactive sphingolipid S1P. Fingolimod, an immune modulatory drug whose structure is similar to S1P, has been approved for treatment in multiple sclerosis (MS: fingolimod reduces the rate of MS relapses by preventing leukocyte egress from the lymph nodes. Here, we examined the ability of S1P and fingolimod to act on the BBB, using an in vitro co-culture model that allowed us to investigate the effects of S1P on endothelial cells, astrocytes, and interactions between the two. Acting selectively on endothelial cells, S1P receptor signaling reduced cell death induced by inflammatory cytokines. When acting on astrocytes, fingolimod treatment induced the release of a factor, granulocyte macrophage colony-stimulating factor (GM-CSF that reduced the effects of cytokines on endothelium. In an in vitro BBB model incorporating shear stress, S1P receptor modulation reduced leukocyte migration across the endothelial barrier, indicating a novel mechanism that might contribute to fingolimod efficacy in MS treatment.

  17. Induction of Neuronal Morphology in the 661W Cone Photoreceptor Cell Line with Staurosporine.

    Directory of Open Access Journals (Sweden)

    Alex F Thompson

    Full Text Available RGC-5 cells undergo differentiation into a neuronal phenotype with low concentrations of staurosporine. Although the RGC-5 cell line was initially thought to be of retinal ganglion cell origin, recent evidence suggests that the RGC-5 line could have been the result of contamination with 661W mouse cone photoreceptor cells. This raised the possibility that a cone photoreceptor cell line could be multipotent and could be differentiated to a neuronal phenotype.661W and RGC-5 cells, non-neuronal retinal astrocytes, retinal endothelial cells, retinal pericytes, M21 melanoma cells, K562 chronic myelogenous leukemia cells, and Daudi Burkitt lymphoma cells, were differentiated with staurosporine. The resulting morphology was quantitated using NeuronJ with respect to neurite counts and topology.Treatment with staurosporine induced similar-appearing morphological differentiation in both 661W and RGC-5 cells. The following measures were not significantly different between 661W and RGC-5 cells: number of neurites per cell, total neurite field length, number of neurite branch points, and cell viability. Neuronal-like differentiation was not observed in the other cell lines tested.661W and RGC-5 cells have virtually identical and distinctive morphology when differentiated with low concentrations of staurosporine. This result demonstrates that a retinal neuronal precursor cell with cone photoreceptor lineage can be differentiated to express a neuronal morphology.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. 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

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

    Science.gov (United States)

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

    2011-09-08

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

  1. Primary cultures of astrocytes

    DEFF Research Database (Denmark)

    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. 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

  3. 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.

  4. Redefining the concept of reactive astrocytes as cells that remain within their unique domains upon reaction to injury

    OpenAIRE

    2006-01-01

    Reactive astrocytes in neurotrauma, stroke, or neurodegeneration are thought to undergo cellular hypertrophy, based on their morphological appearance revealed by immunohistochemical detection of glial fibrillary acidic protein, vimentin, or nestin, all of them forming intermediate filaments, a part of the cytoskeleton. Here, we used a recently established dye-filling method to reveal the full three-dimensional shape of astrocytes assessing the morphology of reactive astrocytes in two neurotra...

  5. The mechanisms of in vitro cytotoxicity of mountain tea, Sideritis scardica, against the C6 glioma cell line.

    Science.gov (United States)

    Jeremic, Ivica; Tadic, Vanja; Isakovic, Andjelka; Trajkovic, Vladimir; Markovic, Ivanka; Redzic, Zoran; Isakovic, Aleksandra

    2013-11-01

    Sideritis scardica (mountain tea) is an endemic plant on the Balkan Peninsula traditionally used for treating different conditions, mainly of inflammatory nature. This study was aimed to examine the cytotoxic activity of different S. scardica extracts against the rat glioma C6 line and rat astrocytes in primary culture. The obtained data revealed that diethyl ether (extract 2) and ethyl acetate (extract 3) extracts of S. scardica exerted a cytotoxic effect on C6 rat glioma cells. Diethyl ether extract induced an increase in reactive oxygen species production, leading to apoptotic and autophagic cell death. Ethyl acetate extract induced G2 M cell cycle arrest and autophagy. None of the tested extracts was cytotoxic to rat astrocytes in primary culture. Cytotoxic effects of S. scardica extracts were, at least in part, mediated by their flavonoid constituents apigenin and luteolin that, when applied alone, induced cell cycle arrest, apoptosis, and autophagy.

  6. Molecular scaffolds underpinning macroglial polarization: an analysis of retinal Müller cells and brain astrocytes in mouse.

    Science.gov (United States)

    Enger, Rune; Gundersen, Georg Andreas; Haj-Yasein, Nadia Nabil; Eilert-Olsen, Martine; Thoren, Anna Elisabeth; Vindedal, Gry Fluge; Petersen, Pétur Henry; Skare, Øivind; Nedergaard, Maiken; Ottersen, Ole Petter; Nagelhus, Erlend A

    2012-12-01

    Key roles of macroglia are inextricably coupled to specialized membrane domains. The perivascular endfoot membrane has drawn particular attention, as this domain contains a unique complement of aquaporin-4 (AQP4) and other channel proteins that distinguishes it from perisynaptic membranes. Recent studies indicate that the polarization of macroglia is lost in a number of diseases, including temporal lobe epilepsy and Alzheimer's disease. A better understanding is required of the molecular underpinning of astroglial polarization, particularly when it comes to the significance of the dystrophin associated protein complex (DAPC). Here, we employ immunofluorescence and immunogold cytochemistry to analyze the molecular scaffolding in perivascular endfeet in macroglia of retina and three regions of brain (cortex, dentate gyrus, and cerebellum), using AQP4 as a marker. Compared with brain astrocytes, Müller cells (a class of retinal macroglia) exhibit lower densities of the scaffold proteins dystrophin and α-syntrophin (a DAPC protein), but higher levels of AQP4. In agreement, depletion of dystrophin or α-syntrophin--while causing a dramatic loss of AQP4 from endfoot membranes of brain astrocytes--had only modest or insignificant effect, respectively, on the AQP4 pool in endfoot membranes of Müller cells. In addition, while polarization of brain macroglia was less affected by dystrophin depletion than by targeted deletion of α-syntrophin, the reverse was true for retinal macroglia. These data indicate that the molecular scaffolding in perivascular endfeet is more complex than previously assumed and that macroglia are heterogeneous with respect to the mechanisms that dictate their polarization.

  7. 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.

  8. Bone morphogenetic protein 7 regulates reactive gliosis in retinal astrocytes and Müller glia

    OpenAIRE

    2014-01-01

    Purpose The focus of this study was to determine whether bone morphogenetic proteins (BMPs) trigger reactive gliosis in retinal astrocytes and/or Müller glial cells. Methods Retinal astrocytes and the Müller glial cell line MIO-M1 were treated with vehicle, BMP7, or BMP4. Samples from the treated cells were analyzed for changes in gliosis markers using reverse transcriptase – quantitative PCR (RT-qPCR) and western blotting. To determine potential similarities and differences in gliosis states...

  9. A phosphatase-independent gain-of-function mutation in PTEN triggers aberrant cell growth in astrocytes through an autocrine IGF-1 loop.

    Science.gov (United States)

    Fernández, S; Genis, L; Torres-Alemán, I

    2014-08-07

    Loss-of-function mutations in the phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome10) contribute to aberrant cell growth in part through upregulation of the mitogenic IGF-1/PI3K/Akt pathway. In turn, this pathway exerts a homeostatic feedback over PTEN. Using mutagenesis analysis to explore a possible impact of this mutual control on astrocyte growth, we found that truncation of the C-terminal region of PTEN (Δ51) associates with a marked increase in NFκB activity, a transcription factor overactivated in astrocyte tumors. Whereas mutations of PTEN are considered to lead to a loss-of-function, PTENΔ51, a truncation that comprises a region frequently mutated in human gliomas, displayed a neomorphic (gain-of-function) activity that was independent of its phosphatase activity. This gain-of-function of PTENΔ51 includes stimulation of IGF-1 synthesis through protein kinase A activation of the IGF-1 promoter. Increased IGF-1 originates an autocrine loop that activates Akt and NFκB. Constitutive activation of NFκB in PTENΔ51-expressing astrocytes leads to aberrant cell growth; astrocytes expressing this mutant PTEN generate colonies in vitro and tumors in vivo. Mutations converting a tumor suppressor such as PTEN into a tumor promoter through a gain-of-function involving IGF-1 production may further our understanding of the role played by this growth factor in glioma growth and help us define druggable targets for personalized therapy.

  10. 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.

  11. 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

  12. Relaxin protects astrocytes from hypoxia in vitro.

    Directory of Open Access Journals (Sweden)

    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.

  13. 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.

  14. 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

  15. Dopamine receptor activation increases glial cell line-derived neurotrophic factor in experimental stroke.

    Science.gov (United States)

    Kuric, Enida; Wieloch, Tadeusz; Ruscher, Karsten

    2013-09-01

    Treatment with levodopa enhances functional recovery after experimental stroke but its mechanisms of action are elusive. Reactive astrocytes in the ischemic hemisphere are involved in mechanisms promoting recovery and also express dopamine 1 (D1) and dopamine 2 (D2) receptors. Here we investigated if the activation of astrocytic dopamine receptors (D1 and D2) regulates the expression of glial cell line-derived neurotrophic factor (GDNF) after combined in vitro hypoxia/aglycemia (H/A) and studied the expression of GDNF in the ischemic brain after treatment with levodopa/benserazide following transient occlusion of the middle cerebral artery (tMCAO) in the rat. Twenty-four hours after H/A, GDNF levels were upregulated in exposed astrocytes compared to normoxic control cultures and further elevated by the addition of the selective D1 receptor agonist (R)-(+)-SKF-38393 hydrochloride while D1 receptor antagonism by R(+)-SCH-23390 hydrochloride significantly reduced GDNF. No effect on GDNF levels was observed by the application of the D2 receptor agonist R(-)-2,10,11-trihydroxy-N-propyl-noraporphine hydrobromide hydrate or S-(-)-eticlopride hydrochloride (D2 receptor antagonist). After tMCAO, GDNF was upregulated in D1 expressing reactive astrocytes in the peri-infarct area. In addition, treatment with levodopa/benserazide significantly increased GDNF levels in the infarct core and peri-infarct area after tMCAO without affecting the expression of glial fibrillar acidic protein (GFAP), an intermediate filament and marker of reactive gliosis. After stroke, GDNF levels increase in the ischemic hemisphere in rats treated with levodopa, implicating GDNF in the mechanisms of tissue reorganization and plasticity and in l-DOPA enhanced recovery of lost brain function. Our results support levodopa treatment as a potential recovery enhancing therapy in stroke patients.

  16. Effects of intrathecal injection of glial cell inhibitor on spinal cord astrocytes following chronic compression of dorsal root ganglia in rats

    Institute of Scientific and Technical Information of China (English)

    Xianhong Zhang; Wen Shen; Mingde Wang; Yinming Zeng

    2009-01-01

    obtained to count astrocytes under an inverted microscope. RESULTS: No significant differences in astrocyte count were detected between the fluorocitrate and model groups. Cell bodies were small with a few processes in the fluorocitrate group, compared with the model group. The astrocyte count decreased significantly in the minocycline group and the minocycline and fluorocitrate group compared with the sham operation, model, PBS and fluorocitrate groups (P < 0.01). The decrease in astrocyte count was mainly found in layers Ⅲ-Ⅳ of the spinal dorsal horn. Cell body volume was smaller and process numbers were fewer in the minocycline group and the minocycline and fluorocitrate group, compared with the model and PBS groups. CONCLUSION: Fluorocitrate can inhibit astrocyte activation, but does not affect astrocyte proliferation. However, minocycline can inhibit the activation and proliferation of astrocytes.

  17. 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

  18. Effects of triptolide on hippocampal microglial cells and astrocytes in the APP/PS1 double transgenic mouse model of Alzheimer’s disease

    Institute of Scientific and Technical Information of China (English)

    Jian-ming Li; Yan Zhang; Liang Tang; Yong-heng Chen; Qian Gao; Mei-hua Bao; Ju Xiang; De-liang Lei

    2016-01-01

    The principal pathology of Alzheimer’s disease includes neuronal extracellular deposition of amyloid-beta peptides and formation of senile pl aques, which in turn induce neuroinlfammation in the brain. Triptolide, a natural extract from the vine-like herb Tripterygium wilfordiiHook F, has potent anti-inlfammatory and immunosuppressive efifcacy. Therefore, we determined if triptolide can inhibit activation and proliferation of microglial cells and astrocytes in the APP/PS1 double transgenic mouse model of Alzheimer’s disease. We used 1 or 5 μg/kg/d triptolide to treat APP/PS1 double transgenic mice (aged 4–4.5 months) for 45 days. Unbiased stereology analysis found that triptolide dose-dependent-ly reduced the total number of microglial cells, and transformed microglial cells into the resting state. Further, triptolide (5 μg/kg/d) also reduced the total number of hippocampal astrocytes. Our in vivo test results indicate that triptolide suppresses activation and proliferation of microglial cells and astrocytes in the hippocampus of APP/PS1 double transgenic mice with Alzheimer’s disease.

  19. Neuron-glia interactions through the Heartless FGF receptor signaling pathway mediate morphogenesis of Drosophila astrocytes.

    Science.gov (United States)

    Stork, Tobias; Sheehan, Amy; Tasdemir-Yilmaz, Ozge E; Freeman, Marc R

    2014-07-16

    Astrocytes are critically important for neuronal circuit assembly and function. Mammalian protoplasmic astrocytes develop a dense ramified meshwork of cellular processes to form intimate contacts with neuronal cell bodies, neurites, and synapses. This close neuron-glia morphological relationship is essential for astrocyte function, but it remains unclear how astrocytes establish their intricate morphology, organize spatial domains, and associate with neurons and synapses in vivo. Here we characterize a Drosophila glial subtype that shows striking morphological and functional similarities to mammalian astrocytes. We demonstrate that the Fibroblast growth factor (FGF) receptor Heartless autonomously controls astrocyte membrane growth, and the FGFs Pyramus and Thisbe direct astrocyte processes to ramify specifically in CNS synaptic regions. We further show that the shape and size of individual astrocytes are dynamically sculpted through inhibitory or competitive astrocyte-astrocyte interactions and Heartless FGF signaling. Our data identify FGF signaling through Heartless as a key regulator of astrocyte morphological elaboration in vivo.

  20. Differentiation-Dependent Energy Production and Metabolite Utilization: A Comparative Study on Neural Stem Cells, Neurons, and Astrocytes

    Science.gov (United States)

    Jády, Attila Gy.; Nagy, Ádám M.; Kőhidi, Tímea; Ferenczi, Szilamér; Tretter, László

    2016-01-01

    While it is evident that the metabolic machinery of stem cells should be fairly different from that of differentiated neurons, the basic energy production pathways in neural stem cells (NSCs) or in neurons are far from clear. Using the model of in vitro neuron production by NE-4C NSCs, this study focused on the metabolic changes taking place during the in vitro neuronal differentiation. O2 consumption, H+ production, and metabolic responses to single metabolites were measured in cultures of NSCs and in their neuronal derivatives, as well as in primary neuronal and astroglial cultures. In metabolite-free solutions, NSCs consumed little O2 and displayed a higher level of mitochondrial proton leak than neurons. In stem cells, glycolysis was the main source of energy for the survival of a 2.5-h period of metabolite deprivation. In contrast, stem cell-derived or primary neurons sustained a high-level oxidative phosphorylation during metabolite deprivation, indicating the consumption of own cellular material for energy production. The stem cells increased O2 consumption and mitochondrial ATP production in response to single metabolites (with the exception of glucose), showing rapid adaptation of the metabolic machinery to the available resources. In contrast, single metabolites did not increase the O2 consumption of neurons or astrocytes. In “starving” neurons, neither lactate nor pyruvate was utilized for mitochondrial ATP production. Gene expression studies also suggested that aerobic glycolysis and rapid metabolic adaptation characterize the NE-4C NSCs, while autophagy and alternative glucose utilization play important roles in the metabolism of stem cell-derived neurons. PMID:27116891

  1. In situ probing of cholesterol in astrocytes at the single-cell level using laser desorption ionization mass spectrometric imaging with colloidal silver.

    Science.gov (United States)

    Perdian, D C; Cha, Sangwon; Oh, Jisun; Sakaguchi, Donald S; Yeung, Edward S; Lee, Young Jin

    2010-04-30

    Mass spectrometric imaging has been utilized to localize individual astrocytes and to obtain cholesterol populations at the single-cell level in laser desorption ionization (LDI) with colloidal silver. The silver ion adduct of membrane-bound cholesterol was monitored to detect individual cells. Good correlation between mass spectrometric and optical images at different cell densities indicates the ability to perform single-cell studies of cholesterol abundance. The feasibility of quantification is confirmed by the agreement between the LDI-MS ion signals and the results from a traditional enzymatic fluorometric assay. We propose that this approach could be an effective tool to study chemical populations at the cellular level.

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

    Directory of Open Access Journals (Sweden)

    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

  3. Difference in Membrane Repair Capacity Between Cancer Cell Lines and a Normal Cell Line.

    Science.gov (United States)

    Frandsen, Stine Krog; McNeil, Anna K; Novak, Ivana; McNeil, Paul L; Gehl, Julie

    2016-08-01

    Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested cancer cell lines (p normal cell line (98 % viable cells) was higher than in the three tested cancer cell lines (81-88 % viable cells). These data suggest more effective membrane repair in normal, primary cells and supplement previous explanations why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective on malignant cells compared to normal cells in cancer treatment.

  4. 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.

  5. Delayed transplantation of precursor cell-derived astrocytes provides multiple benefits in a rat model of Parkinsons.

    Science.gov (United States)

    Proschel, Christoph; Stripay, Jennifer L; Shih, Chung-Hsuan; Munger, Joshua C; Noble, Mark D

    2014-04-01

    In addition to dopaminergic neuron loss, it is clear that Parkinson disease includes other pathological changes, including loss of additional neuronal populations. As a means of addressing multiple pathological changes with a single therapeutically-relevant approach, we employed delayed transplantation of a unique class of astrocytes, GDAs(BMP), that are generated in vitro by directed differentiation of glial precursors. GDAs(BMP) produce multiple agents of interest as treatments for PD and other neurodegenerative disorders, including BDNF, GDNF, neurturin and IGF1. GDAs(BMP) also exhibit increased levels of antioxidant pathway components, including levels of NADPH and glutathione. Delayed GDA(BMP) transplantation into the 6-hydroxydopamine lesioned rat striatum restored tyrosine hydroxylase expression and promoted behavioral recovery. GDA(BMP) transplantation also rescued pathological changes not prevented in other studies, such as the rescue of parvalbumin(+) GABAergic interneurons. Consistent with expression of the synaptic modulatory proteins thrombospondin-1 and 2 by GDAs(BMP), increased expression of the synaptic protein synaptophysin was also observed. Thus, GDAs(BMP) offer a multimodal support cell therapy that provides multiple benefits without requiring prior genetic manipulation.

  6. Generation and properties of a new human ventral mesencephalic neural stem cell line

    Energy Technology Data Exchange (ETDEWEB)

    Villa, Ana; Liste, Isabel; Courtois, Elise T.; Seiz, Emma G.; Ramos, Milagros [Center of Molecular Biology ' Severo Ochoa' , Autonomous University of Madrid-C.S.I.C., Campus Cantoblanco 28049-Madrid (Spain); Meyer, Morten [Department of Anatomy and Neurobiology, Institute of Medical Biology, University of Southern Denmark, Winslowparken 21,st, DK-500, Odense C (Denmark); Juliusson, Bengt; Kusk, Philip [NsGene A/S, Ballerup (Denmark); Martinez-Serrano, Alberto, E-mail: amserrano@cbm.uam.es [Center of Molecular Biology ' Severo Ochoa' , Autonomous University of Madrid-C.S.I.C., Campus Cantoblanco 28049-Madrid (Spain)

    2009-07-01

    Neural stem cells (NSCs) are powerful research tools for the design and discovery of new approaches to cell therapy in neurodegenerative diseases like Parkinson's disease. Several epigenetic and genetic strategies have been tested for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new stable cell line of human neural stem cells derived from ventral mesencephalon (hVM1) based on v-myc immortalization. The cells expressed neural stem cell and radial glia markers like nestin, vimentin and 3CB2 under proliferation conditions. After withdrawal of growth factors, proliferation and expression of v-myc were dramatically reduced and the cells differentiated into astrocytes, oligodendrocytes and neurons. hVM1 cells yield a large number of dopaminergic neurons (about 12% of total cells are TH{sup +}) after differentiation, which also produce dopamine. In addition to proneural genes (NGN2, MASH1), differentiated cells show expression of several genuine mesencephalic dopaminergic markers such as: LMX1A, LMX1B, GIRK2, ADH2, NURR1, PITX3, VMAT2 and DAT, indicating that they retain their regional identity. Our data indicate that this cell line and its clonal derivatives may constitute good candidates for the study of development and physiology of human dopaminergic neurons in vitro, and to develop tools for Parkinson's disease cell replacement preclinical research and drug testing.

  7. 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

  8. 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

  9. 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

  10. 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.

  11. Astrocyte-Synapse Structural Plasticity

    Directory of Open Access Journals (Sweden)

    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. Characterizing Mystery Cell Lines: Student-driven Research Projects in an Undergraduate Neuroscience Laboratory Course.

    Science.gov (United States)

    Lemons, Michele L

    2012-01-01

    Inquiry-based projects promote discovery and retention of key concepts, increase student engagement, and stimulate interest in research. Described here are a series of lab exercises within an undergraduate upper level neuroscience course that train students to design, execute and analyze their own hypothesis-driven research project. Prior to developing their own projects, students learn several research techniques including aseptic cell culture, cell line maintenance, immunocytochemistry and fluorescent microscopy. Working in groups, students choose how to use these techniques to characterize and identify a "mystery" cell line. Each lab group is given a unique cell line with either a neural, astrocyte, or Schwann cell origin. Working together, students plan and execute experiments to determine the cellular origin and other unique characteristics of their mystery cell line. Students generate testable hypotheses, design interpretable experiments, generate and analyze data, and report their findings in both oral and written formats. Students receive instructor and peer feedback throughout the entire project. In summary, these labs train students the process of scientific research. This series of lab exercises received very strong positive feedback from the students. Reflections on student feedback and plans for future improvements are discussed.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. False leukemia-lymphoma cell lines: an update on over 500 cell lines.

    Science.gov (United States)

    Drexler, H G; Dirks, W G; Matsuo, Y; MacLeod, R A F

    2003-02-01

    Human leukemia-lymphoma (LL) cell lines represent an extremely important resource for research in a variety of fields and disciplines. As the cell lines are used as in vitro model systems in lieu of primary cell material, it is crucial that the cells in the culture flasks faithfully correspond to the purported objects of study. Obviously, proper authentication of cell line derivation and precise characterization are indispensable requirements to use as model systems. A number of studies has shown an unacceptable level of LL cell lines to be false. We present here the results of authenticating a comprehensively large sample (n = 550) of LL cell lines mainly by DNA fingerprinting and cytogenetic evaluation. Surprisingly, near-identical incidences (ca 15%) of false cell lines were observed among cell lines obtained directly from original investigators (59/395: 14.9%) and from secondary sources (23/155: 14.8%) implying that most cross-contamination is perpetrated by originators, presumably during establishment. By comparing our data with those published, we were further able to subclassify the false cell lines as (1) virtual: cross-contaminated with and unretrievably overgrown by other cell lines during initiation, never enjoying independent existence; (2) misidentified: cross-contaminated subsequent to establishment so that an original prototype may still exist; or (3) misclassified: unwittingly established from an unintended (often normal) cell type. Prolific classic leukemia cell lines were found to account for the majority of cross-contaminations, eg CCRF-CEM, HL-60, JURKAT, K-562 and U-937. We discuss the impact of cross-contaminations on scientific research, the reluctance of scientists to address the problem, and consider possible solutions. These findings provide a rationale for mandating the procurement of reputably sourced LL cell lines and their regular authentication thereafter.

  15. Loss of Sparc in p53-null Astrocytes Promotes Macrophage Activation and Phagocytosis Resulting in Decreased Tumor Size and Tumor Cell Survival.

    Science.gov (United States)

    Thomas, Stacey L; Schultz, Chad R; Mouzon, Ezekiell; Golembieski, William A; El Naili, Reima; Radakrishnan, Archanna; Lemke, Nancy; Poisson, Laila M; Gutiérrez, Jorge A; Cottingham, Sandra; Rempel, Sandra A

    2015-07-01

    Both the induction of SPARC expression and the loss of the p53 tumor suppressor gene are changes that occur early in glioma development. Both SPARC and p53 regulate glioma cell survival by inverse effects on apoptotic signaling. Therefore, during glioma formation, the upregulation of SPARC may cooperate with the loss of p53 to enhance cell survival. This study determined whether the loss of Sparc in astrocytes that are null for p53 would result in reduced cell survival and tumor formation and increased tumor immunogenicity in an in vivo xenograft brain tumor model. In vitro, the loss of Sparc in p53-null astrocytes resulted in an increase in cell proliferation, but a loss of tumorigenicity. At 7 days after intracranial implantation, Sparc-null tumors had decreased tumor cell survival, proliferation and reduced tumor size. The loss of Sparc promoted microglia/macrophage activation and phagocytosis of tumor cells. Our results indicate that the loss of p53 by deletion/mutation in the early stages of glioma formation may cooperate with the induction of SPARC to potentiate cancer cell survival and escape from immune surveillance.

  16. 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...

  17. 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

  18. Virus Discovery Using Tick Cell Lines

    Science.gov (United States)

    Bell-Sakyi, Lesley; Attoui, Houssam

    2016-01-01

    While ticks have been known to harbor and transmit pathogenic arboviruses for over 80 years, the application of high-throughput sequencing technologies has revealed that ticks also appear to harbor a diverse range of endogenous tick-only viruses belonging to many different families. Almost nothing is known about these viruses; indeed, it is unclear in most cases whether the identified viral sequences are derived from actual replication-competent viruses or from endogenous virus elements incorporated into the ticks’ genomes. Tick cell lines play an important role in virus discovery and isolation through the identification of novel viruses chronically infecting such cell lines and by acting as host cells to aid in determining whether or not an entire replication-competent, infective virus is present in a sample. Here, we review recent progress in tick-borne virus discovery and comment on the actual and potential applications for tick cell lines in this emerging research area. PMID:27679414

  19. Anions Govern Cell Volume: A Case Study of Relative Astrocytic and Neuronal Swelling in Spreading Depolarization.

    Directory of Open Access Journals (Sweden)

    Niklas Hübel

    Full Text Available Cell volume changes are ubiquitous in normal and pathological activity of the brain. Nevertheless, we know little about the dynamics of cell and tissue swelling, and the differential changes in the volumes of neurons and glia during pathological states such as spreading depolarizations (SD under ischemic and non-ischemic conditions, and epileptic seizures. By combining the Hodgkin-Huxley type spiking dynamics, dynamic ion concentrations, and simultaneous neuronal and astroglial volume changes into a comprehensive model, we elucidate why glial cells swell more than neurons in SD and the special case of anoxic depolarization (AD, and explore the relative contributions of the two cell types to tissue swelling. Our results demonstrate that anion channels, particularly Cl-, are intrinsically connected to cell swelling and blocking these currents prevents changes in cell volume. The model is based on a simple and physiologically realistic description. We introduce model extensions that are either derived purely from first physical principles of electroneutrality, osmosis, and conservation of particles, or by a phenomenological combination of these principles and known physiological facts. This work provides insights into numerous studies related to neuronal and glial volume changes in SD that otherwise seem contradictory, and is broadly applicable to swelling in other cell types and conditions.

  20. 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.

  1. Quantifying Filopodia in Cultured Astrocytes by an Algorithm.

    Science.gov (United States)

    Aumann, Georg; Friedländer, Felix; Thümmler, Matthias; Keil, Fabian; Brunkhorst, Robert; Korf, Horst-Werner; Derouiche, Amin

    2017-02-27

    Astrocytes in vivo extend thin processes termed peripheral astrocyte processes (PAPs), in particular around synapses where they can mediate glia-neuronal communication. The relation of PAPs to synapses is not based on coincidence, but it is not clear which stimuli and mechanisms lead to their formation and are active during process extension/ retraction in response to neuronal activity. Also, the molecular basis of the extremely fine PAP morphology (often 50 to 100 nm) is not understood. These open questions can be best investigated under in vitro conditions studying glial filopodia. We have previously analyzed filopodial mechanisms (Lavialle et al. PNAS 108:12915) applying an automated method for filopodia morphometry, which is now described in greater detail. The Filopodia Specific Shape Factor (FSSF) developed integrates number and length of filopodia. It quantifies filopodia independent of overall astrocytic shape or size, which can be intricate in itself. The algorithm supplied here permits automated image processing and measurements using ImageJ. Cells have to be sampled in higher numbers to obtain significant results. We validate the FSSF, and characterize the systematic influence of thresholding and camera pixel grid on measurements. We provide exemplary results of substance-induced filopodia dynamics (glutamate, mGluR agonists, EGF), and show that filopodia formation is highly sensitive to medium pH (CO2) and duration of cell culture. Although the FSSF was developed to study astrocyte filopodia with focus on the perisynaptic glial sheath, we expect that this parameter can also be applied to neuronal growth cones, non-neural cell types, or cell lines.

  2. 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

  3. Inflammatory cytokines IL-1β and TNF-α regulate p75NTR expression in CNS neurons and astrocytes by distinct cell-type-specific signalling mechanisms

    Directory of Open Access Journals (Sweden)

    Wilma J Friedman

    2009-05-01

    Full Text Available The p75NTR (where NTR is neurotrophin receptor can mediate many distinct cellular functions, including cell survival and apoptosis, axonal growth and cell proliferation, depending on the cellular context. This multifunctional receptor is widely expressed in the CNS (central nervous system during development, but its expression is restricted in the adult brain. However, p75NTR is induced by a variety of pathophysiological insults, including seizures, lesions and degenerative disease. We have demonstrated previously that p75NTR is induced by seizures in neurons, where it induces apoptosis, and in astrocytes, where it may regulate proliferation. In the present study, we have investigated whether the inflammatory cytokines IL (interleukin-1β and TNF-α (tumour necrosis factor-α, that are commonly elevated in these pathological conditions, mediate the regulation of p75NTR in neurons and astrocytes. We have further analysed the signal transduction pathways by which these cytokines induce p75NTR expression in the different cell types, specifically investigating the roles of the NF-κB (nuclear factor κB and p38 MAPK (mitogen-activated protein kinase pathways. We have demonstrated that both cytokines regulate p75NTR expression; however, the mechanisms governing this regulation are cytokine- and cell-type specific. The distinct mechanisms of cytokine-mediated p75NTR regulation that we demonstrate in the present study may facilitate therapeutic intervention in regulation of this receptor in a cell-selective manner.

  4. Achillolide A Protects Astrocytes against Oxidative Stress by Reducing Intracellular Reactive Oxygen Species and Interfering with Cell Signaling

    Directory of Open Access Journals (Sweden)

    Anat Elmann

    2016-03-01

    Full Text Available Achillolide A is a natural sesquiterpene lactone that we have previously shown can inhibit microglial activation. In this study we present evidence for its beneficial effects on astrocytes under oxidative stress, a situation relevant to neurodegenerative diseases and brain injuries. Viability of brain astrocytes (primary cultures was determined by lactate dehydrogenase (LDH activity, intracellular ROS levels were detected using 2′,7′-dichlorofluorescein diacetate, in vitro antioxidant activity was measured by differential pulse voltammetry, and protein phosphorylation was determined using specific ELISA kits. We have found that achillolide A prevented the H2O2-induced death of astrocytes, and attenuated the induced intracellular accumulation of reactive oxygen species (ROS. These activities could be attributed to the inhibition of the H2O2-induced phosphorylation of MAP/ERK kinase 1 (MEK1 and p44/42 mitogen-activated protein kinases (MAPK, and to the antioxidant activity of achillolide A, but not to H2O2 scavenging. This is the first study that demonstrates its protective effects on brain astrocytes, and its ability to interfere with MAPK activation. We propose that achillolide A deserves further evaluation for its potential to be developed as a drug for the prevention/treatment of neurodegenerative diseases and brain injuries where oxidative stress is part of the pathophysiology.

  5. Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines.

    Science.gov (United States)

    Romano, Paolo; Manniello, Assunta; Aresu, Ottavia; Armento, Massimiliano; Cesaro, Michela; Parodi, Barbara

    2009-01-01

    The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http://bioinformatics.istge.it/hypercldb/) is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (http://bioinformatics.istge.it/clima/), that allows to link authentication data to actual cell lines.

  6. Ecdysterone protects gerbil brain from temporal global cerebral ischemia/reperfusion injury via preventing neuron apoptosis and deactivating astrocytes and microglia cells.

    Science.gov (United States)

    Wang, Wei; Wang, Tao; Feng, Wan-Yu; Wang, Zhan-You; Cheng, Mao-Sheng; Wang, Yun-Jie

    2014-01-01

    Ecdysterone (EDS), a common derivative of ecdysteroid, has shown its effects on alleviating cognitive impairment and improving the cognition and memory. However, the mechanisms remain unknown. Using temporal global forebrain ischemia and reperfusion-induced brain injury as a model system, we investigated the roles of EDS in improving cognitive impairment in gerbil. Our results demonstrated that intraperitoneal injection of EDS obviously increased the number of surviving neuron cells by Nissl and neuronal nuclei (NeuN) staining. Indeed, the protecting effects of EDS are because of its ability to prevent the apoptosis of neuron cells as evidenced by TUNEL staining and caspase-3 deactivation in the brain of temporal global forebrain ischemia/reperfusion-treated gerbil. Moreover, EDS administration suppressed the ischemia stimulated activity of astrocytes and microglia cells by inhibiting the production of tumor necrosis alpha (TNF-α) in the brain of gerbil. More importantly, these actions of neurons and astrocytes/microglia cells in response to EDS treatment played pivotal roles in ameliorating the cognitive impairment in the ischemia/reperfusion-injured gerbil. In view of these observations, we not only decipher the mechanisms of EDS in reducing the syndrome of ischemia, but also provide novel perspectives to combat ischemic stroke.

  7. Co-culture with microglia promotes neural stem cells differentiation into astrocytes

    Institute of Scientific and Technical Information of China (English)

    GU Feng; WANG Juan; FU Li; MA Yong-jie

    2011-01-01

    Background Neural stem cells (NSCs) are a self-renewing and multipotent population of the central nervous system (CNS),which are active during development and maintain homeostasis and tissue integrity throughout life.Microglias are an immune cell population resident in the CNS,which have crucial physiological functions in the developing and adult CNS.This study aimed to investigate that whether microglia co-cultured with NSCs could promote astrogliogenesis from NSCs.Methods Microglia and NSCs were co-cultured in 24-well insert plates.NSCs were plated in the bottom of the well and microglia in the insert.Fluorescent staining,Western blotting and RT-PCR were used to determine the effect of microglia on NSCs differentiation.Results Co-culture of microglia and NSCs promoted astrogliogenesis from NSCs.Several key genes,such as Notch 1,Notch 2,Notch 3,Hes 5,and NRSFwera downregulated,while the critical genes Id1 and Id2 were upregulated.BMP2 and FGF2 were upregulated.Conclusion Microglias act as a regulator of NSCs astrogliogenesis.

  8. 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.

  9. 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.

  10. Radiation sensitivity of Merkel cell carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Leonard, J.H.; Ramsay, J.R.; Birrell, G.W. [Queensland Institute of Medical Research (Australia)] [and others

    1995-07-30

    Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

  11. Handling of Copper and Copper Oxide Nanoparticles by Astrocytes.

    Science.gov (United States)

    Bulcke, Felix; Dringen, Ralf

    2016-02-01

    Copper is an essential trace element for many important cellular functions. However, excess of copper can impair cellular functions by copper-induced oxidative stress. In brain, astrocytes are considered to play a prominent role in the copper homeostasis. In this short review we summarise the current knowledge on the molecular mechanisms which are involved in the handling of copper by astrocytes. Cultured astrocytes efficiently take up copper ions predominantly by the copper transporter Ctr1 and the divalent metal transporter DMT1. In addition, copper oxide nanoparticles are rapidly accumulated by astrocytes via endocytosis. Cultured astrocytes tolerate moderate increases in intracellular copper contents very well. However, if a given threshold of cellular copper content is exceeded after exposure to copper, accelerated production of reactive oxygen species and compromised cell viability are observed. Upon exposure to sub-toxic concentrations of copper ions or copper oxide nanoparticles, astrocytes increase their copper storage capacity by upregulating the cellular contents of glutathione and metallothioneins. In addition, cultured astrocytes have the capacity to export copper ions which is likely to involve the copper ATPase 7A. The ability of astrocytes to efficiently accumulate, store and export copper ions suggests that astrocytes have a key role in the distribution of copper in brain. Impairment of this astrocytic function may be involved in diseases which are connected with disturbances in brain copper metabolism.

  12. 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

  13. Stem cell characteristics in prostate cancer cell lines.

    NARCIS (Netherlands)

    Pfeiffer, M.J.; Schalken, J.A.

    2010-01-01

    BACKGROUND: Recent studies indicate the presence of a small, stem-like cell population in several human cancers that is crucial for the tumour (re)population. OBJECTIVE: Six established prostate cancer (PCa) cell lines-DU145, DuCaP, LAPC-4, 22Rv1, LNCaP, and PC-3-were examined for their stem cell pr

  14. 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.

  15. Astrocyte Hypertrophy Contributes to Aberrant Neurogenesis after Traumatic Brain Injury

    Directory of Open Access Journals (Sweden)

    Clark Robinson

    2016-01-01

    Full Text Available Traumatic brain injury (TBI is a widespread epidemic with severe cognitive, affective, and behavioral consequences. TBIs typically result in a relatively rapid inflammatory and neuroinflammatory response. A major component of the neuroinflammatory response is astrocytes, a type of glial cell in the brain. Astrocytes are important in maintaining the integrity of neuronal functioning, and it is possible that astrocyte hypertrophy after TBIs might contribute to pathogenesis. The hippocampus is a unique brain region, because neurogenesis persists in adults. Accumulating evidence supports the functional importance of these newborn neurons and their associated astrocytes. Alterations to either of these cell types can influence neuronal functioning. To determine if hypertrophied astrocytes might negatively influence immature neurons in the dentate gyrus, astrocyte and newborn neurons were analyzed at 30 days following a TBI in mice. The results demonstrate a loss of radial glial-like processes extending through the granule cell layer after TBI, as well as ectopic growth and migration of immature dentate neurons. The results further show newborn neurons in close association with hypertrophied astrocytes, suggesting a role for the astrocytes in aberrant neurogenesis. Future studies are needed to determine the functional significance of these alterations to the astrocyte/immature neurons after TBI.

  16. 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

  17. An astrocyte regenerative response from vimentin-containing cells in the spinal cord of amyotrophic lateral sclerosis's disease-like transgenic (G93A SOD1) mice.

    Science.gov (United States)

    Zhou, Yiyi; Lu, Yi; Fang, Xin; Zhang, Jie; Li, Jiao; Li, Shujuan; Deng, Xia; Yu, Yaqing; Xu, Renshi

    2015-01-01

    The reason for regeneration in the adult spinal cord during motor neuron degeneration in amyotrophic lateral sclerosis (ALS) remains largely unknown. To this end, we studied the alteration of vimentin (a neural precursor cells marker in CNS)-containing cells (VCCs) in spinal cord during different stages of ALS used C57BL/6J G93A SOD1 transgenic mice mimicking ALS. Results showed that VCCs were mostly distributed in the ependymal zone (EZ) surrounding the central canal of spinal cord in SOD1 wild type mice; a few of VCCs were sparsely distributed in other regions. However, the number of VCCs significantly increased in the spinal cord during the onset and progression stages of ALS. They were extensively distributed in the EZ, the anterior, the lateral and the posterior horn of grey matter, particularly in the posterior horn region at the progression stage. A majority of VCCs in the anterior, the lateral and the posterior horn of grey matter (outside of EZ) generated astrocytes, but no neurons, oligodendrocytes and microgliocytes. Our results suggested that there was a potential astrocyte regenerative response to motor neuron degeneration in motor neurons-degenerated regions in the adult spinal cord during the onset and progression stages of ALS-like disease. The regenerative responses in the adult spinal cord of ALS-like mice may be a potential pathway in attempting to repair the degenerated motor neurons and restore the dysfunctional neural circuitry.

  18. 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.

  19. Acrylonitrile-induced oxidative DNA damage in rat astrocytes.

    Science.gov (United States)

    Pu, Xinzhu; Kamendulis, Lisa M; Klaunig, James E

    2006-10-01

    Chronic administration of acrylonitrile results in a dose-related increase in astrocytomas in rat brain, but the mechanism of acrylonitrile carcinogenicity is not fully understood. The potential of acrylonitrile or its metabolites to induce direct DNA damage as a mechanism for acrylonitrile carcinogenicity has been questioned, and recent studies indicate that the mechanism involves the induction of oxidative stress in rat brain. The present study examined the ability of acrylonitrile to induce DNA damage in the DI TNC1 rat astrocyte cell line using the alkaline Comet assay. Oxidized DNA damage also was evaluated using formamidopyrimidine DNA glycosylase treatment in the modified Comet assay. No increase in direct DNA damage was seen in astrocytes exposed to sublethal concentrations of acrylonitrile (0-1.0 mM) for 24 hr. However, acrylonitrile treatment resulted in a concentration-related increase in oxidative DNA damage after 24 hr. Antioxidant supplementation in the culture media (alpha-tocopherol, (-)-epigallocathechin-3 gallate, or trolox) reduced acrylonitrile-induced oxidative DNA damage. Depletion of glutathione using 0.1 mM DL-buthionine-[S,R]-sulfoximine increased acrylonitrile-induced oxidative DNA damage (22-46%), while cotreatment of acrylonitrile with 2.5 mM L-2-oxothiazolidine-4-carboxylic acid, a precursor for glutathione biosynthesis, significantly reduced acrylonitrile-induced oxidative DNA damage (7-47%). Cotreatment of acrylonitrile with 0.5 mM 1-aminobenzotriazole, a suicidal inhibitor of cytochrome P450, prevented the oxidative DNA damage produced by acrylonitrile. Cyanide (0.1-0.5 mM) increased oxidative DNA damage (44-160%) in astrocytes. These studies demonstrate that while acrylonitrile does not directly damage astrocyte DNA, it does increase oxidative DNA damage. The oxidative DNA damage following acrylonitrile exposure appears to arise mainly through the P450 metabolic pathway; moreover, glutathione depletion may contribute to the

  20. Cellular radiosensitivity of small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Krarup, M; Poulsen, H S; Spang-Thomsen, M

    1997-01-01

    PURPOSE: The objective of this study was to determine the radiobiological characteristics of a panel of small-cell lung cancer (SCLC) cell lines by use of a clonogenic assay. In addition, we tested whether comparable results could be obtained by employing a growth extrapolation method based...

  1. Susceptibility testing of fish cell lines for virus isolation

    DEFF Research Database (Denmark)

    Ariel, Ellen; Skall, Helle Frank; Olesen, Niels Jørgen

    2009-01-01

    compare susceptibility between cell lines and between lineages within a laboratory and between laboratories (Inter-laboratory Proficiency Test). The objective being that the most sensitive cell line and lineages are routinely selected for diagnostic purposes.In comparing cell lines, we simulated "non......-cell-culture-adapted" virus by propagating the virus in heterologous cell lines to the one tested. A stock of test virus was produced and stored at - 80 °C and tests were conducted biannually. This procedure becomes complicated when several cell lines are in use and does not account for variation among lineages. In comparing...... cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test...

  2. Induced pluripotent stem cell lines derived from human somatic cells.

    Science.gov (United States)

    Yu, Junying; Vodyanik, Maxim A; Smuga-Otto, Kim; Antosiewicz-Bourget, Jessica; Frane, Jennifer L; Tian, Shulan; Nie, Jeff; Jonsdottir, Gudrun A; Ruotti, Victor; Stewart, Ron; Slukvin, Igor I; Thomson, James A

    2007-12-21

    Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

  3. 77 FR 5489 - Identification of Human Cell Lines Project

    Science.gov (United States)

    2012-02-03

    ... Genetics Group Web site at http://www.nist.gov/mml/biochemical/genetics/index.cfm . Once the total number... methods for human cell line authentication the identity of a cell line need no longer be in doubt. NIST...

  4. Expression of Cyclooxygenase-2 in Ovarian Cancer Cell Lines

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To investigate the expression of cyclooxygenase-2 (COX-2) in ovarian cancer cell lines,RT-PCR and immunocytochemistry were used to detect the expression of COX-2 in 5 ovarian cancer cell lines. The expression of COX-2 mRNA and protein was detected in all 5 cell lines. It is suggested that COX-2 is expressed in ovarian cancer cell lines, which provides a basis for the chemoprevention of ovarian cancer.

  5. Astrocyte Cultures Mimicking Brain Astrocytes in Gene Expression, Signaling, Metabolism and K(+) Uptake and Showing Astrocytic Gene Expression Overlooked by Immunohistochemistry and In Situ Hybridization.

    Science.gov (United States)

    Hertz, Leif; Chen, Ye; Song, Dan

    2017-01-01

    Based on differences in gene expression between cultured astrocytes and freshly isolated brain astrocytes it has been claimed that cultured astrocytes poorly reflect the characteristics of their in vivo counterparts. This paper shows that this is not the case with the cultures of mouse astrocytes we have used since 1978. The culture is prepared following guidelines provided by Drs. Monique Sensenbrenner and John Booher, with the difference that dibutyryl cyclic AMP is added to the culture medium from the beginning of the third week. This addition has only minor effects on glucose and glutamate metabolism, but it is crucial for effects by elevated K(+) concentrations and for Ca(2+) homeostasis, important aspects of astrocyte function. Work by Liang Peng and her colleagues has shown identity between not only gene expression but also drug-induced gene upregulations and editings in astrocytes cultured by this method and astrocytes freshly isolated from brains of drug-treated animals. Dr. Norenberg's laboratory has demonstrated identical upregulation of the cotransporter NKCC1 in ammonia-exposed astrocytes and rats with liver failure. Similarity between cultured and freshly isolated astrocytes has also been shown in metabolism, K(+) uptake and several aspects of signaling. However, others have shown that the gene for the glutamate transporter GLT1 is not expressed, and rat cultures show some abnormalities in K(+) effects. Nevertheless, the overall reliability of the cultured cells is important because immunohistochemistry and in situ hybridization poorly demonstrate many astrocytic genes, e.g., those of nucleoside transporters, and even microarray analysis of isolated cells can be misleading.

  6. The pivotal role of astrocytes in an in-vitro stroke model of the blood-brain barrier

    Directory of Open Access Journals (Sweden)

    Winfried eNeuhaus

    2014-10-01

    Full Text Available Stabilization of the blood-brain barrier during and after stroke can lead to less adverse outcome. For elucidation of underlying mechanisms and development of novel therapeutic strategies validated in-vitro disease models of the blood-brain barrier could be very helpful. To mimic in-vitro stroke conditions we have established a blood-brain barrier in-vitro model based on mouse cell line cerebEND and applied oxygen/glucose deprivation (OGD. The role of astrocytes in this disease model was investigated by using cell line C6. Transwell studies pointed out that addition of astrocytes during OGD increased the barrier damage significantly in comparison to the endothelial monoculture shown by changes of transendothelial electrical resistance as well as fluorescein permeability data. Analysis on mRNA and protein levels by qPCR, western blotting and immunofluorescence microscopy of tight junction molecules claudin-3,-5,-12, occludin and ZO-1 revealed that their regulation and localisation is associated with the functional barrier breakdown. Furthermore, soluble factors of astrocytes, OGD and their combination were able to induce changes of functionality and expression of ABC-transporters Abcb1a (P-gp, Abcg2 (bcrp and Abcc4 (mrp4. Moreover, the expression of proteases (matrixmetalloproteinases MMP-2, MMP-3 and MMP-9 and t-PA as well as of their endogenous inhibitors (TIMP-1, TIMP-3, PAI-1 was altered by astrocyte factors and OGD which resulted in significant changes of total MMP and t-PA activity. Morphological rearrangements induced by OGD and treatment with astrocyte factors were confirmed at a nanometer scale using atomic force microscopy. In conclusion, astrocytes play a major role in blood-brain barrier breakdown during OGD in vitro.

  7. Radiosensitivity of Human Melanoma Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

    Bergoc, R. M.; Medina, V.; Cricco, G.; Mohamed, N.; Martin, G.; Nunez, M.; Croci, M.; Crescenti, E. J.; Rivera, E. S.

    2004-07-01

    Cutaneous melanoma is a skin cancer resulting from the malign transformation of skin-pigment cells, the melanocytes. The radiotherapy, alone or in combination with other treatment, is an important therapy for this disease. the objective of this paper was to determine in vitro the radiosensitivity of two human melanoma cell lines with different metastatic capability: WM35 and MI/15, and to study the effect of drugs on radiobiological parameters. The Survival Curves were adjusted to the mathematical Linear-quadratic model using GrapsPad Prism software. Cells were seeded in RPMI medium (3000-3500 cells/flask), in triplicate and irradiated 24 h later. The irradiation was performed using an IBL 437C H Type equipment (189 TBq, 7.7 Gy/min) calibrated with a TLD 700 dosimeter. The range of Doses covered from 0 to 10 Gy and the colonies formed were counted at day 7th post-irradiation. Results obtained were: for WM35, {alpha}=0.37{+-}0.07 Gy''-1 and {beta}=0.06{+-}0.02 Gy''-2, for M1/15m {alpha}=0.47{+-}0.03 Gy''-1 and {beta}=0.06{+-}0.01 Gy''-2. The {alpha}/{beta} values WM35: {alpha}/{beta} values WM35: {alpha}/{beta}=6.07 Gy and M1/15: {alpha}/{beta}{sub 7}.33 Gy were similar, independently of their metastatic capabillity and indicate that both lines exhibit high radioresistance. Microscopic observation of irradiated cells showed multinuclear cells with few morphologic changes non-compatible with apoptosis. By means of specific fluorescent dyes and flow cytometry analysis we determined the intracellular levels of the radicals superoxide and hydrogen peroxide and their modulation in response to ionizing radiation. The results showed a marked decreased in H{sub 2}O{sub 2} intracellular levels with a simultaneous increase in superoxide that will be part of a mechanism responsible for induction of cell radioresistance. This response triggered by irradiated cells could not be abrogated by different treatments like histamine or the

  8. 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

  9. 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.

  10. 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.

  11. Zirconium oxide ceramic foam: a promising supporting biomaterial for massive production of glial cell line-derived neurotrophic factor.

    Science.gov (United States)

    Liu, Zhong-wei; Li, Wen-qiang; Wang, Jun-kui; Ma, Xian-cang; Liang, Chen; Liu, Peng; Chu, Zheng; Dang, Yong-hui

    2014-12-01

    This study investigated the potential application of a zirconium oxide (ZrO2) ceramic foam culturing system to the production of glial cell line-derived neurotrophic factor (GDNF). Three sets of ZrO2 ceramic foams with different pore densities of 10, 20, and 30 pores per linear inch (PPI) were prepared to support a 3D culturing system. After primary astrocytes were cultured in these systems, production yields of GDNF were evaluated. The biomaterial biocompatibility, cell proliferation and activation of cellular signaling pathways in GDNF synthesis and secretion in the culturing systems were also assessed and compared with a conventional culturing system. In this study, we found that the ZrO2 ceramic foam culturing system was biocompatible, using which the GDNF yields were elevated and sustained by stimulated cell proliferation and activation of signaling pathways in astrocytes cultured in the system. In conclusion, the ZrO2 ceramic foam is promising for the development of a GDNF mass production device for Parkinson's disease treatment.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. 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.

  14. In vitro study of uptake and synthesis of creatine and its precursors by cerebellar granule cells and astrocytes suggests some hypotheses on the physiopathology of the inherited disorders of creatine metabolism

    Directory of Open Access Journals (Sweden)

    Carducci Claudia

    2012-04-01

    Full Text Available Abstract Background The discovery of the inherited disorders of creatine (Cr synthesis and transport in the last few years disclosed the importance of blood Cr supply for the normal functioning of the brain. These putatively rare diseases share a common pathogenetic mechanism (the depletion of brain Cr and similar phenotypes characterized by mental retardation, language disturbances, seizures and movement disorders. In the effort to improve our knowledge on the mechanisms regulating Cr pool inside the nervous tissue, Cr transport and synthesis and related gene transcripts were explored in primary cultures of rat cerebellar granule cells and astrocytes. Methods Cr uptake and synthesis were explored in vitro by incubating monotypic primary cultures of rat type I astrocytes and cerebellar granule cells with: a D3-Creatine (D3Cr and D3Cr plus β-guanidinopropionate (GPA, an inhibitor of Cr transporter, and b labelled precursors of Guanidinoacetate (GAA and Cr (Arginine, Arg; Glycine, Gly. Intracellular D3Cr and labelled GAA and Cr were assessed by ESI-MS/MS. Creatine transporter (CT1, L-arginine:glycine amidinotransferase (AGAT, and S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT gene expression was assessed in the same cells by real time PCR. Results D3Cr signal was extremely high in cells incubated with this isotope (labelled/unlabelled Cr ratio reached about 10 and 122, respectively in cerebellar granule cells and astrocytes and was reduced by GPA. Labelled Arg and Gly were taken up by the cells and incorporated in GAA, whose concentration paralleled that of these precursors both in the extracellular medium and inside the cells (astrocytes. In contrast, the increase of labelled Cr was relatively much more limited since labelled Cr after precursors' supplementation did not exceed 2,7% (cerebellar granule cells and 21% (astrocytes of unlabelled Cr. Finally, AGAT, GAMT and SLC6A8 were expressed in both kind of cells. Conclusions Our

  15. Down-Regulation of Neurocan Expression in Reactive Astrocytes Promotes Axonal Regeneration and Facilitates the Neurorestorative Effects of Bone Marrow Stromal Cells in the Ischemic Rat Brain

    Institute of Scientific and Technical Information of China (English)

    LI HONG SHEN; YI LI; QI GAO; SMITA SAVANT-BHONSALE; MICHAEL CHOPP

    2008-01-01

    脑卒中后缺血组织边界形成胶质疤痕,抑制轴突再生.神经蛋白聚糖是一种轴突延长抑制分子,在卒中后胶质疤痕中表达上调.骨髓基质干细胞(BMSCs)可降低胶质疤痕壁的厚度,加速缺血周边区的轴突重塑.为了进一步明确BMSCs在轴突再生中的作用及机制,本文重点研究脑缺血组织中BMSCs对神经蛋白聚糖表达的作用.31只成年雄性Wistar大鼠大脑中动脉阻塞(MCAo)2 h,24 h后从中选择16只给予尾静脉注射3 × 106鼠BMSCs(BMSCs组),15只注射磷酸盐缓冲生理盐水(对照组).缺血后8 d处死实验大鼠,免疫染色表明反应性星形胶质细胞是神经蛋白聚糖的原始来源,且BMSCs组缺血半暗带脑组织的神经聚糖表达明显低于对照组,生长相关蛋白43表达高于对照组,这在蛋白印迹分析中得到确认.为了进一步检测BMSCs在星形胶质细胞神经蛋白聚糖表达中的作用,用激光捕获显微切割法从缺血周边区收集单纯的反应性星形胶质细胞.BMSCs组的神经蛋白聚糖基因表达明显下调(n=4/组).原代培养的星形胶质细胞也表现出相同改变,糖氧剥离的星形胶质细胞再给氧时与BMSCs共培养会抑制神经蛋白聚糖基因的表达上调(n=3/组).本研究表明BMSCs通过下调梗死周边星形胶质细胞中神经蛋白聚糖的表达来促进轴突再生.%The glial scar, a primarily astrocytic structure bordering the infarct tissue inhibits axonal regeneration after stroke. Neurocan, an axonal extension inhibitory molecule, is upregulated in the scar region after stroke. Bone marrow stromal cells (BMSCs) reduce the thickness of glial scar wall and facilitate axonal remodeling in the ischemic boundary zone. To further clarify the role of BMSCs in axonal regeneration and its underlying mechanism, the current study focused on the effect of BMSCs on neurocan expression in the ischemic brain. Thirty-one adult male Wistar rats were subjected to 2 h of middle

  16. A stereological analysis of NPY, POMC, Orexin, GFAP astrocyte, and Iba1 microglia cell number and volume in diet-induced obese male mice.

    Science.gov (United States)

    Lemus, Moyra B; Bayliss, Jacqueline A; Lockie, Sarah H; Santos, Vanessa V; Reichenbach, Alex; Stark, Romana; Andrews, Zane B

    2015-05-01

    The hypothalamic arcuate nucleus (ARC) contains 2 key neural populations, neuropeptide Y (NPY) and proopiomelanocortin (POMC), and, together with orexin neurons in the lateral hypothalamus, plays an integral role in energy homeostasis. However, no studies have examined total neuronal number and volume after high-fat diet (HFD) exposure using sophisticated stereology. We used design-based stereology to estimate NPY and POMC neuronal number and volume, as well as glial fibrillary acidic protein (astrocyte marker) and ionized calcium-binding adapter molecule 1 (microglia marker) cell number in the ARC; as well as orexin neurons in the lateral hypothalamus. Stereological analysis indicated approximately 8000 NPY and approximately 9000 POMC neurons in the ARC, and approximately 7500 orexin neurons in the lateral hypothalamus. HFD exposure did not affect total neuronal number in any population. However, HFD significantly increased average NPY cell volume and affected NPY and POMC cell volume distribution. HFD reduced orexin cell volume but had a bimodal effect on volume distribution with increased cells at relatively small volumes and decreased cells with relatively large volumes. ARC glial fibrillary acidic protein cells increased after 2 months on a HFD, although no significant difference after 6 months on chow diet or HFD was observed. No differences in ARC ionized calcium-binding adapter molecule 1 cell number were observed in any group. Thus, HFD affects ARC NPY or POMC neuronal cell volume number not cell number. Our results demonstrate the importance of stereology to perform robust unbiased analysis of cell number and volume. These data should be an empirical baseline reference to which future studies are compared.

  17. Astrocytes Control Neuronal Excitability in the Nucleus Accumbens

    Directory of Open Access Journals (Sweden)

    Tommaso Fellin

    2007-01-01

    Full Text Available Though accumulating evidence shows that the metabotropic glutamate receptor 5 (mGluR5 mediates some of the actions of extracellular glutamate after cocaine use, the cellular events underlying this action are poorly understood. In this review, we will discuss recent results showing that mGluR5 receptors are key regulators of astrocyte activity. Synaptic release of glutamate activates mGluR5 expressed in perisynaptic astrocytes and generates intense Ca2+ signaling in these cells. Ca2+ oscillations, in turn, trigger the release from astrocytes of the gliotransmitter glutamate, which modulates neuronal excitability by activating NMDA receptors. By integrating these results with the most recent evidence demonstrating the importance of astrocytes in the regulation of neuronal excitability, we propose that astrocytes are involved in mediating some of the mGluR5-dependent drug-induced behaviors.

  18. Superantigen presenting capacity of human astrocytes

    DEFF Research Database (Denmark)

    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...

  19. Synaptic modulation by astrocytes via Ca2+-dependent glutamate release.

    Science.gov (United States)

    Santello, M; Volterra, A

    2009-01-12

    In the past 15 years the classical view that astrocytes play a relatively passive role in brain function has been overturned and it has become increasingly clear that signaling between neurons and astrocytes may play a crucial role in the information processing that the brain carries out. This new view stems from two seminal observations made in the early 1990s: 1. astrocytes respond to neurotransmitters released during synaptic activity with elevation of their intracellular Ca2+ concentration ([Ca2+]i); 2. astrocytes release chemical transmitters, including glutamate, in response to [Ca2+]i elevations. The simultaneous recognition that astrocytes sense neuronal activity and release neuroactive agents has been instrumental for understanding previously unknown roles of these cells in the control of synapse formation, function and plasticity. These findings open a conceptual revolution, leading to rethink how brain communication works, as they imply that information travels (and is processed) not just in the neuronal circuitry but in an expanded neuron-glia network. In this review we critically discuss the available information concerning: 1. the characteristics of the astrocytic Ca2+ responses to synaptic activity; 2. the basis of Ca2+-dependent glutamate exocytosis from astrocytes; 3. the modes of action of astrocytic glutamate on synaptic function.

  20. Simultaneous neuron- and astrocyte-specific fluorescent marking

    Energy Technology Data Exchange (ETDEWEB)

    Schulze, Wiebke [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Hayata-Takano, Atsuko [Molecular Research Center for Children' s Mental Development, United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of Fukui, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kamo, Toshihiko [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Nakazawa, Takanobu, E-mail: takanobunakazawa-tky@umin.ac.jp [iPS Cell-based Research Project on Brain Neuropharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Nagayasu, Kazuki [iPS Cell-based Research Project on Brain Neuropharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kasai, Atsushi; Seiriki, Kaoru [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Interdisciplinary Program for Biomedical Sciences, Institute for Academic Initiatives, Osaka University, 1-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Shintani, Norihito [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Ago, Yukio [Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Farfan, Camille [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); and others

    2015-03-27

    Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignment of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains. - Highlights: • We develop a method for the specific fluorescent labeling of neurons and astrocytes. • Neuron-specific labeling is achieved using Scg10 and synapsin promoters. • Astrocyte-specific labeling is generated using the minimal GFAP promoter. • Nuclear localization of fluorescent proteins is achieved with histone 2B protein.

  1. EXPRESSION OF Fas LIGAND IN HUMAN COLON CANCER CELL LINES

    Institute of Scientific and Technical Information of China (English)

    张建军; 丁尔迅; 王强; 陈学云; 付志仁

    2001-01-01

    To investigate the expression of Fas ligand in human colon carcinoma cell lines. Methods: A total of six human colon cancer cell lines were examined for the expression of Fas ligand mRNA and cell surface protein by using RT-PCR and flow cytometry respectively. Results: The results showed that Fas ligand mRNA was expressed in all of the six cancer cell lines and Fas ligand cell surface protein was expressed in part of them. Conclusion: These data suggest that Fas ligand was expressed, at least in part, in human colon cancer cell lines and might facilitate to escape from immune surveillance of the host.

  2. Biological characteristics of cell lines of human dental alveolus

    Institute of Scientific and Technical Information of China (English)

    陈世璋; 黄靖香; 孙明学; 赵斌

    2003-01-01

    Objective To investigate the biological characteristics of cell lines of healthy and diseased human dental alveoli. Methods Primary cell lines from either healthy or diseased human dental alveoli were obtained. Two cell lines, H-258 and H-171 derived from healthy and diseased human tissues respectively, were selected for morphological study and research on their growth and aging, using cell counting, and histochemical and immunohistochemical staining. Results Primary cell lines were successfully established from innormal dental alveoli. After freezing and thawing for three times, cell growth was continued and no morphological alterations were observed. The doubling time was 53.4 hours and mean division index (MDI) was 4‰. Cells were kept normal after twenty generations with no obvious reduction of doubling time and MDI. Of twenty-six primary cell lines derived from healthy human dental alveoli, only three cell lines achieved generation. After freezing and thawing for twice, cultured cells were still alive at a decreased growth speed, with doubling time of 85.9 hours and MDI of 3‰. Both cell lines, H-171 and H-258, shared the characteristics of osteoblast. Conclusions Primary cell lines of diseased human dental alveoli show greater growth potential. All cell lines of dental alveoli share characteristics of osteoblast. The technique we developed may be put into practice for the treatment of abnormal dental alveoli.

  3. Molluscan cells in culture: primary cell cultures and cell lines

    OpenAIRE

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as bi...

  4. In vitro radiosensitivity of human leukemia cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Weichselbaum, R.R.; Greenberger, J.S.; Schmidt, A.; Karpas, A.; Moloney, W.C.; Little, J.B.

    1981-05-01

    The in vitro radiobiologic survival values (anti n, D/sub 0/) of four tumor lines derived from human hematopoietic tumors were studied. These cell lines were HL60 promyelocytic leukemia; K562 erythroleukemia; 45 acute lymphocytic leukemia; and 176 acute monomyelogenous leukemia. More cell lines must be examined before the exact relationship between in vitro radiosensitivity and clinical radiocurability is firmly established.

  5. 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.

  6. Astrocyte proliferation following stroke in the mouse depends on distance from the infarct.

    Directory of Open Access Journals (Sweden)

    George E Barreto

    Full Text Available Reactive gliosis is a hallmark of brain pathology and the injury response, yet the extent to which astrocytes proliferate, and whether this is central to astrogliosis is still controversial. We determined the fraction of mature astrocytes that proliferate in a mouse stroke model using unbiased stereology as a function of distance from the infarct edge. Cumulatively 11.1±1.2% of Aldh1l1(+ astrocytes within 400 µm in the cortical penumbra incorporate BrdU in the first week following stroke, while the overall number of astrocytes does not change. The number of astrocytes proliferating fell sharply with distance with more than half of all proliferating astrocytes found within 100 µm of the edge of the infarct. Despite extensive cell proliferation primarily of microglia and neutrophils/monocytes in the week following stroke, few mature astrocytes re-enter cell cycle, and these are concentrated close to the infarct boundary.

  7. Astrocyte proliferation following stroke in the mouse depends on distance from the infarct.

    Science.gov (United States)

    Barreto, George E; Sun, Xiaoyun; Xu, Lijun; Giffard, Rona G

    2011-01-01

    Reactive gliosis is a hallmark of brain pathology and the injury response, yet the extent to which astrocytes proliferate, and whether this is central to astrogliosis is still controversial. We determined the fraction of mature astrocytes that proliferate in a mouse stroke model using unbiased stereology as a function of distance from the infarct edge. Cumulatively 11.1±1.2% of Aldh1l1(+) astrocytes within 400 µm in the cortical penumbra incorporate BrdU in the first week following stroke, while the overall number of astrocytes does not change. The number of astrocytes proliferating fell sharply with distance with more than half of all proliferating astrocytes found within 100 µm of the edge of the infarct. Despite extensive cell proliferation primarily of microglia and neutrophils/monocytes in the week following stroke, few mature astrocytes re-enter cell cycle, and these are concentrated close to the infarct boundary.

  8. [In vitro co-cultivation of Toxoplasma gondii tachyzoites with rat brain astrocytes].

    Science.gov (United States)

    Li, Dong-na; Liang, You-sheng; Zhou, Yong-hua; Zhang, Huan-xiang; Sheng, Hai-ying; Luo, Wei; Gong, Wei; Zhuge, Hong-xiang

    2010-08-01

    Purified astrocytes were cultured in plates. When astrocytes grew over 80% of the plate, tachyzoites of Toxoplasma gondii RH strain were added for co-culture. In the period of 0-72 h, change of the astrocytes and tachyzoites was observed after Giemsa staining. In 0-48 h, monodansylcadaverine (MDC) was used to study the action of autophagy in the process of tachyzoites invading astrocytes. At 1 h co-culture, tachyzoites had entered in astrocytes and the autophagosomes appeared. At 4 h, the autophagosomes increased pronouncedly. However, after 12 h, number of autophagosomes considerably decreased and damage of the cells occurred. 48 h later, autophagosomes disappeared and more astrocytes were destroyed. At 72 h most cells destroyed and tachyzoites were released. The result showed that autophagy is inhibited when the astrocytes were in vitro infected by tachyzoites.

  9. Astrocytes Mediate In Vivo Cholinergic-Induced Synaptic Plasticity

    OpenAIRE

    2012-01-01

    In vivo and in vitro studies reveal that astrocytes, classically considered supportive cells for neurons, regulate synaptic plasticity in the mouse hippocampus and are directly involved in information storage.

  10. Cloning of aminopeptidase Npromoter and its activity in hematopoietic cell and different tumor cell lines

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Aminopeptidase N (APN) promoter region was cloned and sequenced from peripheral blood mononuclear cells. The recombinant reporter construct containing the promoter and luciferase gene, designated pXP1-APNLuc, was introduced into myeloblastic cell line, T lymphocyte cell line and various tumor cell lines. Luciferase assay showed that APN upstream promoter is myeloid-specific for high expression in myeloblastic cell line and much lower expres sion in T lymphocyte cell line. The promoter activity was relatively high in lung adenoma cell line compared with other tumor cell lines including hepatoma cell line, tong cancer cell line and esophageal cancer cell line in which the promoter activity significantly diminished or was almost undetectable. The characteristics of APN promoter may pro vide a new strategy for specific myeloprotection while tumor patients are being treated with chemotherapy and/or radio therapy.

  11. Dual Phases of Respiration Chain Defect-Augmented mROS-Mediated mCa2+ Stress during Oxidative Insult in Normal and ρ0 RBA1 Astrocytes

    Directory of Open Access Journals (Sweden)

    Tsung-I Peng

    2013-01-01

    Full Text Available Mitochondrial respiratory chain (RC deficits, resulting in augmented mitochondrial ROS (mROS generation, underlie pathogenesis of astrocytes. However, mtDNA-depleted cells (ρ0 lacking RC have been reported to be either sensitive or resistant to apoptosis. In this study, we sought to determine the effects of RC-enhanced mitochondrial stress following oxidative insult. Using noninvasive fluorescence probe-coupled laser scanning imaging microscopy, the ability to resist oxidative stress and levels of mROS formation and mitochondrial calcium (mCa2+ were compared between two different astrocyte cell lines, control and ρ0 astrocytes, over time upon oxidative stress. Our results showed that the cytoplasmic membrane becomes permeated with YO-PRO-1 dye at 150 and 130 minutes in RBA-1 and ρ0 astrocytes, respectively. In contrast to RBA-1, 30 minutes after 20 mM H2O2 exposure, ρ0 astrocytes formed marked plasma membrane blebs, lost the ability to retain Mito-R, and showed condensation of nuclei. Importantly, H2O2-induced ROS and accompanied mCa2+ elevation in control showed higher levels than ρ0 at early time point but vice versa at late time point. Our findings underscore dual phase of RC-defective cells harboring less mitochondrial stress due to low RC activity during short-term oxidative stress but augmented mROS-mediated mCa2+ stress during severe oxidative insult.

  12. Dual phases of respiration chain defect-augmented mROS-mediated mCa 2+ stress during oxidative insult in normal and ρ 0 RBA1 astrocytes.

    Science.gov (United States)

    Peng, Tsung-I; Lin, Muh-Shi; Jou, Mei-Jie

    2013-01-01

    Mitochondrial respiratory chain (RC) deficits, resulting in augmented mitochondrial ROS (mROS) generation, underlie pathogenesis of astrocytes. However, mtDNA-depleted cells (ρ (0)) lacking RC have been reported to be either sensitive or resistant to apoptosis. In this study, we sought to determine the effects of RC-enhanced mitochondrial stress following oxidative insult. Using noninvasive fluorescence probe-coupled laser scanning imaging microscopy, the ability to resist oxidative stress and levels of mROS formation and mitochondrial calcium (mCa(2+)) were compared between two different astrocyte cell lines, control and ρ (0) astrocytes, over time upon oxidative stress. Our results showed that the cytoplasmic membrane becomes permeated with YO-PRO-1 dye at 150 and 130 minutes in RBA-1 and ρ (0) astrocytes, respectively. In contrast to RBA-1, 30 minutes after 20 mM H2O2 exposure, ρ (0) astrocytes formed marked plasma membrane blebs, lost the ability to retain Mito-R, and showed condensation of nuclei. Importantly, H2O2-induced ROS and accompanied mCa(2+) elevation in control showed higher levels than ρ (0) at early time point but vice versa at late time point. Our findings underscore dual phase of RC-defective cells harboring less mitochondrial stress due to low RC activity during short-term oxidative stress but augmented mROS-mediated mCa(2+) stress during severe oxidative insult.

  13. 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.

  14. 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.

  15. Involvement of the Notch pathway in terminal astrocytic differentiation: role of PKA

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    Carla Angulo‑Rojo

    2013-12-01

    Full Text Available The Notch pathway is a highly conserved signaling system essential for modulating neurogenesis and promoting astrogenesis. Similarly, the cAMP signaling cascade can promote astrocytic commitment in several cell culture models, such as the C6 glioma cell line. These cells have the capacity to differentiate into oligodendrocytes or astrocytes, characteristics that allow their use as a glial progenitor model. In this context, we explore here the plausible involvement of cAMP in Notch-dependent signal transactions. The exposure of C6 cells to a non-hydrolysable cAMP analogue resulted in a sustained augmentation of Notch activity, as detected by nuclear translocation of its intracellular domain portion (NICD and transcriptional activity. The cAMP effect is mediated through the activation of the γ-secretase complex, responsible for Notch cleavage and is sensitive to inhibitors of the cAMP-dependent protein kinase, PKA. As expected, Notch cleavage and nuclear translocation resulted in the up-regulation of the mRNA levels of one of its target genes, the transcription factor Hair and enhancer of split 5. Moreover, the glutamate uptake activity, as well as the expression of astrocytic markers such as glial fibrillary acidic protein, S100β protein and GLAST was also enhanced in cAMP-exposed cells. Our results clearly suggest that during the process of C6 astrocytic differentiation, cAMP activates the PKA/γ-secretase/NICD/RBPJκ pathway and Notch1 expression, leading to transcriptional activation of the genes responsible for glial progenitor cell fate decision.

  16. Involvement of the Notch Pathway in Terminal Astrocytic Differentiation: Role of PKA

    Directory of Open Access Journals (Sweden)

    Carla Angulo-Rojo

    2013-11-01

    Full Text Available The Notch pathway is a highly conserved signaling system essential for modulating neurogenesis and promoting astrogenesis. Similarly, the cAMP signaling cascade can promote astrocytic commitment in several cell culture models, such as the C6 glioma cell line. These cells have the capacity to differentiate into oligodendrocytes or astrocytes, characteristics that allow their use as a glial progenitor model. In this context, we explore here the plausible involvement of cAMP in Notch-dependent signal transactions. The exposure of C6 cells to a non-hydrolysable cAMP analogue resulted in a sustained augmentation of Notch activity, as detected by nuclear translocation of its intracellular domain portion (NICD and transcriptional activity. The cAMP effect is mediated through the activation of the γ-secretase complex, responsible for Notch cleavage and is sensitive to inhibitors of the cAMP-dependent protein kinase, PKA. As expected, Notch cleavage and nuclear translocation resulted in the up-regulation of the mRNA levels of one of its target genes, the transcription factor Hair and enhancer of split 5. Moreover, the glutamate uptake activity, as well as the expression of astrocytic markers such as glial fibrillary acidic protein, S100β protein and GLAST was also enhanced in cAMP-exposed cells. Our results clearly suggest that during the process of C6 astrocytic differentiation, cAMP activates the PKA/γ-secretase/NICD/RBPJK pathway and Notch1 expression, leading to transcriptional activation of the genes responsible for glial progenitor cell fate decision.

  17. MODERATE CYTOTOXICITY OF PROANTHOCYANIDINS TO HUMAN TUMOR-CELL LINES

    NARCIS (Netherlands)

    KOLODZIEJ, H; HABERLAND, C; WOERDENBAG, HJ; KONINGS, AWT

    1995-01-01

    In the present study the cytotoxicity of 16 proanthocyanidins was evaluated in GLC(4), a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC50 values ranging from 18 to >200 mu m following continuous

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

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    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

  19. 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

  20. Transfer of mitochondria from astrocytes to neurons after stroke

    Science.gov (United States)

    Hayakawa, Kazuhide; Esposito, Elga; Wang, Xiaohua; Terasaki, Yasukazu; Liu, Yi; Xing, Changhong; Ji, Xunming; Lo, Eng H.

    2016-01-01

    Recently, it was suggested that neurons can release and transfer damaged mitochondria to astrocytes for disposal and recycling 1. This ability to exchange mitochondria may represent a potential mode of cell-cell signaling in the central nervous system (CNS). Here, we show that astrocytes can also release functional mitochondria that enter into neurons. Astrocytic release of extracellular mitochondria particles was mediated by a calcium-dependent mechanism involving CD38/cyclic ADP ribose signaling. Transient focal cerebral ischemia in mice induced astrocytic mitochondria entry to adjacent neurons that amplified cell survival signals. Suppression of CD38 signaling with siRNA reduced extracellular mitochondria transfer and worsened neurological outcomes. These findings suggest a new mitochondrial mechanism of neuroglial crosstalk that may contribute to endogenous neuroprotective and neurorecovery mechanisms after stroke. PMID:27466127

  1. Versatile and simple approach to determine astrocyte territories in mouse neocortex and hippocampus.

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    Antje Grosche

    Full Text Available BACKGROUND: Besides their neuronal support functions, astrocytes are active partners in neuronal information processing. The typical territorial structure of astrocytes (the volume of neuropil occupied by a single astrocyte is pivotal for many aspects of glia-neuron interactions. METHODS: Individual astrocyte territorial volumes are measured by Golgi impregnation, and astrocyte densities are determined by S100β immunolabeling. These data are compared with results from conventionally applied methods such as dye filling and determination of the density of astrocyte networks by biocytin loading. Finally, we implemented our new approach to investigate age-related changes in astrocyte territories in the cortex and hippocampus of 5- and 21-month-old mice. RESULTS: The data obtained by our simplified approach based on Golgi impregnation were compared to previously published dye filling experiments, and yielded remarkably comparable results regarding astrocyte territorial volumes. Moreover, we found that almost all coupled astrocytes (as indicated by biocytin loading were immunopositive for S100β. A first application of this new experimental approach gives insight in age-dependent changes in astrocyte territorial volumes. They increased with age, while cell densities remained stable. In 5-month-old mice, the overlap factor was close to 1, revealing little or no interdigitation of astrocyte territories. However, in 21-month-old mice, the overlap factor was more than 2, suggesting that processes of adjacent astrocytes interdigitate. CONCLUSION: Here we verified the usability of a simple, versatile method for assessing astrocyte territories and the overlap factor between adjacent territories. Second, we found that there is an age-related increase in territorial volumes of astrocytes that leads to loss of the strict organization in non-overlapping territories. Future studies should elucidate the physiological relevance of this adaptive reaction of

  2. POTENTIAL CELL LINE TOXICITY OF ENVIRONMENTAL NANOPARTICLES

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    Mohan Durga

    2012-01-01

    Full Text Available In India, the unprecedented growth rate and urbanization along with the rapid increase in motor vehicle activity and industrialization are contributing to high levels of urban air pollution. The population is mainly exposed to high air pollution concentrations, where motor vehicle emissions constitute the main source of fine and ultrafine particles. Motor exhaust emissions is a mixture of gases and Particulate Matter (PM. Diesel and petrol fuels in vehicles produce combustion-derived particles as a result of combustion. Vehicle exhaust particles are the main constituents of environmental nanoparticles. In the present investigation, environmental nanoparticles such as Diesel Exhaust Particles (DEP and Petrol Exhaust Particles (PEP were collected from on-road vehicles using a specially designed collection chamber. The surface morphology of the collected particles was analyzed through Transmission Electron Microscope (TEM, and the elemental mapping was performed through EDAX analysis. Results indicated the presence of nanometer-size particles in both the categories of vehicle exhaust. These small-size particles of respirable range can enter the respiratory tract of humans and get deposited in the lungs and cause various effects inside the human body. The aim of this study is to assess the cytotoxicity of the collected Diesel Exhaust Nanoparticles (DENPs and Petrol Exhaust Nanoparticles (PENPs. Cytotoxicity endpoint, such as IC50 (50% Inhibitory Concentration, was determined after a 24-h exposure. Results of this study indicated that all five cell lines were sensitive to these vehicle exhaust nanoparticles at varying levels.

  3. Derivation and Utilization of Functional CD8(+) Dendritic Cell Lines.

    Science.gov (United States)

    Pigni, Matteo; Ashok, Devika; Acha-Orbea, Hans

    2016-01-01

    It is notoriously difficult to obtain large quantities of non-activated dendritic cells ex vivo. For this reason, we produced and characterized a mouse model expressing the large T oncogene under the CD11c promoter (Mushi mice), in which CD8α(+) dendritic cells transform after 4 months. We derived a variety of stable cell lines from these primary lines. These cell lines reproducibly share with freshly isolated dendritic cells most surface markers, mRNA and protein expression, and all tested biological functions. Cell lines can be derived from various strains and knockout mice and can be easily transduced with lentiviruses. In this article, we describe the derivation, culture, and lentiviral transduction of these dendritic cell lines.

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

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    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.

  5. The Refsum disease marker phytanic acid, a branched chain fatty acid, affects Ca2+ homeostasis and mitochondria, and reduces cell viability in rat hippocampal astrocytes.

    Science.gov (United States)

    Kahlert, Stefan; Schönfeld, Peter; Reiser, Georg

    2005-02-01

    The saturated branched chain fatty acid, phytanic acid, a degradation product of chlorophyll, accumulates in Refsum disease, an inherited peroxisomal disorder with neurological clinical features. To elucidate the pathogenic mechanism, we investigated the influence of phytanic acid on cellular physiology of rat hippocampal astrocytes. Phytanic acid (100 microM) induced an immediate transient increase in cytosolic Ca2+ concentration, followed by a plateau. The peak of this biphasic Ca2+ response was largely independent of extracellular Ca2+, indicating activation of cellular Ca2+ stores by phytanic acid. Phytanic acid depolarized mitochondria without causing in situ swelling of mitochondria. The slow decrease of mitochondrial potential is not consistent with fast and simultaneous opening of the mitochondrial permeability transition pore. However, phytanic acid induced substantial generation of reactive oxygen species. Phytanic acid caused astroglia cell death after a few hours of exposure. We suggest that the cytotoxic effect of phytanic acid seems to be due to a combined action on Ca2+ regulation, mitochondrial depolarization, and increased ROS generation in brain cells.

  6. Investigation of the selenium metabolism in cancer cell lines

    DEFF Research Database (Denmark)

    Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan

    2011-01-01

    incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size...... except MeSeA. Speciation analysis showed that MeSeA was completely transformed during the incubations, while metabolic conversion of the other Se compounds was limited. Production of volatile dimethyl diselenide was observed for MeSeA and MeSeCys. MeSeA, MeSeCys and selenite showed noticeable protein......The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line...

  7. Imaging of mitochondrial Ca2+ dynamics in astrocytes using cell-specific mitochondria-targeted GCaMP5G/6s: mitochondrial Ca2+ uptake and cytosolic Ca2+ availability via the endoplasmic reticulum store.

    Science.gov (United States)

    Li, Hailong; Wang, Xiaowan; Zhang, Nannan; Gottipati, Manoj K; Parpura, Vladimir; Ding, Shinghua

    2014-12-01

    Mitochondrial Ca(2+) plays a critical physiological role in cellular energy metabolism and signaling, and its overload contributes to various pathological conditions including neuronal apoptotic death in neurological diseases. Live cell mitochondrial Ca(2+) imaging is an important approach to understand mitochondrial Ca(2+) dynamics. Recently developed GCaMP genetically-encoded Ca(2+) indicators provide unique opportunity for high sensitivity/resolution and cell type-specific mitochondrial Ca(2+) imaging. In the current study, we implemented cell-specific mitochondrial targeting of GCaMP5G/6s (mito-GCaMP5G/6s) and used two-photon microscopy to image astrocytic and neuronal mitochondrial Ca(2+) dynamics in culture, revealing Ca(2+) uptake mechanism by these organelles in response to cell stimulation. Using these mitochondrial Ca(2+) indicators, our results show that mitochondrial Ca(2+) uptake in individual mitochondria in cultured astrocytes and neurons can be seen after stimulations by ATP and glutamate, respectively. We further studied the dependence of mitochondrial Ca(2+) dynamics on cytosolic Ca(2+) changes following ATP stimulation in cultured astrocytes by simultaneously imaging mitochondrial and cytosolic Ca(2+) increase using mito-GCaMP5G and a synthetic organic Ca(2+) indicator, x-Rhod-1, respectively. Combined with molecular intervention in Ca(2+) signaling pathway, our results demonstrated that the mitochondrial Ca(2+) uptake is tightly coupled with inositol 1,4,5-trisphosphate receptor-mediated Ca(2+) release from the endoplasmic reticulum and the activation of G protein-coupled receptors. The current study provides a novel approach to image mitochondrial Ca(2+) dynamics as well as Ca(2+) interplay between the endoplasmic reticulum and mitochondria, which is relevant for neuronal and astrocytic functions in health and disease.

  8. 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.

  9. Effects of huperzine A on secretion of nerve growth factor in cultured rat cortical astrocytes and neurite outgrowth in rat PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Li-li TANG; Rui WANG; Xi-can TANG

    2005-01-01

    Aim: To study the effects of huperzine A (HupA) on neuritogenic activity and the expression of nerve growth factor (NGF). Methods: After being treated with 10 μmol/L HupA, neurite outgrowth of PC12 cells was observed and counted under phase-contrast microscopy. Mitogenic activity was assayed by [3H]thymidine incorporation. Cell cytotoxicity was evaluated by lactate dehydrogenase (LDH)release. AChE activity, mRNA and protein expression were measured by the Ellman's method, RT-PCR, and Western blot, respectively. NGF mRNA and protein levels were determined by RT-PCR and ELISA assays. Results: Treatment of PC 12 cells with 10 μmol/L HupA for 48 h markedly increased the number of neuritebearing cells, but caused no significant alteration in cell viability or other signs of cytotoxicity. In addition to inhibiting AChE activity, 10 μmol/L HupA also increased the mRNA and protein levels of this enzyme. In addition, following 2 h exposure of the astrocytes to 10 μmol/L HupA, there was a significant up-regulation of mRNA for NGF and P75 low-affinity NGF receptor. The protein level of NGF was also increased after 24 h treatment with HupA. Conclusion: Our findings demonstrate for the first time that HupA has a direct or indirect neurotrophic activity, which might be beneficial in treatment of neurodegenerative disorders such as Alzheimer disease.

  10. 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.

  11. YAP stabilizes SMAD1 and promotes BMP2-induced neocortical astrocytic differentiation.

    Science.gov (United States)

    Huang, Zhihui; Hu, Jinxia; Pan, Jinxiu; Wang, Ying; Hu, Guoqing; Zhou, Jiliang; Mei, Lin; Xiong, Wen-Cheng

    2016-07-01

    ‪YAP (yes-associated protein), a key transcriptional co-factor that is negatively regulated by the Hippo pathway, is crucial for the development and size control of multiple organs, including the liver. However, its role in the brain remains unclear. Here, we provide evidence for YAP regulation of mouse neocortical astrocytic differentiation and proliferation. YAP was undetectable in neurons, but selectively expressed in neural stem cells (NSCs) and astrocytes. YAP in NSCs was required for neocortical astrocytic differentiation, with no apparent role in self-renewal or neural differentiation. However, YAP in astrocytes was necessary for astrocytic proliferation. Yap (Yap1) knockout, Yap(nestin) conditional knockout and Yap(GFAP) conditional knockout mice displayed fewer neocortical astrocytes and impaired astrocytic proliferation and, consequently, death of neocortical neurons. Mechanistically, YAP was activated by BMP2, and the active/nuclear YAP was crucial for BMP2 induction and stabilization of SMAD1 and astrocytic differentiation. Expression of SMAD1 in YAP-deficient NSCs partially rescued the astrocytic differentiation deficit in response to BMP2. Taken together, these results identify a novel function of YAP in neocortical astrocytic differentiation and proliferation, and reveal a BMP2-YAP-SMAD1 pathway underlying astrocytic differentiation in the developing mouse neocortex.

  12. 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.

  13. Authentication of the R06E Fruit Bat Cell Line

    Directory of Open Access Journals (Sweden)

    Ingo Jordan

    2012-05-01

    Full Text Available Fruit bats and insectivorous bats are believed to provide a natural reservoir for a wide variety of infectious diseases. Several lines of evidence, including the successful isolation of infectious viruses, indicate that Marburg virus and Ravn virus have found a major reservoir in colonies of the Egyptian rousette (Rousettus aegyptiacus. To facilitate molecular studies on virus-reservoir host interactions and isolation of viruses from environmental samples, we established cell lines from primary cells of this animal. The cell lines were given to several laboratories until we realized that a contamination with Vero cells in one of the cultures had occurred. Here we describe a general diagnostic procedure for identification of cross-species contamination with the focus on Vero and Rousettus cell lines, and summarize newly discovered properties of the cell lines that may pertain to pathogen discovery.

  14. Abundance of Flt3 and its ligand in astrocytic tumors

    Directory of Open Access Journals (Sweden)

    Eßbach C

    2013-05-01

    Full Text Available C Eßbach,1 N Andrae,1 D Pachow,1 J-P Warnke,2 A Wilisch-Neumann,1 E Kirches,1 C Mawrin11Department of Neuropathology, Otto-von-Guericke University, Magdeburg, 2Department of Neurosurgery, Paracelsus Hospital, Zwickau, GermanyBackground: Molecular targeted therapies for astrocytic tumors are the subject of growing research interest, due to the limited response of these tumors, especially glioblastoma multiforme, to conventional chemotherapeutic regimens. Several of these approaches exploit the inhibition of receptor tyrosine kinases. To date, it has not been elucidated if fms-like tyrosine kinase-3 (Flt3 and its natural ligand (Flt3L are expressed in astrocytic tumors, although some of the clinically intended small-molecule receptor tyrosine kinase inhibitors affect Flt3, while others do not. More importantly, the recent proof of principle for successful stimulation of the immune system against gliomas in preclinical models via local Flt3L application requires elucidation of this receptor tyrosine kinase pathway in these tumors in more detail. This therapy is based on recruitment of Flt3-positive dendritic cells, but may be corroborated by activity of this signaling pathway in glioma cells.Methods: Receptor and ligand expression was analyzed by real-time polymerase chain reaction in 31 astrocytic tumors (six diffuse and 11 anaplastic astrocytomas, 14 glioblastomas derived from patients of both genders and in glioblastoma cell lines. The two most common activating mutations of the Flt3 gene, ie, internal tandem duplication and D835 point mutation, were assessed by specific polymerase chain reaction.Results: A relatively high abundance of Flt3L mRNA (4%–6% of the reference, β2 microglobulin could be demonstrated in all tumor samples. Flt3 expression could generally be demonstrated by 40 specific polymerase chain reaction cycles and gel electrophoresis in 87% of the tumors, including all grades, although the small quantities of the receptor did

  15. 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

  16. Effect of leptin on cell proliferation and apoptosis of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29

    Institute of Scientific and Technical Information of China (English)

    Chang-Wen Yu; Bi-Sheng Zhu

    2016-01-01

    Objective:To explore effect of leptin on cell proliferation and apoptosis of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29.Methods: MTT and flow cytometry were adopted for detecting the effect of exogenous leptin on cell cycle of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29.Results: Leptin with mass concentration (0 ng/mL, 5 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL) could stimulate the growth of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29; exogenous leptin with mass concentration (5 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL) could inhibit cell growth of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29 after 72 h; among which, inhibiting effects of cell line-SGC7901 and cell line-HT-29 were the most significant under the effect of exogenous leptin with mass concentration-200 ng/mL.Conclusion:Within a certain concentration and action time, exogenous leptin can stimulate the growth of gastric adenocarcinoma cell line and colon cancer cell line, and then promot the tumor cell proliferation and/or inhibit the tumor cell apoptosis.

  17. Mild brain ischemia induces unique physiological properties in striatal astrocytes.

    Science.gov (United States)

    Wang, Li-Ping; Cheung, Giselle; Kronenberg, Golo; Gertz, Karen; Ji, Shengbo; Kempermann, Gerd; Endres, Matthias; Kettenmann, Helmut

    2008-07-01

    We studied the properties of GFAP-expressing cells in adult mouse striatum using acute brain slices from transgenic animals expressing EGFP under GFAP promoter. Under physiological conditions, two distinct populations of GFAP-EGFP cells could be identified: (1) brightly fluorescent cells had bushy processes, passive membrane properties, glutamate transporter activity, and high gap junction coupling rate typical for classical astrocytes; (2) weakly fluorescent cells were characterized by thin, clearly distinguishable processes, voltage-gated currents, complex responses to kainate, and low coupling rate reminiscent of an astrocyte subtype recently described in the hippocampus. Mild focal cerebral ischemia confers delayed neuronal cell death and astrogliosis in the striatum. Following middle cerebral artery occlusion and reperfusion, brightly fluorescent cells were the dominant GFAP-EGFP population observed within the ischemic lesion. Interestingly, the majority of these cells expressed voltage-gated channels, showed complex responses to kainate, and a high coupling rate exceeding that of brightly fluorescent control cells. A minority of cells had passive membrane properties and was coupled less compared with passive control cells. We conclude that, in the adult striatum, astrocytes undergo distinct pathophysiological changes after ischemic insults. The dominant population in the ischemic lesion constitutes a novel physiological phenotype unlike any normal astrocyte and generates a large syncytium which might be a neuroprotective response of reactive astrocytes.

  18. Cancer and inflammation studies using zebrafish cell lines

    NARCIS (Netherlands)

    He, Shuning

    2010-01-01

    As the zebrafish, Danio rerio, has been increasingly used as an animal model for biomedical research, we aimed to establish zebrafish cell line models for inflammation and cancer studies in this thesis. Several zebrafish cell lines were characterized and their genetic and physiological properties we

  19. Derivation of the human embryonic stem cell line RCM1

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    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  20. Trichloroethylene toxicity in a human hepatoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Thevenin, E.; McMillian, J. [Medical Univ. of Charleston South Carolina, SC (United States)

    1994-12-31

    The experiments conducted in this study were designed to determine the usefullness of hepatocyte cultures and a human hepatoma cell line as model systems for assessing human susceptibility to hepatocellular carcinoma due to exposure to trichloroethylene. The results from these studies will then be analyzed to determine if human cell lines can be used to conduct future experiments of this nature.

  1. Beryllium-stimulated apoptosis in macrophage cell lines.

    Science.gov (United States)

    Sawyer, R T; Fadok, V A; Kittle, L A; Maier, L A; Newman, L S

    2000-08-21

    In vitro stimulation of bronchoalveolar lavage cells from patients with chronic beryllium disease (CBD) induces the production of TNF-alpha. We tested the hypothesis that beryllium (Be)-stimulated TNF-alpha might induce apoptosis in mouse and human macrophage cell lines. These cell lines were selected because they produce a range of Be-stimulated TNF-alpha. The mouse macrophage cell line H36.12j produces high levels of Be-stimulated TNF-alpha. The mouse macrophage cell line P388D.1 produces low, constitutive, levels of TNF-alpha and does not up-regulate Be-stimulated TNF-alpha production. The DEOHS-1 human CBD macrophage cell line does not produce constitutive or Be-stimulated TNF-alpha. Apoptosis was determined by microscopic observation of propidium iodide stained fragmented nuclei in unstimulated and BeSO(4)-stimulated macrophage cell lines. BeSO(4) induced apoptosis in all macrophage cell lines tested. Beryllium-stimulated apoptosis was dose-responsive and maximal after 24 h of exposure to 100 microM BeSO(4). In contrast, unstimulated and Al(2)(SO(4))(3)-stimulated macrophage cell lines did not undergo apoptosis. The general caspase inhibitor BD-fmk inhibited Be-stimulated macrophage cell line apoptosis at concentrations above 50 microM. Our data show that Be-stimulated macrophage cell line apoptosis was caspase-dependent and not solely dependent on Be-stimulated TNF-alpha levels. We speculate that the release of Be-antigen from apoptotic macrophages may serve to re-introduce Be material back into the lung microenvironment, make it available for uptake by new macrophages, and thereby amplify Be-stimulated cytokine production, promoting ongoing inflammation and granuloma maintenance in CBD.

  2. Regulated expression of erythropoietin by two human hepatoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Goldberg, M.A.; Glass, G.A.; Cunningham, J.M.; Bunn, H.F.

    1987-11-01

    The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. The authors have screened multiple renal and hepatic cell lines for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10/sup 6/ cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities < 3.3 x 10/sup 5/ cells per cm/sup 2/, there was little constitutive release of Epo in the medium. With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 ..mu..M cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

  3. Human embryonic stem cell lines derived from the Chinese population

    Institute of Scientific and Technical Information of China (English)

    Zhen Fu FANG; Fan JIN; Hui GAI; Ying CHEN; Li WU; Ai Lian LIU; Bin CHEN; Hui Zhen SHENG

    2005-01-01

    Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF,Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.

  4. Motoneuron differentiation of immortalized human spinal cord cell lines.

    Science.gov (United States)

    Li, R; Thode, S; Zhou, J; Richard, N; Pardinas, J; Rao, M S; Sah, D W

    2000-02-01

    Human motoneuron cell lines will be valuable tools for spinal cord research and drug discovery. To create such cell lines, we immortalized NCAM(+)/neurofilament(+) precursors from human embryonic spinal cord with a tetracycline repressible v-myc oncogene. Clonal NCAM(+)/neurofilament(+) cell lines differentiated exclusively into neurons within 1 week. These neurons displayed extensive processes, exhibited immunoreactivity for mature neuron-specific markers such as tau and synaptophysin, and fired action potentials upon current injection. Moreover, a clonal precursor cell line gave rise to multiple types of spinal cord neurons, including ChAT(+)/Lhx3(+)/Lhx4(+) motoneurons and GABA(+) interneurons. These neuronal restricted precursor cell lines will expedite the elucidation of molecular mechanisms that regulate the differentiation, maturation and survival of specific subsets of spinal cord neurons, and the identification and validation of novel drug targets for motoneuron diseases and spinal cord injury.

  5. Dynamic volume changes in astrocytes are an intrinsic phenomenon mediated by bicarbonate ion flux.

    Directory of Open Access Journals (Sweden)

    Clare M Florence

    Full Text Available Astrocytes, the major type of non-neuronal cells in the brain, play an important functional role in extracellular potassium ([K(+](o and pH homeostasis. Pathological brain states that result in [K(+](o and pH dysregulation have been shown to cause astrocyte swelling. However, whether astrocyte volume changes occur under physiological conditions is not known. In this study we used two-photon imaging to visualize real-time astrocyte volume changes in the stratum radiatum of the hippocampus CA1 region. Astrocytes were observed to swell by 19.0±0.9% in response to a small physiological increase in the concentration of [K(+](o (3 mM. Astrocyte swelling was mediated by the influx of bicarbonate (HCO(3- ions as swelling was significantly decreased when the influx of HCO(3- was reduced. We found: 1 in HCO(3- free extracellular solution astrocytes swelled by 5.4±0.7%, 2 when the activity of the sodium-bicarbonate cotransporter (NBC was blocked the astrocytes swelled by 8.3±0.7%, and 3 in the presence of an extracellular carbonic anhydrase (CA inhibitor astrocytes swelled by 11.4±0.6%. Because a significant HCO(3- efflux is known to occur through the γ-amino-butyric acid (GABA channel, we performed a series of experiments to determine if astrocytes were capable of HCO(3- mediated volume shrinkage with GABA channel activation. Astrocytes were found to shrink -7.7±0.5% of control in response to the GABA(A channel agonist muscimol. Astrocyte shrinkage from GABA(A channel activation was significantly decreased to -5.0±0.6% of control in the presence of the membrane-permeant CA inhibitor acetazolamide (ACTZ. These dynamic astrocyte volume changes may represent a previously unappreciated yet fundamental mechanism by which astrocytes regulate physiological brain functioning.

  6. Derivation of human embryonic stem cell line Genea022

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    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea022 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea022 was demonstrated with 84% of cells expressed Nanog, 98% Oct4, 55% Tra1–60 and 97% SSEA4, gave a Pluritest Pluripotency score of 42.95, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

  7. Astrocytes, Synapses and Brain Function: A Computational Approach

    Science.gov (United States)

    Nadkarni, Suhita

    2006-03-01

    Modulation of synaptic reliability is one of the leading mechanisms involved in long- term potentiation (LTP) and long-term depression (LTD) and therefore has implications in information processing in the brain. A recently discovered mechanism for modulating synaptic reliability critically involves recruitments of astrocytes - star- shaped cells that outnumber the neurons in most parts of the central nervous system. Astrocytes until recently were thought to be subordinate cells merely participating in supporting neuronal functions. New evidence, however, made available by advances in imaging technology has changed the way we envision the role of these cells in synaptic transmission and as modulator of neuronal excitability. We put forward a novel mathematical framework based on the biophysics of the bidirectional neuron-astrocyte interactions that quantitatively accounts for two distinct experimental manifestation of recruitment of astrocytes in synaptic transmission: a) transformation of a low fidelity synapse transforms into a high fidelity synapse and b) enhanced postsynaptic spontaneous currents when astrocytes are activated. Such a framework is not only useful for modeling neuronal dynamics in a realistic environment but also provides a conceptual basis for interpreting experiments. Based on this modeling framework, we explore the role of astrocytes for neuronal network behavior such as synchrony and correlations and compare with experimental data from cultured networks.

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

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    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.

  9. Further characterization of the first seminoma cell line TCam-2.

    Science.gov (United States)

    de Jong, Jeroen; Stoop, Hans; Gillis, Ad J M; Hersmus, Remko; van Gurp, Ruud J H L M; van de Geijn, Gert-Jan M; van Drunen, Ellen; Beverloo, H Berna; Schneider, Dominik T; Sherlock, Jon K; Baeten, John; Kitazawa, Sohei; van Zoelen, E Joop; van Roozendaal, Kees; Oosterhuis, J Wolter; Looijenga, Leendert H J

    2008-03-01

    Testicular germ cell tumors of adolescents and adults (TGCTs) can be classified into seminomatous and nonseminomatous tumors. Various nonseminomatous cell lines, predominantly embryonal carcinoma, have been established and proven to be valuable for pathobiological and clinical studies. So far, no cell lines have been derived from seminoma which constitutes more than 50% of invasive TGCTs. Such a cell line is essential for experimental investigation of biological characteristics of the cell of origin of TGCTs, i.e., carcinoma in situ of the testis, which shows characteristics of a seminoma cell. Before a cell line can be used as model, it must be verified regarding its origin and characteristics. Therefore, a multidisciplinary approach was undertaken on TCam-2 cells, supposedly the first seminoma cell line. Fluorescence in situ hybridization, array comparative genomic hybridization, and spectral karyotyping demonstrated an aneuploid DNA content, with gain of 12p, characteristic for TGCTs. Genome wide mRNA and microRNA expression profiling supported the seminoma origin, in line with the biallelic expression of imprinted genes IGF2/H19 and associated demethylation of the imprinting control region. Moreover, the presence of specific markers, demonstrated by immunohistochemistry, including (wild type) KIT, stem cell factor, placental alkaline phosphatase, OCT3/4 (also demonstrated by a specific Q-PCR) and NANOG, and the absence of CD30, SSX2-4, and SOX2, confirms that TCam-2 is a seminoma cell line. Although mutations in oncogenes and tumor suppressor genes are rather rare in TGCTs, TCam-2 had a mutated BRAF gene (V600E), which likely explains the fact that these cells could be propagated in vitro. In conclusion, TCam-2 is the first well-characterized seminoma-derived cell line, with an exceptional mutation, rarely found in TGCTs.

  10. Establishment of Germ-line Competent C57BL/6J Embryonic Stem Cell Lines

    Institute of Scientific and Technical Information of China (English)

    Gui-jun YAN; Zheng GU; Jian WANG; Jia-ke TSO

    2004-01-01

    Objective To establish C57BL/6J embryonic stem (ES) cell lines with potential germline contribution Methods ES cells were isolated from blastocyst inner cell mass of C57BL/6J mice, and cultured for 15 passages, and then injected into blastococels of lCR mice blastocysts to establish chimeric mice.Results Three ES cell lines (mC57ESl,mC57ES3, mC57ES7) derived from the inner cell mass of C57BL/6J mice blastocysts were established. They were characteristic of undifferentiated state, including normal XY karyotype, expression of a specific cell surface marker "stage-specific embryonic antigen-1" and alkaline phosphatase in continuous passage. When injected into immunodeficient mice, mC5 7ES1 cells consis tently differentiated into derivatives of all three embryonic germ layers. When mC57ES1cells were transferred into ICR mice blastocysts, 4 chimeric mice have been obtained.One male of them revealed successful germ-line transmission. Conclussion We have obtained C57BL/6J ES cell lines with a potential germ-line contribution, which can be used to generate transgenic and gene knock-out mice.

  11. Cytotoxinic Mechanism of Hydroxyapatite Nanoparticles on Human Hepatoma Cell Lines

    Institute of Scientific and Technical Information of China (English)

    CAO Xian-ying; QI Zhi-tao; DAI Hong-lian; YAN Yu-hua; LI Shi-pu

    2003-01-01

    Stable and single-dispersed HAP nanoparticles were synthesized with chemical method assisted by ultrasonic treatment.HAP nanoparticles were surveyed by AFM and Zataplus.The effect on the Bel-7402 human hepatoma cell lines treated with HAP nanoparticles was investigated by the MTT methods and observation of morphology,and the mechanism was studied in changes of cell cycle and ultrastructure.The result shows that inhibition of HAP nanoparticles on the Bel-7402 human hepatoma cell lines is obviously in vitro.HAP nanoparticles the entered cancer cytoplasm,and cell proliferation is stopped at G1 phase of cell cycle,thus,cancer cells die directly.

  12. Deriving cell lines from zebrafish embryos and tumors.

    Science.gov (United States)

    Choorapoikayil, Suma; Overvoorde, John; den Hertog, Jeroen

    2013-09-01

    Over the last two decades the zebrafish has emerged as a powerful model organism in science. The experimental accessibility, the broad range of zebrafish mutants, and the highly conserved genetic and biochemical pathways between zebrafish and mammals lifted zebrafish to become one of the most attractive vertebrate models to study gene function and to model human diseases. Zebrafish cell lines are highly attractive to investigate cell biology and zebrafish cell lines complement the experimental tools that are available already. We established a straightforward method to culture cells from a single zebrafish embryo or a single tumor. Here we describe the generation of fibroblast-like cell lines from wild-type and ptenb(-/-) embryos and an endothelial-like cell line from a tumor of an adult ptena(+/-)ptenb(-/-) zebrafish. This protocol can easily be adapted to establish stable cell lines from any mutant or transgenic zebrafish line and the average time to obtain a pro-stable cell line is 3-5 months.

  13. Recombinant protein production from stable mammalian cell lines and pools.

    Science.gov (United States)

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced.

  14. Physiopathologic dynamics of vesicle traffic in astrocytes.

    Science.gov (United States)

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

    2011-02-01

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

  15. Establishment of human embryonic stem cell line from gamete donors

    Institute of Scientific and Technical Information of China (English)

    LI Tao; ZHOU Can-quan; MAI Qing-yun; ZHUANG Guang-lun

    2005-01-01

    Background Human embryonic stem (HES) cell derived from human blastocyst can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent. It has exciting potential in human developmental biology, drug discovery, and transplantation medicine. But there are insufficient HES cell lines for further study. Methods Three oocyte donors were studied, and 3 in vitro fertilization (IVF) cycles were carried out to get blastocysts for the establishment of HES cell line. Isolated from blastocysts immunosurgically, inner cell mass (ICM) was cultured and propagated on mouse embryonic fibroblasts (MEFs). Once established, morphology, cell surface markers, karyotype and differentiating ability of the cell line were thoroughly analyzed.Results Four ICMs from 7 blastocysts were cultured on MEFs. After culture, one cell line (cHES-1) was established and met the criteria for defining human pluripotent stem cells including a series of markers used to identify pluripotent stem cells, morphological similarity to primate embryonic stem cells and HES reported else where. Normal and stable karyotype maintained over 60 passages, and demonstrated ability to differentiate into a wide variety of cell types.Conclusions HES cell lines can be established from gamete donors at a relatively highly efficient rate. The establishment will exert a widespread impact on biomedical research.

  16. Susceptibility of various cell lines to Neospora caninum tachyzoites cultivation

    Directory of Open Access Journals (Sweden)

    Khordadmehr, M.,

    2014-05-01

    Full Text Available Neospora caninum is a coccidian protozoan parasite which is a major cause of bovine abortions and neonatal mortality in cattle, sheep, goat and horse. Occasionally, cultured cells are used for isolation and multiplication of the agent in vitro with several purposes. In this study the tachyzoite yields of N. caninum were compared in various cell cultures as the host cell lines. Among the cell cultures tested, two presented good susceptibility to the agent: cell lines Vero and MA-104. SW742 and TLI (in vitro suspension culture of lymphoid cells infected with Theileria lestoquardi showed moderate sensitivity. No viable tachyzoite were detected in the culture of MDCK and McCoy cell lines. These results demonstrate that MA-104 and SW742 cells present adequate susceptibility to N. caninum compared to Vero cells, which have been largely used to multiply the parasite in vitro. Moreover, these have easy manipulation, fast multiplication and relatively low nutritional requirements. In addition, the result of this study showed that TLI cell line as a suspension cell culture is susceptible to Nc-1 tachyzoites infection and could be used as an alternative host cell line for tachyzoites culture in vitro studies.

  17. Tricyclic Antidepressant Amitriptyline-induced Glial Cell Line-derived Neurotrophic Factor Production Involves Pertussis Toxin-sensitive Gαi/o Activation in Astroglial Cells.

    Science.gov (United States)

    Hisaoka-Nakashima, Kazue; Miyano, Kanako; Matsumoto, Chie; Kajitani, Naoto; Abe, Hiromi; Okada-Tsuchioka, Mami; Yokoyama, Akinobu; Uezono, Yasuhito; Morioka, Norimitsu; Nakata, Yoshihiro; Takebayashi, Minoru

    2015-05-29

    Further elaborating the mechanism of antidepressants, beyond modulation of monoaminergic neurotransmission, this study sought to elucidate the mechanism of amitriptyline-induced production of glial cell line-derived neurotrophic factor (GDNF) in astroglial cells. Previous studies demonstrated that an amitriptyline-evoked matrix metalloproteinase (MMP)/FGF receptor (FGFR)/FGFR substrate 2α (FRS2α)/ERK cascade is crucial for GDNF production, but how amitriptyline triggers this cascade remains unknown. MMP is activated by intracellular mediators such as G proteins, and this study sought to clarify the involvement of G protein signaling in amitriptyline-evoked GDNF production in rat C6 astroglial cells (C6 cells), primary cultured rat astrocytes, and normal human astrocytes. Amitriptyline-evoked GDNF mRNA expression and release were inhibited by pertussis toxin (PTX), a Gα(i/o) inhibitor, but not by NF449, a Gα(s) inhibitor, or YM-254890, a Gαq inhibitor. The activation of the GDNF production cascade (FGFR/FRS2α/ERK) was also inhibited by PTX. Deletion of Gα(ο1) and Gα(i3) by RNAi demonstrated that these G proteins play important roles in amitriptyline signaling. G protein activation was directly analyzed by electrical impedance-based biosensors (CellKey(TM) assay), using a label-free (without use of fluorescent proteins/probes or radioisotopes) and real time approach. Amitriptyline increased impedance, indicating Gα(i/o) activation that was suppressed by PTX treatment. The impedance evoked by amitriptyline was not affected by inhibitors of the GDNF production cascade. Furthermore, FGF2 treatment did not elicit any effect on impedance, indicating that amitriptyline targets PTX-sensitive Gα(i/o) upstream of the MMP/FGFR/FRS2α/ERK cascade. These results suggest novel targeting for the development of antidepressants.

  18. Extracellular poly(ADP-ribose) is a neurotrophic signal that upregulates glial cell line-derived neurotrophic factor (GDNF) levels in vitro and in vivo.

    Science.gov (United States)

    Nakajima, Hidemitsu; Itakura, Masanori; Sato, Keishi; Nakamura, Sunao; Azuma, Yasu-Taka; Takeuchi, Tadayoshi

    2017-03-04

    Synthesis of poly(ADP-ribose) (PAR) is catalyzed by PAR polymerase-1 (PARP-1) in neurons. PARP1 plays a role in various types of brain damage in neurodegenerative disorders. In neurons, overactivation of PARP-1 during oxidative stress induces robust PAR formation, which depletes nicotinamide adenine dinucleotide levels and leads to cell death. However, the role of the newly-formed PAR in neurodegenerative disorders remains elusive. We hypothesized that the effects of PAR could occur in the extracellular space after it is leaked from damaged neurons. Here we report that extracellular PAR (EC-PAR) functions as a neuroprotective molecule by inducing the synthesis of glial cell line-derived neurotrophic factor (GDNF) in astrocytes during neuronal cell death, both in vitro and in vivo. In primary rat astrocytes, exogenous treatment with EC-PAR produced GDNF but not other neurotrophic factors. The effect was concentration-dependent and did not affect cell viability in rat C6 astrocytoma cells. Topical injection of EC-PAR into rat striatum upregulated GDNF levels in activated astrocytes and improved pathogenic rotation behavior in a unilateral 6-hydroxydopamine model of Parkinson disease in rats. These findings indicate that EC-PAR acts as a neurotrophic enhancer by upregulating GDNF levels. This effect protects the remaining neurons following oxidative stress-induced brain damage, such as that seen with Parkinson disease.

  19. Astrocyte morphology, heterogeneity and density in the developing African Giant Rat (Cricetomys gambianus

    Directory of Open Access Journals (Sweden)

    James Olukayode Olopade

    2015-05-01

    Full Text Available Astrocyte morphologies and heterogeneity were described in male African giant rats (AGR (Cricetomys gambianus, Waterhouse across three age groups (5 neonates, 5 juveniles and 5 adults using Silver impregnation method and immunohistochemistry against glia fibrillary acidic protein (GFAP. Immunopositive cell signaling, cell size and population were least in neonates, followed by adults and juveniles respectively. In neonates, astrocyte processes were mostly detected within the glia limitans of the mid and hind brain; their cell bodies measuring 32±4.8 µm in diameter against 91±5.4µm and 75± 1.9µm in juveniles and adults respectively. Astrocyte heterogeneity in juvenile and adult groups revealed eight subtypes to include fibrous astrocytes chiefly in the corpus callosum and brain stem, protoplasmic astrocytes in the cortex and dentate gyrus (DG; radial glia were found along the olfactory bulb (OB and subventricular zone (SVZ; velate astrocytes were mainly found in the cerebellum and hippocampus; marginal astrocytes close to the pia mater; Bergmann glia in the molecular layer of the cerebellum; perivascular and periventricular astrocytes in the cortex and third ventricle respectively. Cell counts from twelve anatomical regions of the brain were significantly higher in juveniles than in adults (p≤0.01 using unpaired student t-test in the cerebral cortex, pia, corpus callosum, rostral migratory stream (RMS, DG and cerebellum. Highest astrocyte count was found in the DG, while the least count was in the brain stem and sub cortex. Astrocytes along the periventricular layer of the OB are believed to be part of the radial glia system that transport newly formed cells towards the hippocampus and play roles in neurogenesis migration and homeostasis in the AGR. Therefore, astrocyte heterogeneity was examined across age groups in the AGR to determine whether age influences astrocytes population in different regions of the AGR brain and discuss

  20. Copper handling by astrocytes: insights into neurodegenerative diseases.

    Science.gov (United States)

    Tiffany-Castiglioni, Evelyn; Hong, Sandra; Qian, Yongchang

    2011-12-01

    Copper (Cu) is an essential trace element in the brain that can be toxic at elevated levels. Cu accumulation is a suspected etiology in several neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and prion-induced disorders. Astrocytes are a proposed depot in the brain for Cu and other metals, including lead (Pb). This article describes the physiological roles of Cu in the central nervous system and in selected neurodegenerative diseases, and reviews evidence that astrocytes accumulate Cu and protect neurons from Cu toxicity. Findings from murine genetic models of Menkes disease and from cell culture models concerning the molecular mechanisms by which astrocytes take up, store, and buffer Cu intracellularly are discussed, as well as potential mechanistic linkages between astrocyte functions in Cu handling and neurodegenerative diseases.

  1. JKT-1 is not a human seminoma cell line.

    Science.gov (United States)

    de Jong, Jeroen; Stoop, Hans; Gillis, Ad J M; van Gurp, Ruud J H L M; van Drunen, Ellen; Beverloo, H Berna; Lau, Yun-Fai Chris; Schneider, Dominik T; Sherlock, Jon K; Baeten, John; Hatakeyama, Shingo; Ohyama, Chikara; Oosterhuis, J Wolter; Looijenga, Leendert H J

    2007-08-01

    The JKT-1 cell line has been used in multiple independent studies as a representative model of human testicular seminoma. However, no cell line for this specific tumour type has been independently confirmed previously; and therefore, the seminomatous origin of JKT-1 must be proven. The genetic constitution of the JKT-1 cells was determined using flow cytometry and spectral karyotyping, as well as array comparative genomic hybridization and fluorescent in situ hybridization. Marker profiling, predominantly based on differentially expressed proteins during normal germ cell development, was performed by immunohistochemistry and Western blot analyses. Moreover, genome wide affymetrix mRNA expression and profiling of 157 microRNAs was performed, and the status of genomic imprinting was determined. A germ cell origin of the JKT-1 cells was in line with genomic imprinting status and marker profile (including positive staining for several cancer-testis antigens). However, the supposed primary tumour, from which the cell line was derived, being indeed a classical seminoma, was molecularly proven not to be the origin of the cell line. The characteristic chromosomal anomalies of seminoma, e.g. gain of the short arm of chromosome 12, as well as the informative marker profile (positive staining for OCT3/4, NANOG, among others) were absent in the various JKT-1 cell lines investigated, irrespective of where the cells were cultured. All results indicate that the JKT-1 cell line is not representative of human seminoma. Although it can originate from an early germ cell, a non-germ cell derivation cannot be excluded.

  2. Investigation of radiosensitivity gene signatures in cancer cell lines.

    Directory of Open Access Journals (Sweden)

    John S Hall

    Full Text Available Intrinsic radiosensitivity is an important factor underlying radiotherapy response, but there is no method for its routine assessment in human tumours. Gene signatures are currently being derived and some were previously generated by expression profiling the NCI-60 cell line panel. It was hypothesised that focusing on more homogeneous tumour types would be a better approach. Two cell line cohorts were used derived from cervix [n = 16] and head and neck [n = 11] cancers. Radiosensitivity was measured as surviving fraction following irradiation with 2 Gy (SF2 by clonogenic assay. Differential gene expression between radiosensitive and radioresistant cell lines (SF2 median was investigated using Affymetrix GeneChip Exon 1.0ST (cervix or U133A Plus2 (head and neck arrays. There were differences within cell line cohorts relating to tissue of origin reflected by expression of the stratified epithelial marker p63. Of 138 genes identified as being associated with SF2, only 2 (1.4% were congruent between the cervix and head and neck carcinoma cell lines (MGST1 and TFPI, and these did not partition the published NCI-60 cell lines based on SF2. There was variable success in applying three published radiosensitivity signatures to our cohorts. One gene signature, originally trained on the NCI-60 cell lines, did partially separate sensitive and resistant cell lines in all three cell line datasets. The findings do not confirm our hypothesis but suggest that a common transcriptional signature can reflect the radiosensitivity of tumours of heterogeneous origins.

  3. Astrocytic modulation of blood brain barrier: perspectives on Parkinson's disease.

    Science.gov (United States)

    Cabezas, Ricardo; Avila, Marcos; Gonzalez, Janneth; El-Bachá, Ramon Santos; Báez, Eliana; García-Segura, Luis Miguel; Jurado Coronel, Juan Camilo; Capani, Francisco; Cardona-Gomez, Gloria Patricia; Barreto, George E

    2014-01-01

    The blood-brain barrier (BBB) is a tightly regulated interface in the Central Nervous System (CNS) that regulates the exchange of molecules in and out from the brain thus maintaining the CNS homeostasis. It is mainly composed of endothelial cells (ECs), pericytes and astrocytes that create a neurovascular unit (NVU) with the adjacent neurons. Astrocytes are essential for the formation and maintenance of the BBB by providing secreted factors that lead to the adequate association between the cells of the BBB and the formation of strong tight junctions. Under neurological disorders, such as chronic cerebral ischemia, brain trauma, Epilepsy, Alzheimer and Parkinson's Diseases, a disruption of the BBB takes place, involving a lost in the permeability of the barrier and phenotypical changes in both the ECs and astrocytes. In this aspect, it has been established that the process of reactive gliosis is a common feature of astrocytes during BBB disruption, which has a detrimental effect on the barrier function and a subsequent damage in neuronal survival. In this review we discuss the implications of astrocyte functions in the protection of the BBB, and in the development of Parkinson's disease (PD) and related disorders. Additionally, we highlight the current and future strategies in astrocyte protection aimed at the development of restorative therapies for the BBB in pathological conditions.

  4. Astrocytic Insulin Signaling Couples Brain Glucose Uptake with Nutrient Availability.

    Science.gov (United States)

    García-Cáceres, Cristina; Quarta, Carmelo; Varela, Luis; Gao, Yuanqing; Gruber, Tim; Legutko, Beata; Jastroch, Martin; Johansson, Pia; Ninkovic, Jovica; Yi, Chun-Xia; Le Thuc, Ophelia; Szigeti-Buck, Klara; Cai, Weikang; Meyer, Carola W; Pfluger, Paul T; Fernandez, Ana M; Luquet, Serge; Woods, Stephen C; Torres-Alemán, Ignacio; Kahn, C Ronald; Götz, Magdalena; Horvath, Tamas L; Tschöp, Matthias H

    2016-08-11

    We report that astrocytic insulin signaling co-regulates hypothalamic glucose sensing and systemic glucose metabolism. Postnatal ablation of insulin receptors (IRs) in glial fibrillary acidic protein (GFAP)-expressing cells affects hypothalamic astrocyte morphology, mitochondrial function, and circuit connectivity. Accordingly, astrocytic IR ablation reduces glucose-induced activation of hypothalamic pro-opio-melanocortin (POMC) neurons and impairs physiological responses to changes in glucose availability. Hypothalamus-specific knockout of astrocytic IRs, as well as postnatal ablation by targeting glutamate aspartate transporter (GLAST)-expressing cells, replicates such alterations. A normal response to altering directly CNS glucose levels in mice lacking astrocytic IRs indicates a role in glucose transport across the blood-brain barrier (BBB). This was confirmed in vivo in GFAP-IR KO mice by using positron emission tomography and glucose monitoring in cerebral spinal fluid. We conclude that insulin signaling in hypothalamic astrocytes co-controls CNS glucose sensing and systemic glucose metabolism via regulation of glucose uptake across the BBB.

  5. Unveiling astrocytic control of cerebral blood flow with optogenetics.

    Science.gov (United States)

    Masamoto, Kazuto; Unekawa, Miyuki; Watanabe, Tatsushi; Toriumi, Haruki; Takuwa, Hiroyuki; Kawaguchi, Hiroshi; Kanno, Iwao; Matsui, Ko; Tanaka, Kenji F; Tomita, Yutaka; Suzuki, Norihiro

    2015-06-16

    Cortical neural activities lead to changes in the cerebral blood flow (CBF), which involves astrocytic control of cerebrovascular tone. However, the manner in which astrocytic activity specifically leads to vasodilation or vasoconstriction is difficult to determine. Here, cortical astrocytes genetically expressing a light-sensitive cation channel, channelrhodopsin-2 (ChR2), were transcranially activated with a blue laser while the spatiotemporal changes in CBF were noninvasively monitored with laser speckle flowgraphy in the anesthetised mouse cortex. A brief photostimulation induced a fast transient increase in CBF. The average response onset time was 0.7 ± 0.7 sec at the activation foci, and this CBF increase spread widely from the irradiation spot with an apparent propagation speed of 0.8-1.1 mm/sec. The broad increase in the CBF could be due to a propagation of diffusible vasoactive signals derived from the stimulated astrocytes. Pharmacological manipulation showed that topical administration of a K(+) channel inhibitor (BaCl2; 0.1-0.5 mM) significantly reduced the photostimulation-induced CBF responses, which indicates that the ChR2-evoked astrocytic activity involves K(+) signalling to the vascular smooth muscle cells. These findings demonstrate a unique model for exploring the role of the astrocytes in gliovascular coupling using non-invasive, time-controlled, cell-type specific perturbations.

  6. Development of a cell line from Echinococcus granulosus germinal layer.

    Science.gov (United States)

    Albani, Clara María; Cumino, Andrea Carina; Elissondo, María Celina; Denegri, Guillermo María

    2013-10-01

    In vitro culture of parasitic helminths provides an important tool to study cell regeneration and physiology, as well as for molecular biology and genetic engineering studies. In the present study, we established in vitro propagation of cells from Echinococcus granulosus germinal cyst layer. E. granulosus germinal cells grew beyond 100 passages and showed no signs of reduced proliferation capacity. Microscopic analysis revealed that cells grew both attached to the substrate and in suspension, forming three-dimensional structures like mammalian stem cell aggregates. Examination of the chromosome number of attached germinal cells showed a high degree of heteroploidy, suggesting the occurrence of transformation during culture. Monolayer cells survived cryopreservation and were able to proliferate after thawing. Based on the characteristics displayed by E. granulosus germinal cells, we establish a cell line from the E. granulosus germinal layer. Furthermore, we propose that this cell line could be useful for drug screening and for obtaining parasite material.

  7. Cell type-specific dependency on the PI3K/Akt signaling pathway for the endogenous Epo and VEGF induction by baicalein in neurons versus astrocytes.

    Directory of Open Access Journals (Sweden)

    Yu-Yo Sun

    Full Text Available The neuroprotective effect of baicalein is generally attributed to inhibition of 12/15-lipoxygenase (12/15-LOX and suppression of oxidative stress, but recent studies showed that baicalein also activates hypoxia-inducible factor-α (HIF1α through inhibition of prolyl hydrolase 2 (PHD2 and activation of the phosphatidylinositide-3 kinase (PI3K/Akt signaling pathway. Yet, the significance and regulation of prosurvival cytokines erythropoietin (Epo and vascular endothelial growth factor (VEGF, two transcriptional targets of HIF1α, in baicalein-mediated neuroprotection in neurons and astrocytes remains unknown. Here we investigated the causal relationship between the PI3K/Akt signaling pathway and Epo/VEGF expression in baicalein-mediated neuroprotection in primary rat cortical neurons and astrocytes. Our results show that baicalein induced Epo and VEGF expression in a HIF1α- and PI3K/Akt-dependent manner in neurons. Baicalein also protected neurons against excitotoxicity in a PI3K- and Epo/VEGF-dependent manner without affecting neuronal excitability. In contrast, at least a 10-fold higher concentration of baicalein was needed to induce Epo/VEGF production and PI3K/Akt activity in astrocytes for protection of neurons. Moreover, only baicalein-induced astrocytic VEGF, but not Epo expression requires HIF1α, while PI3K/Akt signaling had little role in baicalein-induced astrocytic Epo/VEGF expression. These results suggest distinct mechanisms of baicalein-mediated Epo/VEGF production in neurons and astrocytes for neuroprotection, and provide new insights into the mechanisms and potential of baicalein in treating brain injury in vivo.

  8. Cell type-specific dependency on the PI3K/Akt signaling pathway for the endogenous Epo and VEGF induction by baicalein in neurons versus astrocytes.

    Science.gov (United States)

    Sun, Yu-Yo; Lin, Shang-Hsuan; Lin, Hung-Cheng; Hung, Chia-Chi; Wang, Chen-Yu; Lin, Yen-Chu; Hung, Kuo-Sheng; Lien, Cheng-Chang; Kuan, Chia-Yi; Lee, Yi-Hsuan

    2013-01-01

    The neuroprotective effect of baicalein is generally attributed to inhibition of 12/15-lipoxygenase (12/15-LOX) and suppression of oxidative stress, but recent studies showed that baicalein also activates hypoxia-inducible factor-α (HIF1α) through inhibition of prolyl hydrolase 2 (PHD2) and activation of the phosphatidylinositide-3 kinase (PI3K)/Akt signaling pathway. Yet, the significance and regulation of prosurvival cytokines erythropoietin (Epo) and vascular endothelial growth factor (VEGF), two transcriptional targets of HIF1α, in baicalein-mediated neuroprotection in neurons and astrocytes remains unknown. Here we investigated the causal relationship between the PI3K/Akt signaling pathway and Epo/VEGF expression in baicalein-mediated neuroprotection in primary rat cortical neurons and astrocytes. Our results show that baicalein induced Epo and VEGF expression in a HIF1α- and PI3K/Akt-dependent manner in neurons. Baicalein also protected neurons against excitotoxicity in a PI3K- and Epo/VEGF-dependent manner without affecting neuronal excitability. In contrast, at least a 10-fold higher concentration of baicalein was needed to induce Epo/VEGF production and PI3K/Akt activity in astrocytes for protection of neurons. Moreover, only baicalein-induced astrocytic VEGF, but not Epo expression requires HIF1α, while PI3K/Akt signaling had little role in baicalein-induced astrocytic Epo/VEGF expression. These results suggest distinct mechanisms of baicalein-mediated Epo/VEGF production in neurons and astrocytes for neuroprotection, and provide new insights into the mechanisms and potential of baicalein in treating brain injury in vivo.

  9. Establishment of Jurkat tet-on cell line

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Tet-control system is developed to tightly control target gene expression in mammalian cells by using the regulatory elements of tetracycline-repressor of the transposor Tn10 from E.Coli.We have transfected reverse tetracycline-controlled transactivator gene (rtTA) into genome of Jurkat cells and established two Jurkat tet-on cell lines.Induction of luciferase reporter activity with doxycycline,a tetracycline derivative,is dose-dependent with a peak value of 32-fold increment.Establishment of Jurkat tet-on cell lines greatly facilitates quantitative studies on target gene functions in the cells.

  10. Establishment and characterization of rat portal myofibroblast cell lines.

    Directory of Open Access Journals (Sweden)

    Michel Fausther

    Full Text Available The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC and portal fibroblasts (PF. In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5'-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myofibroblasts and their contribution to the progression of liver fibrosis.

  11. 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.

  12. Regulation of neurotrophic factors and energy metabolism by antidepressants in astrocytes

    KAUST Repository

    Martin, Jean Luc

    2013-09-01

    There is growing evidence that astrocytes are involved in the neuropathology of major depression. In particular, decreases in glial cell density observed in the cerebral cortex of individuals with major depressive disorder are accompanied by a reduction of several astrocytic markers suggesting that astrocyte dysfunction may contribute to the pathophysiology of major depression. In rodents, glial loss in the prefrontal cortex is sufficient to induce depressive-like behaviors and antidepressant treatment prevents the stress-induced reduction of astrocyte number in the hippocampus. Collectively, these data support the existence of a link between astrocyte loss or dysfunction, depressive-like behavior and antidepressant treatment. Astrocytes are increasingly recognized to play important roles in neuronal development, neurotransmission, synaptic plasticity and maintenance of brain homeostasis. It is also well established that astrocytes provide trophic, structural, and metabolic support to neurons. In this article, we review evidence that antidepressants regulate energy metabolism and neurotrophic factor expression with particular emphasis on studies in astrocytes. These observations support a role for astrocytes as new targets for antidepressants. The contribution of changes in astrocyte glucose metabolism and neurotrophic factor expression to the therapeutic effects of antidepressants remains to be established. © 2013 Bentham Science Publishers.

  13. Calcium Imaging of Living Astrocytes in the Mouse Spinal Cord following Sensory Stimulation

    Directory of Open Access Journals (Sweden)

    Giovanni Cirillo

    2012-01-01

    Full Text Available Astrocytic Ca2+ dynamics have been extensively studied in ex vivo models; however, the recent development of two-photon microscopy and astrocyte-specific labeling has allowed the study of Ca2+ signaling in living central nervous system. Ca2+ waves in astrocytes have been described in cultured cells and slice preparations, but evidence for astrocytic activation during sensory activity is lacking. There are currently few methods to image living spinal cord: breathing and heart-beating artifacts have impeded the widespread application of this technique. We here imaged the living spinal cord by two-photon microscopy in C57BL6/J mice. Through pressurized injection, we specifically loaded spinal astrocytes using the red fluorescent dye sulforhodamine 101 (SR101 and imaged astrocytic Ca2+ levels with Oregon-Green BAPTA-1 (OGB. Then, we studied astrocytic Ca2+ levels at rest and after right electrical hind paw stimulation. Sensory stimulation significantly increased astrocytic Ca2+ levels within the superficial dorsal horn of the spinal cord compared to rest. In conclusion, in vivo morphofunctional imaging of living astrocytes in spinal cord revealed that astrocytes actively participate to sensory stimulation.

  14. Fibrous and protoplasmic astrocytes express GABAA receptors that differ in benzodiazepine pharmacology.

    Science.gov (United States)

    Rosewater, K; Sontheimer, H

    1994-02-04

    Astrocytes cultured from spinal cord contain two morphologically distinguishable types of astrocytes: fibrous and protoplasmic cells. Both astrocyte subtypes, in culture, are able to express GABAA receptors, and their activation results in inward currents at the resting potential. Using patch-clamp electrophysiology we characterized their basic receptor pharmacology and compared it to spinal cord neurons that were also present in small numbers in these cultures. As in neuronal GABAA receptors, the local anesthetic pentobarbital effectively potentiated GABA-induced currents in both astrocyte subtypes. Similarly, the benzodiazepine diazepam, on average doubled GABA-induced currents in both astrocytes subtypes. In contrast to these effects that were similar in both astrocytes types and similar to spinal cord neurons, the response to the convulsant methyl-4-ethyl-6,7-dimethoxy-beta-carboline-3-carboxylate (DMCM), which is an inverse benzodiazepine agonist differs between astrocyte subtypes. DMCM reduced GABA-induced currents by about 50% in fibrous astrocytes as we also observed with spinal cord neurons. In contrast, DMCM increased GABA currents in protoplasmic astrocytes by up to 150%, an effect never observed in neurons. DMCM potentiations of GABA currents have recently been attributed to differences in receptor subunit composition. Our results thus indicate that subtypes of astrocytes express GABAA receptors that differ pharmacologically and likely differ also in subunit composition.

  15. Mercury specifically induces LINE-1 activity in a human neuroblastoma cell line.

    Science.gov (United States)

    Habibi, Laleh; Shokrgozar, Mohammad Ali; Tabrizi, Mina; Modarressi, Mohammad Hossein; Akrami, Seyed Mohammad

    2014-01-01

    L1 retro-elements comprise 17% of the human genome. Approximately 100 copies of these autonomous mobile elements are active in our DNA and can cause mutations, gene disruptions, and genomic instability. Therefore, human cells control the activities of L1 elements, in order to prevent their deleterious effects through different mechanisms. However, some toxic agents increase the retrotransposition activity of L1 elements in somatic cells. In order to identify specific effects of neurotoxic metals on L1 activity in neuronal cells, we studied the effects of mercury and cobalt on L1-retroelement activity by measuring levels of cellular transcription, protein expression, and genomic retrotransposition in a neuroblastoma cell line compared with the effects in three non-neuronal cell lines. Our results show that mercury increased the expression of L1 RNA, the activity of the L1 5'UTR, and L1 retrotransposition exclusively in the neuroblastoma cell line but not in non-neuronal cell lines. However, cobalt increased the expression of L1 RNA in neuroblastoma cells, HeLa cells, and wild-type human fibroblasts, and also increased the activity of the L1 5'UTR as well as the SV40 promoter in HeLa cells but not in neuroblastoma cells. Exposure to cobalt did not result in increased retrotransposition activity in HeLa cells or neuroblastoma cells. We conclude that non-toxic levels of the neurotoxic agent mercury could influence DNA by increasing L1 activities, specifically in neuronal cells, and may make these cells susceptible to neurodegeneration over time.

  16. Apoptotic effect of noscapine in breast cancer cell lines.

    Science.gov (United States)

    Quisbert-Valenzuela, Edwin O; Calaf, Gloria M

    2016-06-01

    Cancer is a public health problem in the world and breast cancer is the most frequently cancer in women. Approximately 15% of the breast cancers are triple-negative. Apoptosis regulates normal growth, homeostasis, development, embryogenesis and appropriate strategy to treat cancer. Bax is a protein pro-apoptotic enhancer of apoptosis in contrast to Bcl-2 with antiapoptotic properties. Initiator caspase-9 and caspase-8 are features of intrinsic and extrinsic apoptosis pathway, respectively. NF-κB is a transcription factor known to be involved in the initiation and progression of breast cancer. Noscapine, an alkaloid derived from opium is used as antitussive and showed antitumor properties that induced apoptosis in cancer cell lines. The aim of the present study was to determine the apoptotic effect of noscapine in breast cancer cell lines compared to breast normal cell line. Three cell lines were used: i) a control breast cell line MCF-10F; ii) a luminal-like adenocarcinoma triple-positive breast cell line MCF-7; iii) breast cancer triple-negative cell line MDA-MB-231. Our results showed that noscapine had lower toxicity in normal cells and was an effective anticancer agent that induced apoptosis in breast cancer cells because it increases Bax gene and protein expression in three cell lines, while decreases Bcl-xL gene expression, and Bcl-2 protein expression decreased in breast cancer cell lines. Therefore, Bax/Bcl-2 ratio increased in the three cell lines. This drug increased caspase-9 gene expression in breast cancer cell lines and caspase-8 gene expression increased in MCF-10F and MDA-MB-231. Furthermore, it increased cleavage of caspase-8, suggesting that noscapine-induced apoptosis is probably due to the involvement of extrinsic and intrinsic apoptosis pathways. Antiapoptotic gene and protein expression diminished and proapoptotic gene and protein expression increased noscapine-induced expression, probably due to decrease in NF-κB gene and protein expression

  17. Differential effects of bisphosphonates on breast cancer cell lines

    NARCIS (Netherlands)

    Verdijk, R.; Franke, H.R.; Wolbers, F.; Vermes, I.

    2007-01-01

    Bisphosphonates may induce direct anti-tumor effects in breast cancers cells in virtro. In this study, six bisphosphonates were administered to three breast caner cell lines. Cell proliferation was measured by quantification of th expressio of Cyclin D1 mRNA. Apoptosis was determined by flow cytome

  18. Astrocytes as a source for Extracellular matrix molecules and cytokines

    Directory of Open Access Journals (Sweden)

    Stefan eWiese

    2012-06-01

    Full Text Available Research of the past 25 years has shown that astrocytes do more than participating and building up the blood brain barrier and detoxify the active synapse by reuptake of neurotransmitters and ions. Indeed, astrocytes express neurotransmitter receptors and, as a consequence, respond to stimuli. Deeper knowledge of the differentiation processes during development of the central nervous system (CNS might help explaining and even help treating neurological diseases like Alzheimer’s disease, Amyotrophic lateral sclerosis (ALS and psychiatric disorders in which astrocytes have been shown to play a role. Astrocytes and oligodendrocytes develop from a multipotent stem cell that prior to this has produced primarily neuronal precursor cells. This switch towards the more astroglial differentiation is regulated by a change in receptor composition on the cell surface and responsiveness of the respective trophic factors Fibroblast growth factor (FGF and Epidermal growth factor (EGF. The glial precursor cell is driven into the astroglial direction by signaling molecules like Ciliary neurotrophic factor (CNTF, Bone Morphogenetic Proteins (BMPs, and EGF. However, the early astrocytes influence their environment not only by releasing and responding to diverse soluble factors but also express a wide range of extracellular matrix (ECM molecules, in particular proteoglycans of the lectican family and tenascins. Lately these ECM molecules have been shown to participate in glial development. In this regard, especially the matrix protein Tenascin C (Tnc proved to be an important regulator of astrocyte precursor cell proliferation and migration during spinal cord development. On the other hand, ECM molecules expressed by reactive astrocytes are also known to act mostly in an inhibitory fashion under pathophysiological conditions. In this regard, we further summarize recent data concerning the role of chondroitin sulfate proteoglycans and Tnc under pathological

  19. Investigation of the selenium metabolism in cancer cell lines.

    Science.gov (United States)

    Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan; Andresen, Lars; Skov, Søren; Gammelgaard, Bente

    2011-02-01

    The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line (Jurkat E6-1) were incubated with five selenium compounds representing inorganic as well as organic Se compounds in different oxidation states. Selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), methylseleninic acid (MeSeA), selenite and selenate in the concentration range 5-100 μM were incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size exclusion chromatography and ICP-MS detection. The selenium compounds exhibited large differences in their ability to induce cell death in the three cell lines and the susceptibilities of the cell lines were different. Full recovery of selenium in the cellular fractions was observed for all Se compounds except MeSeA. Speciation analysis showed that MeSeA was completely transformed during the incubations, while metabolic conversion of the other Se compounds was limited. Production of volatile dimethyl diselenide was observed for MeSeA and MeSeCys. MeSeA, MeSeCys and selenite showed noticeable protein binding. Correlations between cell death induction and the Se compounds transformations could not be demonstrated.

  20. Global Conservation of Protein Status between Cell Lines and Xenografts

    Directory of Open Access Journals (Sweden)

    Julian Biau

    2016-08-01

    Full Text Available Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery.

  1. Phenotypes and karyotypes of human malignant mesothelioma cell lines.

    Directory of Open Access Journals (Sweden)

    Vandana Relan

    Full Text Available BACKGROUND: Malignant mesothelioma is an aggressive tumour of serosal surfaces most commonly pleura. Characterised cell lines represent a valuable tool to study the biology of mesothelioma. The aim of this study was to develop and biologically characterise six malignant mesothelioma cell lines to evaluate their potential as models of human malignant mesothelioma. METHODS: Five lines were initiated from pleural biopsies, and one from pleural effusion of patients with histologically proven malignant mesothelioma. Mesothelial origin was assessed by standard morphology, Transmission Electron Microscopy (TEM and immunocytochemistry. Growth characteristics were assayed using population doubling times. Spectral karyotyping was performed to assess chromosomal abnormalities. Authentication of donor specific derivation was undertaken by DNA fingerprinting using a panel of SNPs. RESULTS: Most of cell lines exhibited spindle cell shape, with some retaining stellate shapes. At passage 2 to 6 all lines stained positively for calretinin and cytokeratin 19, and demonstrated capacity for anchorage-independent growth. At passage 4 to 16, doubling times ranged from 30-72 hours, and on spectral karyotyping all lines exhibited numerical chromosomal abnormalities ranging from 41 to 113. Monosomy of chromosomes 8, 14, 22 or 17 was observed in three lines. One line displayed four different karyotypes at passage 8, but only one karyotype at passage 42, and another displayed polyploidy at passage 40 which was not present at early passages. At passages 5-17, TEM showed characteristic features of mesothelioma ultrastructure in all lines including microvilli and tight intercellular junctions. CONCLUSION: These six cell lines exhibit varying cell morphology, a range of doubling times, and show diverse passage-dependent structural chromosomal changes observed in malignant tumours. However they retain characteristic immunocytochemical protein expression profiles of

  2. Laminins containing the β2 and γ3 chains regulate astrocyte migration and angiogenesis in the retina.

    Science.gov (United States)

    Gnanaguru, Gopalan; Bachay, Galina; Biswas, Saptarshi; Pinzón-Duarte, Germán; Hunter, Dale D; Brunken, William J

    2013-05-01

    Pathologies of retinal blood vessels are among the major causes of blindness worldwide. A key cell type that regulates retinal vascular development is the astrocyte. Generated extrinsically to the retina, astrocytes migrate into the retina through the optic nerve head. Even though there is a strong correlation between astrocyte distribution and retinal vascular development, the factors that guide astrocytes into the retina remain unclear. In this study, we show that astrocytes migrate within a laminin-containing basement membrane - the inner limiting membrane. Genetic deletion of the laminin β2 and γ3 chains affects astrocyte migration and spatial distribution. We show that laminins act as haptotactic factors in vitro in an isoform-specific manner, inducing astrocyte migration and promoting astrocyte differentiation. The addition of exogenous laminins to laminin-null retinal explants rescues astrocyte migration and spatial patterning. Furthermore, we show that the loss of laminins reduces β1 integrin expression in astrocytes. Culturing laminin-null retinal astrocytes on laminin substrates restores focal localization of β1 integrin. Finally, we show that laminins containing β2 and γ3 chains regulate subsequent retinal blood vessel growth and maintain vascular integrity. These in vivo and in vitro studies demonstrate clearly that laminins containing β2 and γ3 chains are indispensable for migration and spatial organization of astrocytes and that they play a crucial role during retinal angiogenesis in vivo.

  3. The similarity of astrocytes number in dentate gyrus and CA3 subfield of rats hippocampus.

    Science.gov (United States)

    Jahanshahi, Mehrdad; Sadeghi, Y; Hosseini, A; Naghdi, N

    2007-01-01

    The dentate gyrus is a part of hippocampal formation that it contains granule cells, which project to the pyramidal cells and interneurons of the CA3 subfield of the hippocampus. Astrocytes play a more active role in neuronal activity, including regulating ion flux currents, energy production, neurotransmitter release and synaptogenesis. Astrocytes are the only cells in the brain that contain the energy molecule glycogen. The close relationship between dentate gyrus and CA3 area can cause the similarity of the number of astrocytes in these areas. In this study 5 male albino wistar rats were used. Rats were housed in large plastic cage in animal house and were maintained under standard conditions, after histological processing, The 7 microm slides of the brains were stained with PTAH staining for showing the astrocytes. This staining is specialized for astrocytes. We showed that the number of astrocytes in different (ant., mid., post) parts of dentate gyrus and CA3 of hippocampus is the same. For example, the anterior parts of two area have the most number of astrocytes and the middle parts of two area have the least number of astrocytes. We concluded that dentate gyrus and CA3 area of hippocampus have the same group of astrocytes.

  4. Pathway-specific differences between tumor cell lines and normal and tumor tissue cells

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    Tozeren Aydin

    2006-11-01

    Full Text Available Abstract Background Cell lines are used in experimental investigation of cancer but their capacity to represent tumor cells has yet to be quantified. The aim of the study was to identify significant alterations in pathway usage in cell lines in comparison with normal and tumor tissue. Methods This study utilized a pathway-specific enrichment analysis of publicly accessible microarray data and quantified the gene expression differences between cell lines, tumor, and normal tissue cells for six different tissue types. KEGG pathways that are significantly different between cell lines and tumors, cell lines and normal tissues and tumor and normal tissue were identified through enrichment tests on gene lists obtained using Significance Analysis of Microarrays (SAM. Results Cellular pathways that were significantly upregulated in cell lines compared to tumor cells and normal cells of the same tissue type included ATP synthesis, cell communication, cell cycle, oxidative phosphorylation, purine, pyrimidine and pyruvate metabolism, and proteasome. Results on metabolic pathways suggested an increase in the velocity nucleotide metabolism and RNA production. Pathways that were downregulated in cell lines compared to tumor and normal tissue included cell communication, cell adhesion molecules (CAMs, and ECM-receptor interaction. Only a fraction of the significantly altered genes in tumor-to-normal comparison had similar expressions in cancer cell lines and tumor cells. These genes were tissue-specific and were distributed sparsely among multiple pathways. Conclusion Significantly altered genes in tumors compared to normal tissue were largely tissue specific. Among these genes downregulation was a major trend. In contrast, cell lines contained large sets of significantly upregulated genes that were common to multiple tissue types. Pathway upregulation in cell lines was most pronounced over metabolic pathways including cell nucleotide metabolism and oxidative

  5. Regulation of Kir4.1 expression in astrocytes and astrocytic tumors: a role for interleukin-1 β

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    Zurolo Emanuele

    2012-12-01

    Full Text Available Abstract Objective Decreased expression of inwardly rectifying potassium (Kir channels in astrocytes and glioma cells may contribute to impaired K+ buffering and increased propensity for seizures. Here, we evaluated the potential effect of inflammatory molecules, such as interleukin-1β (IL-1β on Kir4.1 mRNA and protein expression. Methods We investigated Kir4.1 (Kcnj10 and IL-1β mRNA expression in the temporal cortex in a rat model of temporal lobe epilepsy 24 h and 1 week after induction of status epilepticus (SE, using real-time PCR and western blot analysis. The U373 glioblastoma cell line and human fetal astrocytes were used to study the regulation of Kir4.1 expression in response to pro-inflammatory cytokines. Expression of Kir4.1 protein was also evaluated by means of immunohistochemistry in surgical specimens of patients with astrocytic tumors (n = 64, comparing the expression in tumor patients with (n = 38 and without epilepsy (n = 26. Results Twenty-four hours after onset of SE, Kir4.1 mRNA and protein were significantly down-regulated in temporal cortex of epileptic rats. This decrease in expression was followed by a return to control level at 1 week after SE. The transient downregulation of Kir4.1 corresponded to the time of prominent upregulation of IL-1β mRNA. Expression of Kir4.1 mRNA and protein in glial cells in culture was downregulated after exposure to IL-1β. Evaluation of Kir4.1 in tumor specimens showed a significantly lower Kir4.1 expression in the specimens of patients with epilepsy compared to patients without epilepsy. This paralleled the increased presence of activated microglial cells, as well as the increased expression of IL-1β and the cytoplasmic translocation of high mobility group box 1 (HMGB1. Conclusions Taken together, these findings indicate that alterations in expression of Kir4.1 occurring in epilepsy-associated lesions are possibly influenced by the local inflammatory environment and in

  6. Determinants of intrinsic radiosensitivity of mammalian cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Radford, I.R. [Peter MacCallum Cancer Institute, East Melbourne, VIC (Australia). Research Division

    1998-12-31

    Differences in the radiosensitivity of normal and cancerous cells could arise in various ways. Although there is no compelling data to support the view, the currently prevailing opinion is that differences in radiosensitivity are related to differences in some aspect of enzymatic DNA repair. A test of the importance of possible differences in enzymatic DNA repair in determining relative radiosensitivity would be to compare lethality in cells containing equivalent numbers of DNA lesions. Six cell lines were used in these studies: two Chinese hamster (CHO and V79) and a monkey (Vero) fibroblast-like line, a mouse melanoma line (B16-F1), and a rat (RUC-2) and a human (SQ-20B) carcinoma line. This group of cell lines displays a wide range of sensitivities to external beam low-LET radiation, ranging from the relatively radiosensitive B16-F1 and Vero lines through to the highly radioresistant RUC-2 line. However, it is important to note that none of the lines has a demonstrated defect in enzymatic DNA repair and that all appear to die by necrosis following a lethal radiation insult. Despite having significantly different radiosensitivities, CHO and V79 cells showed comparable responses to DNA-associated {sup 125}I-decays with D{sub o} values of around 65. More surprisingly, the radiosensitive B16-F1 line and the radioresistant RUC-2 line both had responses with D{sub o} values of around 133 {sup 125}I-decays. The factor of two difference between the D{sub o} values for these two pairs of cell lines is probably attributable to CHO and V79 cells being pseudo-diploid whereas B 16-F1 and RUC2 appear to have derived from tetraploid cells. The generality of the above result, for DNA lesions of different quality, was tested by comparing the sensitivities of CHO and V79 cells to DNA-associated {sup 3}H-decays. Again, consistent with the {sup 125}I-decay data, there was no significant difference in the D{sub o} values for these lines. Our {sup 3}H- and {sup 125}I-decay data are

  7. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren

    2016-01-01

    Stem cell banks curating and distributing human embryonic stem cells have been established in a number of countries and by a number of private institutions. This paper identifies and critically discusses a number of arguments that are used to justify the importance of such banks in policy...... are curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being...

  8. 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.

  9. Cold storage and cryopreservation of tick cell lines

    Directory of Open Access Journals (Sweden)

    Lallinger Gertrud

    2010-04-01

    Full Text Available Abstract Background Tick cell lines are now available from fifteen ixodid and argasid species of medical and veterinary importance. However, some tick cell lines can be difficult to cryopreserve, and improved protocols for short- and long-term low temperature storage will greatly enhance their use as tools in tick and tick-borne pathogen research. In the present study, different protocols were evaluated for cold storage and cryopreservation of tick cell lines derived from Rhipicephalus (Boophilus decoloratus, Rhipicephalus (Boophilus microplus, Ixodes ricinus and Ixodes scapularis. For short-term cold storage, cells were kept under refrigeration at 6°C for 15, 30 and 45 days. For cryopreservation in liquid nitrogen, use of a sucrose-phosphate-glutamate freezing buffer (SPG as cryoprotectant was compared with dimethylsulfoxide (DMSO supplemented with sucrose. Cell viability was determined by the trypan blue exclusion test and cell morphology was evaluated in Giemsa-stained cytocentrifuge smears. Results Cold storage at 6°C for up to 30 days was successful in preserving R. (B. microplus, R. (B. decoloratus, I. ricinus and I. scapularis cell lines; lines from the latter three species could be easily re-cultivated after 45 days under refrigeration. While cell lines from all four tick species cryopreserved with 6% DMSO were successfully resuscitated, the R. (B. decoloratus cells did not survive freezing in SPG and of the other three species, only the R. (B. microplus cells resumed growth during the observation period. Conclusions This constitutes the first report on successful short-term refrigeration of cells derived from R. (B. decoloratus, R. (B. microplus, and I. ricinus, and use of SPG as an alternative to DMSO for cryopreservation, thus making an important contribution to more reliable and convenient tick cell culture maintenance.

  10. [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.

  11. Astrocytes from familial and sporadic ALS patients are toxic to motor neurons.

    Science.gov (United States)

    Haidet-Phillips, Amanda M; Hester, Mark E; Miranda, Carlos J; Meyer, Kathrin; Braun, Lyndsey; Frakes, Ashley; Song, SungWon; Likhite, Shibi; Murtha, Matthew J; Foust, Kevin D; Rao, Meghan; Eagle, Amy; Kammesheidt, Anja; Christensen, Ashley; Mendell, Jerry R; Burghes, Arthur H M; Kaspar, Brian K

    2011-08-10

    Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, with astrocytes implicated as contributing substantially to motor neuron death in familial (F)ALS. However, the proposed role of astrocytes in the pathology of ALS derives in part from rodent models of FALS based upon dominant mutations within the superoxide dismutase 1 (SOD1) gene, which account for 90% of ALS patients, remains to be established. Using astrocytes generated from postmortem tissue from both FALS and SALS patients, we show that astrocytes derived from both patient groups are similarly toxic to motor neurons. We also demonstrate that SOD1 is a viable target for SALS, as its knockdown significantly attenuates astrocyte-mediated toxicity toward motor neurons. Our data highlight astrocytes as a non-cell autonomous component in SALS and provide an in vitro model system to investigate common disease mechanisms and evaluate potential therapies for SALS and FALS.

  12. Metabolic aspects of Neuronal – Oligodendrocytic - Astrocytic (NOA interactions

    Directory of Open Access Journals (Sweden)

    Ana I Amaral

    2013-05-01

    Full Text Available Whereas astrocytes have been in the limelight on the metabolic glucose interaction scene for a while, oligodendrocytes are still waiting for a place. We would like to call oligodendrocyte interaction with astrocytes and neurons: NOA (neuron – oligodendrocyte – astrocyte interactions. One of the reasons to find out more about oligodendrocyte interaction with neurons and astrocytes is to detect markers of healthy oligodendrocyte metabolism, to be used in diagnosis and treatment assessment in diseases such as Perinatal hypoxic-ischemic encephalopathy and multiple sclerosis in which oligodendrocyte function is impaired, possibly due to glutamate toxicity. Glutamate receptors are expressed in oligodendrocytes and also vesicular glutamate release in the white matter has received considerable attention. It is also important to establish if the glial precursor cells recruited to damaged areas are developing oligodendrocyte characteristics or those of astrocytes. Thus, it is important to study astrocytes and oligodendrocytes separately to be able to differentiate between them. This is of particular importance in the white matter where the number of oligodendrocytes is considerable. The present review summarizes the not very extensive information published on glucose metabolism in oligodendrocytes in an attempt to stimulate research into this important field.

  13. Are astrocytes central players in the pathophysiology of multiple sclerosis?

    NARCIS (Netherlands)

    De Keyser, J; Zeinstra, E; Frohman, E

    2003-01-01

    An interaction between antimyelin T cells and antigen-presenting glial cells is a crucial step in the cascade of immune events that lead to the inflammatory lesions in multiple sclerosis (MS). One of the most debated and controversial issues is whether microglial cells or astrocytes are the key play

  14. Establishment of a new bovine leukosis virus producing cell line.

    Science.gov (United States)

    Beier, D; Riebe, R; Blankenstein, P; Starick, E; Bondzio, A; Marquardt, O

    2004-11-01

    Due to the prevalence of different bovine leukosis virus (BLV) species in the cattle population in Europe, problems may arise in the serological diagnosis of BLV infections. In addition, earlier investigations demonstrated that contamination of the BLV antigen-producing cell culture systems by bovine viral diarrhea virus (BVDV) may give rise to misinterpretation of serological test results after BVDV vaccination of cattle. By co-cultivation of peripheral leukocytes of a BLV-infected cow with a permanent sheep kidney cell line, a new BLV-producing cell line named PO714 was established. This line carries a BLV provirus of the Belgian species and has been tested to be free of a variety of possibly contaminating viruses and mycoplasms. Investigations of a panel of well-characterised sera by agar gel immunodiffusion (AGID) and capture ELISA (cELISA) tests using antigen prepared from this new cell line in comparison with antigen of the well-known cell line FLK/BLV yielded comparable results. False positive results caused by BVDV cross-reactions could be eliminated when tests were carried out with antigen derived from the new cell line.

  15. Expressional patterns of chaperones in ten human tumor cell lines

    Directory of Open Access Journals (Sweden)

    Slavc Irene

    2004-12-01

    Full Text Available Abstract Background Chaperones (CH play an important role in tumor biology but no systematic work on expressional patterns has been reported so far. The aim of the study was therefore to present an analytical method for the concomitant determination of several CH in human tumor cell lines, to generate expressional patterns in the individual cell lines and to search for tumor and non-tumor cell line specific CH expression. Human tumor cell lines of neuroblastoma, colorectal and adenocarcinoma of the ovary, osteosarcoma, rhabdomyosarcoma, malignant melanoma, lung, cervical and breast cancer, promyelocytic leukaemia were homogenised, proteins were separated on two-dimensional gel electrophoresis with in-gel digestion of proteins and MALDI-TOF/TOF analysis was carried out for the identification of CH. Results A series of CH was identified including the main CH groups as HSP90/HATPas_C, HSP70, Cpn60_TCP1, DnaJ, Thioredoxin, TPR, Pro_isomerase, HSP20, ERP29_C, KE2, Prefoldin, DUF704, BAG, GrpE and DcpS. Conclusions The ten individual tumor cell lines showed different expression patterns, which are important for the design of CH studies in tumor cell lines. The results can serve as a reference map and form the basis of a concomitant determination of CH by a protein chemical rather than an immunochemical method, independent of antibody availability or specificity.

  16. Metronidazole decreases viability of DLD-1 colorectal cancer cell line.

    Science.gov (United States)

    Sadowska, Anna; Krętowski, Rafał; Szynaka, Beata; Cechowska-Pasko, Marzanna; Car, Halina

    2013-10-01

    The aim of our study was to evaluate the impact of metronidazole (MTZ) on DLD-1 colorectal cancer cell (CRC) line. Toxicity of MTZ was determined by MTT test. Cells were incubated with MTZ used in different concentrations for 24, 48, and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. The morphological changes in human DLD-1 cell line were defined by transmission electron microscope OPTON 900. The influence of MTZ on the apoptosis of DLD-1 cell lines was detected by flow cytometry and fluorescence microscopy, while cell concentration, volume, and diameter were displayed by Scepter Cell Counter from Millipore. Our results show that cell viability was diminished in all experimental groups in comparison with the control, and the differences were statistically significant. We did not find any significant differences in [3H]-thymidine incorporation in all experimental groups and times of observation. Cytofluorimetric assays demonstrated a statistically significant increase of apoptotic rate in MTZ concentrations 10 and 50 μg/mL after 24 hours; 0.1, 10, 50, and 250 μg/mL after 48 hours; and in all concentrations after 72 hours compared with control groups. In the ultrastructural studies, necrotic or apoptotic cells were occasionally seen. In conclusion, MTZ affects human CRC cell line viability. The reduction of cell viability was consistent with the apoptotic test.

  17. Markers of murine embryonic and neural stem cells, neurons and astrocytes: reference points for developmental neurotoxicity testing

    Science.gov (United States)

    Developmental neurotoxicity (DNT) is a significant concern for environmental chemicals, as well as for food and drug constituents. The sensitivity of animal-based DNT models is unclear, and they are expensive and time consuming. Murine embryonic stem cells (mESC) recapitulate sev...

  18. In vitro radiosensitivity of human leukemia cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Weichselbaum, R.R.; Greenberger, J.S.; Schmidt, A.; Karpas, A.; Moloney, W.C.; Little, J.B.

    1981-05-01

    The in vitro radiobiologic survival values (n, D0) of four tumor lines derived from human hematopoietic tumors were studied. These cell lines were HL50 (n . 1.3, D0 . 117 rad(1.17 Gy)), promyelocytic leukemia; K562 (n . 1.4, D0 . 165 rad(1.65 Gy)), erythroleukemia; 45 (n . 1.1, D0 . 147 rad(1.47 Gy)), acute lymphocyte leukemia; and 176 (n . 4.0, D0 . 76 rad(0.76 Gy)), acute monomyelogenous leukemia. More cell lines must be examined before the exact relationship between in vitro radiosensitivity and clinical radiocurability is firmly established.

  19. Effect of failures and repairs on multiple cell production lines

    Energy Technology Data Exchange (ETDEWEB)

    Legato, P.; Bobbio, A.; Roberti, L.

    1989-01-01

    This paper examines a production line composed of multiple stages, or cells, which are passed in sequential order to arrive to the final product. Two possible coordination disciplines are considered, namely: the classical tandem arrangement of sequential working centers with input buffer and the kanban scheme, considered the Japanese shop floor realization of the Just-In-Time (JIT) manifacturing approach. The production line is modelled and analysed by means of Stochastic Petri Nets (SPN). Finally an analysis is made of the possibility that the working cells can incur failure/repair cycles perturbing the production flow of the line and thus reduce performance indices.

  20. Efficacy of ribavirin against malignant glioma cell lines

    Science.gov (United States)

    OGINO, AKIYOSHI; SANO, EMIKO; OCHIAI, YUSHI; YAMAMURO, SHUN; TASHIRO, SHINYA; YACHI, KAZUNARI; OHTA, TAKASHI; FUKUSHIMA, TAKAO; OKAMOTO, YUTAKA; TSUMOTO, KOUHEI; UEDA, TAKUYA; YOSHINO, ATSUO; KATAYAMA, YOICHI

    2014-01-01

    Ribavirin (1-β-D-ribofuranosy-1,2,4-triazole-3-carboxamide) has been widely administered as an antiviral agent against RNA and DNA viruses. Ribavirin, in combination with interferon, has predominantly been applied in the treatment of the hepatitis C virus infection and its potential antitumor efficacy has recently become a point of interest. The aim of the present study was to evaluate the effect of ribavirin on the growth of malignant glioma cells, to identify novel predictive genes in malignant glioma cells (by analyzing gene expression profiles) and to assess the influence of ribavirin on the cell cycle of malignant glioma cells. The present study evaluated the antitumor efficacy of ribavirin against various malignant glioma cell lines (A-172, AM-38, T98G, U-87MG, U-138MG, U-251MG and YH-13). After culturing the cells in ribavirin-containing culture medium (final concentration, 0–1,000 μM) for 72 h, the viable proliferated cells were harvested and counted. The half maximal inhibitory concentration of ribavirin, with regard to the growth of the malignant glioma cell lines, was determined from the concentration of ribavirin required for 50% growth inhibition in comparison to the untreated control cells. Furthermore, the current study identified the genes in which the gene expression levels correlated with the ribavirin sensitivity of the malignant glioma cells lines, using a high-density oligonucleotide array. Finally, cell cycle analysis was performed on the U-87MG cell line. It was identified that ribavirin inhibited the growth of all of the malignant glioma cell lines in a dose-dependent manner, although the ribavirin sensitivity varied between each cell line. Of the extracted genes, PDGFRA demonstrated the strongest positive correlation between gene expression level and ribavirin sensitivity. Cell cycle analysis of the U-87MG cell line demonstrated that ribavirin treatment induces G0/G1 arrest and thus may be an effective agent for inhibiting malignant

  1. Comparative analysis of cell death induction by Taurolidine in different malignant human cancer cell lines

    Directory of Open Access Journals (Sweden)

    Ritter Peter R

    2010-03-01

    Full Text Available Abstract Background Taurolidine (TRD represents an anti-infective substance with anti-neoplastic activity in many malignant cell lines. So far, the knowledge about the cell death inducing mechanisms and pathways activated by TRD is limited. The aim of this study was therefore, to perform a comparative analysis of cell death induction by TRD simultaneously in different malignant cell lines. Materials and methods Five different malignant cell lines (HT29/Colon, Chang Liver/Liver, HT1080/fibrosarcoma, AsPC-1/pancreas and BxPC-3/pancreas were incubated with increasing concentrations of TRD (100 μM, 250 μM and 1000 μM for 6 h and 24 h. Cell viability, apoptosis and necrosis were analyzed by FACS analysis (Propidiumiodide/AnnexinV staining. Additionally, cells were co-incubated with the caspase Inhibitor z-VAD, the radical scavenger N-Acetylcystein (NAC and the Gluthation depleting agent BSO to examine the contribution of caspase activation and reactive oxygen species in TRD induced cell death. Results All cell lines were susceptible to TRD induced cell death without resistance toward this anti-neoplastic agent. However, the dose response effects were varying largely between different cell lines. The effect of NAC and BSO co-treatment were highly different among cell lines - suggesting a cell line specific involvement of ROS in TRD induced cell death. Furthermore, impact of z-VAD mediated inhibition of caspases was differing strongly among the cell lines. Conclusion This is the first study providing a simultaneous evaluation of the anti-neoplastic action of TRD across several malignant cell lines. The involvement of ROS and caspase activation was highly variable among the five cell lines, although all were susceptible to TRD induced cell death. Our results indicate, that TRD is likely to provide multifaceted cell death mechanisms leading to a cell line specific diversity.

  2. Simultaneous neuron- and astrocyte-specific fluorescent marking.

    Science.gov (United States)

    Schulze, Wiebke; Hayata-Takano, Atsuko; Kamo, Toshihiko; Nakazawa, Takanobu; Nagayasu, Kazuki; Kasai, Atsushi; Seiriki, Kaoru; Shintani, Norihito; Ago, Yukio; Farfan, Camille; Hashimoto, Ryota; Baba, Akemichi; Hashimoto, Hitoshi

    2015-03-27

    Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignment of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains.

  3. Exometabolom analysis of breast cancer cell lines: Metabolic signature.

    Science.gov (United States)

    Willmann, Lucas; Erbes, Thalia; Halbach, Sebastian; Brummer, Tilman; Jäger, Markus; Hirschfeld, Marc; Fehm, Tanja; Neubauer, Hans; Stickeler, Elmar; Kammerer, Bernd

    2015-08-21

    Cancer cells show characteristic effects on cellular turnover and DNA/RNA modifications leading to elevated levels of excreted modified nucleosides. We investigated the molecular signature of different subtypes of breast cancer cell lines and the breast epithelial cell line MCF-10A. Prepurification of cell culture supernatants was performed by cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. Samples were analyzed by application of reversed phase chromatography coupled to a triple quadrupole mass spectrometer. Collectively, we determined 23 compounds from RNA metabolism, two from purine metabolism, five from polyamine/methionine cycle, one from histidine metabolism and two from nicotinate and nicotinamide metabolism. We observed major differences of metabolite excretion pattern between the breast cancer cell lines and MCF-10A, just as well as between the different breast cancer cell lines themselves. Differences in metabolite excretion resulting from cancerous metabolism can be integrated into altered processes on the cellular level. Modified nucleosides have great potential as biomarkers in due consideration of the heterogeneity of breast cancer that is reflected by the different molecular subtypes of breast cancer. Our data suggests that the metabolic signature of breast cancer cell lines might be a more subtype-specific tool to predict breast cancer, rather than a universal approach.

  4. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren

    2016-01-01

    are curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being...

  5. Characterisation of thyroid medullary carcinoma TT cell line.

    Science.gov (United States)

    Zabel, M; Grzeszkowiak, J

    1997-01-01

    TT cell line is the best known stabilized cell line derived from the human medullary thyroid carcinoma. The ultrastructural characteristics of these cells include well developed rough endoplasmic reticulum, a prominent Golgi apparatus and a considerable number of secretory granules. Numerous hormones were immunocytochemically demonstrated in TT cells of which calcitonin and calcitonin gene-related peptide (CGRP) are the products of the same gene but an alternative RNA processing. TT cells were found to produce some other hormones as well, namely ACTH, neurotensin, enkephalin, PTHrP, gastrin-releasing peptide (GRP), serotonin but also functional proteins of the chromogranin group, synaptophysin, NSE, calbindin and tyrosine hydroxylase. Some marker proteins have been detected in the cytosol (CEA) and in the cytoskeleton (alpha-tubulin, cytokeratin). The influence of numerous factors on the secretory activity of these cells has been demonstrated so far, including effects of 1,25-dihydroxycholecalciferol, glucocorticoids, sex steroids, cAMP, gastrin-releasing peptide, sodium butyrate, phorbol esters, ionomycin and forskolin. The investigators performed on the TT cell line demonstrate that this is the most reliable model system for the human parafollicular cells developed so far, in comparison to other cell lines derived from the medullary carcinoma of the thyroid.

  6. Neurohypophysial Receptor Gene Expression by Thymic T Cell Subsets and Thymic T Cell Lymphoma Cell Lines

    Directory of Open Access Journals (Sweden)

    I. Hansenne

    2004-01-01

    transcribed in thymic epithelium, while immature T lymphocytes express functional neurohypophysial receptors. Neurohypophysial receptors belong to the G protein-linked seven-transmembrane receptor superfamily and are encoded by four distinct genes, OTR, V1R, V2R and V3R. The objective of this study was to identify the nature of neurohypophysial receptor in thymic T cell subsets purified by immunomagnetic selection, as well as in murine thymic lymphoma cell lines RL12-NP and BW5147. OTR is transcribed in all thymic T cell subsets and T cell lines, while V3R transcription is restricted to CD4+ CD8+ and CD8+ thymic cells. Neither V1R nor V2R transcripts are detected in any kind of T cells. The OTR protein was identified by immunocytochemistry on thymocytes freshly isolated from C57BL/6 mice. In murine fetal thymic organ cultures, a specific OTR antagonist does not modify the percentage of T cell subsets, but increases late T cell apoptosis further evidencing the involvement of OT/OTR signaling in the control of T cell proliferation and survival. According to these data, OTR and V3R are differentially expressed during T cell ontogeny. Moreover, the restriction of OTR transcription to T cell lines derived from thymic lymphomas may be important in the context of T cell leukemia pathogenesis and treatment.

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

    Science.gov (United States)

    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.

  8. Effect of type-2 astrocytes on the viability of dorsal root ganglion neurons and length of neuronal processes

    Institute of Scientific and Technical Information of China (English)

    Chunling Fan; Hui Wang; Dan Chen; Xiaoxin Cheng; Kun Xiong; Xuegang Luo; Qilin Cao

    2014-01-01

    The role of type-2 astrocytes in the repair of central nervous system injury remains poorly un-derstood. In this study, using a relatively simple culture condition in vitro, type-2 astrocytes, differentiated from oligodendrocyte precursor cells by induction with bone morphogenetic pro-tein-4, were co-cultured with dorsal root ganglion neurons. We examined the effects of type-2 astrocytes differentiated from oligodendrocyte precursor cells on the survival and growth of dorsal root ganglion neurons. Results demonstrated that the number of dorsal root ganglion neurons was higher following co-culture of oligodendrocyte precursor cells and type-2 astrocytes than when cultured alone, but lower than that of neurons co-cultured with type-1 astrocytes. The length of the longest process and the length of all processes of a single neuron were shortest in neurons cultured alone, followed by neurons co-cultured with type-2 astrocytes, then neurons co-cultured with oligodendrocyte precursor cells, and longest in neurons co-cultured with type-1 astrocytes. These results indicate that co-culture with type-2 astrocytes can increase neuronal survival rate and process length. However, compared with type-1 astrocytes and oligodendrocyte precursor cells, the promotion effects of type-2 astrocytes on the growth of dorsal root ganglion neurons were weaker.

  9. Membrane lipidome of an epithelial cell line

    DEFF Research Database (Denmark)

    Sampaio, Julio L; Gerl, Mathias J; Klose, Christian

    2011-01-01

    Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology...... to an epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin...... to glycosphingolipid, together with an increase in plasmalogen, phosphatidylethanolamine, and cholesterol content, whereas the opposite changes took place during an epithelial-to-mesenchymal transition. Moreover, during polarization, the sphingolipids became longer, more saturated, and more hydroxylated as required...

  10. Redox state alteration modulates astrocyte glucuronidation.

    Science.gov (United States)

    Heurtaux, T; Benani, A; Bianchi, A; Moindrot, A; Gradinaru, D; Magdalou, J; Netter, P; Minn, A

    2004-10-01

    We have investigated the effects of mild oxidative conditions on drug-metabolizing enzyme activity in rat cultured astrocytes. These experimental conditions promoting an oxidative environment were obtained by short exposure to a low concentration of menadione (5 microM) for a short duration (15 min). This resulted in the rapid and transient production of reactive oxygen species (+130%), associated with a decrease in GSH cellular content (-24%), and an increase in total protein oxidation (+26%), but promoted neither PGE(2) nor NO production. This treatment induced a rapid and persistent decrease in astrocyte glucuronidation activities, which was totally prevented by N-acetyl-l-cysteine. These oxidative conditions also affected the specific UGT1A6 activity measured in transfected V79-1A6 cells. Finally, the subsequent recovery of astrocyte glucuronidation activity may result from upregulation of UGT1A6 expression (+62%) as shown by RT-PCR and gene reporter assay. These results show that the catalytic properties and expression of cerebral UGT1A6 are highly sensitive to the redox environment. The protective effect of N-acetyl-l-cysteine suggests both a direct action of reactive oxygen species on the protein and a more delayed action on the transcriptional regulation of UGT1A6. These results suggest that cerebral metabolism can be altered by physiological or pathological redox modifications.

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

    Science.gov (United States)

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

    2016-05-01

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

  12. Heterozygous embryonic stem cell lines derived from nonhuman primate parthenotes.

    Science.gov (United States)

    Dighe, Vikas; Clepper, Lisa; Pedersen, Darlene; Byrne, James; Ferguson, Betsy; Gokhale, Sumita; Penedo, M Cecilia T; Wolf, Don; Mitalipov, Shoukhrat

    2008-03-01

    Monoparental parthenotes represent a potential source of histocompatible stem cells that should be isogenic with the oocyte donor and therefore suitable for use in cell or tissue replacement therapy. We generated five rhesus monkey parthenogenetic embryonic stem cell (PESC) lines with stable, diploid female karyotypes that were morphologically indistinguishable from biparental controls, expressed key pluripotent markers, and generated cell derivatives representative of all three germ layers following in vivo and in vitro differentiation. Interestingly, high levels of heterozygosity were observed at the majority of loci that were polymorphic in the oocyte donors. Some PESC lines were also heterozygous in the major histocompatibility complex region, carrying haplotypes identical to those of the egg donor females. Expression analysis revealed transcripts from some imprinted genes that are normally expressed from only the paternal allele. These results indicate that limitations accompanying the potential use of PESC-derived phenotypes in regenerative medicine, including aberrant genomic imprinting and high levels of homozygosity, are cell line-dependent and not always present. PESC lines were derived in high enough yields to be practicable, and their derivatives are suitable for autologous transplantation into oocyte donors or could be used to establish a bank of histocompatible cell lines for a broad spectrum of patients.

  13. Transportation characteristics of nolatrexed in three tumor cell lines

    Institute of Scientific and Technical Information of China (English)

    LI Yi-lei; ZHAO Ai-guo; WU Shu-guang

    2002-01-01

    Objective:To investigate the association of the transportation characteristics of nolatrexed in tumor cells with its drug sensitivity. Methods: The sensitivity of 3 tumor cell lines, C6, SRS82 and LoVo, to nolatrexed were determined by growth inhibition study. After exposure to 20 μmol/L nolatrexed at different time intervals ranging from 0 to 30 min, or to nolatrexed at different concentrations ranging from 0 to 40μmol/L for 10 min, the intracellular drug concentration was measured using high-performance liquid chromatography. Results: C6 was the most sensitive cell line among the three, with sensitivity 6. 8-fold and 13.8-fold those of SRS-82 and LoVo cells respectively. Transportation of nolatrexed in the 3 cell lines were qualitatively similar, which rapidly achieved steady-state within 5 min, and linear relationship between the intracellular and extracellular drug concentration was observed. The intracellular steady-state level achieved in C6 was significantly higher than those in the other two cell lines, the latter having comparable levels. Conclusion: Nolatrexed enters the cell very quickly and different transport capacities are involved in the generation of varied sensitivity to nolatrexed in tumor cells.

  14. Reactive astrocytes express NADPH diaphorase in vivo after transient ischemia.

    Science.gov (United States)

    Endoh, M; Maiese, K; Pulsinelli, W A; Wagner, J A

    1993-05-14

    In the hippocampus, ten minutes of transient global ischemia results in the death of CA1 pyramidal cells after a period of one to three days. The neurons in the CA1 region constitutively express NADPH-D (NADPH diaphorase activity). In contrast, astrocytes in the hippocampus do not normally express NADPH-D; but a population of reactive astrocytes (GFAP+ cells) begin to express of NADPH-D one day after transient global ischemia. NADPH-D is thought to be a histological marker for Nitric Oxide Synthase (NOS), the enzyme that is responsible for the synthesis of NO, a potent neurotoxin. We suggest that this increase in NADPH-D/NOS expression is an important element in the sequence of changes that occurs after ischemia, and that NO derived from reactive astrocytes or from neurons may play a causal role in neural cell death after ischemia in the hippocampus.

  15. 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.

  16. Continuous production of erythropoietin by an established human renal carcinoma cell line: development of the cell line

    Energy Technology Data Exchange (ETDEWEB)

    Sherwood, J.B.; Shouval, D.

    1986-01-01

    Establishment of a stable, transformed human renal carcinoma cell line that produces erythropoietin in vitro and has maintained this function continuously since 1981 and for > 150 passages in monolayer culture was accomplished by transplantation of human renal clear cell carcinoma tissue from a patient with erythrocytosis into an immunosuppressed athymic mouse. In addition to its immunocrossreactivity with native human urinary erythropoietin, the tumor erythropoietin demonstrates biological activity in the in vitro mouse erythroid colony-forming unit assay and in tumor-bearing nude mice. The cloned renal carcinoma cell line has an abnormal human karyotype and has ultrastructural features characteristic of human renal clear cell carcinoma. This cell line provides a reproducible model system for the production of an erythropoietin-like material and for the study of its synthesis and secretion.

  17. Channel-Mediated Lactate Release by K+-Stimulated Astrocytes

    KAUST Repository

    Sotelo-Hitschfeld, T.

    2015-03-11

    Excitatory synaptic transmission is accompanied by a local surge in interstitial lactate that occurs despite adequate oxygen availability, a puzzling phenomenon termed aerobic glycolysis. In addition to its role as an energy substrate, recent studies have shown that lactate modulates neuronal excitability acting through various targets, including NMDA receptors and G-protein-coupled receptors specific for lactate, but little is known about the cellular and molecular mechanisms responsible for the increase in interstitial lactate. Using a panel of genetically encoded fluorescence nanosensors for energy metabolites, we show here that mouse astrocytes in culture, in cortical slices, and in vivo maintain a steady-state reservoir of lactate. The reservoir was released to the extracellular space immediately after exposure of astrocytes to a physiological rise in extracellular K+ or cell depolarization. Cell-attached patch-clamp analysis of cultured astrocytes revealed a 37 pS lactate-permeable ion channel activated by cell depolarization. The channel was modulated by lactate itself, resulting in a positive feedback loop for lactate release. A rapid fall in intracellular lactate levels was also observed in cortical astrocytes of anesthetized mice in response to local field stimulation. The existence of an astrocytic lactate reservoir and its quick mobilization via an ion channel in response to a neuronal cue provides fresh support to lactate roles in neuronal fueling and in gliotransmission.

  18. Derivation of human embryonic stem cell line Genea019

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea019 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype, female Allele pattern and unaffected Htt CAG repeat length, compared to HD affected sibling Genea020. Pluripotency of Genea019 was demonstrated with 75% of cells expressing Nanog, 89% Oct4, 48% Tra1-60 and 85% SSEA4, a Pluritest Pluripotency score of 22.97, Novelty score of 1.42, tri-lineage teratoma formation and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination.

  19. Guidelines for the use of cell lines in biomedical research.

    Science.gov (United States)

    Geraghty, R J; Capes-Davis, A; Davis, J M; Downward, J; Freshney, R I; Knezevic, I; Lovell-Badge, R; Masters, J R W; Meredith, J; Stacey, G N; Thraves, P; Vias, M

    2014-09-09

    Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise.

  20. Drosophila astrocytes cover specific territories of the CNS neuropil and are instructed to differentiate by Prospero, a key effector of Notch.

    Science.gov (United States)

    Peco, Emilie; Davla, Sejal; Camp, Darius; Stacey, Stephanie M; Landgraf, Matthias; van Meyel, Don J

    2016-04-01

    Astrocytes are crucial in the formation, fine-tuning, function and plasticity of neural circuits in the central nervous system. However, important questions remain about the mechanisms instructing astrocyte cell fate. We have studied astrogenesis in the ventral nerve cord of Drosophila larvae, where astrocytes exhibit remarkable morphological and molecular similarities to those in mammals. We reveal the births of larval astrocytes from a multipotent glial lineage, their allocation to reproducible positions, and their deployment of ramified arbors to cover specific neuropil territories to form a stereotyped astroglial map. Finally, we unraveled a molecular pathway for astrocyte differentiation in which the Ets protein Pointed and the Notch signaling pathway are required for astrogenesis; however, only Notch is sufficient to direct non-astrocytic progenitors toward astrocytic fate. We found that Prospero is a key effector of Notch in this process. Our data identify an instructive astrogenic program that acts as a binary switch to distinguish astrocytes from other glial cells.

  1. Effects of aspartame metabolites on astrocytes and neurons.

    Science.gov (United States)

    Rycerz, Karol; Jaworska-Adamu, Jadwiga Elżbieta

    2013-01-01

    Aspartame, a widespread sweetener used in many food products, is considered as a highly hazardous compound. Aspartame was discovered in 1965 and raises a lot of controversy up to date. Astrocytes are glial cells, the presence and functions of which are closely connected with the central nervous system (CNS). The aim of this article is to demonstrate the direct and indirect role of astrocytes participating in the harmful effects of aspartame metabolites on neurons. The artificial sweetener is broken down into phenylalanine (50%), aspartic acid (40%) and methanol (10%) during metabolism in the body. The excess of phenylalanine blocks the transport of important amino acids to the brain contributing to reduced levels of dopamine and serotonin. Astrocytes directly affect the transport of this amino acid and also indirectly by modulation of carriers in the endothelium. Aspartic acid at high concentrations is a toxin that causes hyperexcitability of neurons and is also a precursor of other excitatory amino acid - glutamates. Their excess in quantity and lack of astrocytic uptake induces excitotoxicity and leads to the degeneration of astrocytes and neurons. The methanol metabolites cause CNS depression, vision disorders and other symptoms leading ultimately to metabolic acidosis and coma. Astrocytes do not play a significant role in methanol poisoning due to a permanent consumption of large amounts of aspartame. Despite intense speculations about the carcinogenicity of aspartame, the latest studies show that its metabolite - diketopiperazine - is cancirogenic in the CNS. It contributes to the formation of tumors in the CNS such as gliomas, medulloblastomas and meningiomas. Glial cells are the main source of tumors, which can be caused inter alia by the sweetener in the brain. On the one hand the action of astrocytes during aspartame poisoning may be advantageous for neuro-protection while on the other it may intensify the destruction of neurons. The role of the glia in

  2. Differential Proteomics in Malignant and Normal Liver Cell Lines

    Institute of Scientific and Technical Information of China (English)

    LIU Zhi-jun; WANG Bin; YAN Zhi-yong; QIAN Dong-meng; SONG Xu-xia; Ding Shou-yi; BAI Zhi-qiang

    2007-01-01

    Objective: To detect differential protein expression in malignant and normal liver cell lines in vitro using the SELDI ProteinChip platform, for investigating the pathogenesis of liver cancer. Methods: Two cell lines, human normal liver cell line L02 and hepatoma cell line SMMC-7721 were cultured routinely, harvested in good condition and lysed. After quantification, the supernatant of the lysate was tested by IMAC3 (Immobilized Mental Affinity Capture) and WCX2 (Weak Cation Exchange) chips on the SELDI-TOF-MS ProteinChip reader. Results: Protein expression differed between the malignant and normal liver cell lines. A total of 20 differentially expressed proteins were found, among which, 7 were captured by the IMAC3 chip and 14 by the WCX2 chip. Peaks at 5,419, 7,979 and 11,265 Da were higher and at 8,103, 8,492, 10,160 and 11,304 Da lower in SMMC-7721 cells by the IMAC3 chip; peaks at 7,517, 7,945 and 7,979 Da were higher and at 5,061, 5,551, 5,818, 7,439, 9,401,10,100, 10,312, 11,621, 11,662, 11,830 and 12,772 Da lower in SMMC-7721 cells by the WCX2 chip. Interestingly, both chips captured the 7,979 Da peak. In addition, the 11,081 Da peak corresponded precisely with the molecular mass of the calcium binding protein S100A10, which may participate in the formation of liver cancer in association with p36. Conclusion: Detecting differential protein expression in malignant and normal liver cell lines using the SELDI ProteinChip platform was simple, sensitive and repeatable. The results we obtained can serve as a basis for investigating the pathogenesis of liver cancer and aid the discovery of new therapeutic targets.

  3. Reconstruction of endometrium from human endometrial side population cell lines.

    Directory of Open Access Journals (Sweden)

    Irene Cervelló

    Full Text Available Endometrial regeneration is mediated, at least in part, by the existence of a specialized somatic stem cell (SSC population recently identified by several groups using the side population (SP technique. We previously demonstrated that endometrial SP displays genotypic, phenotypic and the functional capability to develop human endometrium after subcutaneous injection in NOD-SCID mice. We have now established seven human endometrial SP (hESP cell lines (ICE 1-7: four from the epithelial and three from the stromal fraction, respectively. SP cell lines were generated under hypoxic conditions based on their cloning efficiency ability, cultured for 12-15 passages (20 weeks and cryopreserved. Cell lines displayed normal 46XX karyotype, intermediate telomerase activity pattern and expressed mRNAs encoding proteins that are considered characteristic of undifferentiated cells (Oct-4, GDF3, DNMT3B, Nanog, GABR3 and those of mesodermal origin (WT1, Cardiac Actin, Enolase, Globin, REN. Phenotype analysis corroborated their epithelial (CD9+ or stromal (vimentin+ cell origin and mesenchymal (CD90+, CD73+ and CD45⁻ attributes. Markers considered characteristic of ectoderm or endoderm were not detected. Cells did not express either estrogen receptor alpha (ERα or progesterone receptor (PR. The hESP cell lines were able to differentiate in vitro into adipocytes and osteocytes, which confirmed their mesenchymal origin. Finally, we demonstrated their ability to generate human endometrium when transplanted beneath the renal capsule of NOD-SCID mice. These findings confirm that SP cells exhibit key features of human endometrial SSC and open up new possibilities for the understanding of gynecological disorders such as endometriosis or Asherman syndrome. Our cell lines can be a valuable model to investigate new targets for endometrium proliferation in endometriosis.

  4. Establishment, immortalisation and characterisation of pteropid bat cell lines.

    Directory of Open Access Journals (Sweden)

    Gary Crameri

    Full Text Available BACKGROUND: Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. METHODOLOGY/FINDINGS: Black flying foxes (Pteropus alecto were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. CONCLUSIONS/SIGNIFICANCE: The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study.

  5. Monoclonal antibodies against the human leukemia cell line K 562.

    Science.gov (United States)

    Böttger, V; Hering, S; Jantscheff, P; Micheel, B

    1985-01-01

    Three monoclonal antibodies raised against K 562, a cell line originally established from a patient with chronic myeloid leukemia (CML) in terminal blast crisis, were selected according to their distinct reaction pattern. Whereas two antibodies (ZIK-C1-A/C5 and ZIK-C1-A/H5 also designated C and H) recognized antigens, present on K 562 cells and other immature and mature hematopoietic cells (cell lines and normal blood and bone marrow cells), antibody ZIK-C1-A/D9 also designated Y showed an exclusive binding to K 562 cells. The results obtained (here and in the following paper) indicate, that antibody ZIK-C1-A/D9 defines an early differentiation antigen of hematopoiesis or a leukemia-associated antigen.

  6. Acute isolation and transcriptome characterization of cortical astrocytes and microglia from young and aged mice

    NARCIS (Netherlands)

    Orre, Marie; Kamphuis, W.; Osborn, Lana M; Melief, Jeroen; Kooijman, Lieneke; Huitinga, I.; Klooster, Jan; Bossers, K.; Hol, Elly M

    2014-01-01

    Astrocytes and microglia become reactive in many neurological disorders resulting in phenotypic and functional alterations. Both cell types might also display functional changes during normal aging. To identify gene signatures and changes in basal cellular functions of astrocytes and microglia in re

  7. Synapses lacking astrocyte appear in the amygdala during consolidation of Pavlovian threat conditioning.

    Science.gov (United States)

    Ostroff, Linnaea E; Manzur, Mustfa K; Cain, Christopher K; Ledoux, Joseph E

    2014-06-15

    There is growing evidence that astrocytes, long held to merely provide metabolic support in the adult brain, participate in both synaptic plasticity and learning and memory. Astrocytic processes are sometimes present at the synaptic cleft, suggesting that they might act directly at individual synapses. Associative learning induces synaptic plasticity and morphological changes at synapses in the lateral amygdala (LA). To determine whether astrocytic contacts are involved in these changes, we examined LA synapses after either threat conditioning (also called fear conditioning) or conditioned inhibition in adult rats by using serial section transmission electron microscopy (ssTEM) reconstructions. There was a transient increase in the density of synapses with no astrocytic contact after threat conditioning, especially on enlarged spines containing both polyribosomes and a spine apparatus. In contrast, synapses with astrocytic contacts were smaller after conditioned inhibition. This suggests that during memory consolidation astrocytic processes are absent if synapses are enlarging but present if they are shrinking. We measured the perimeter of each synapse and its degree of astrocyte coverage, and found that only about 20-30% of each synapse was ensheathed. The amount of synapse perimeter surrounded by astrocyte did not scale with synapse size, giving large synapses a disproportionately long astrocyte-free perimeter and resulting in a net increase in astrocyte-free perimeter after threat conditioning. Thus astrocytic processes do not mechanically isolate LA synapses, but may instead interact through local signaling, possibly via cell-surface receptors. Our results suggest that contact with astrocytic processes opposes synapse growth during memory consolidation.

  8. NT2 derived neuronal and astrocytic network signalling.

    Directory of Open Access Journals (Sweden)

    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.

  9. Effects of cultured astrocytes from rat cerebral cortex onthe neurite development of PC12 cells%星形神经胶质细胞对PC12神经元突起生长发育的影响

    Institute of Scientific and Technical Information of China (English)

    莫永炎; 邵紫韫; 陈瑗; 周玫; 张宝

    2004-01-01

    背景:星形细胞对神经元有提供营养、支持及调节突触活性作用,但它对神经元发育的影响还尚不清楚.目的:探讨体外培养的Sprague Dawley大鼠大脑皮质星形细胞对PC12神经元突起生长发育的作用.设计:完全随机设计,对照实验研究.方法:以培养的星形细胞与PC12神经元按不同细胞数目比例(50:1~1:1)共同培养,并用其制备的条件培养液培养PG12细胞.主要观察指标:用快速灵敏的MTT'比色法测定PC12神经元的细胞活力,用光学相差显微镜观察PC12细胞形态学变化.结果:①星形细胞条件培养液可增强PG12细胞活力(MTT测定的A值由0.255±0.012提高到0.510±0.036,P<0.001),却不能促使PC12神经元突起的生出.②当将星形细胞与PC12细胞按30:1~1:1的比例共同培养时,既可提高PC12细胞折光性和光晕又可促使其突起的生长;但按50:1~40:1的比例共同培养时,只观察到提高PC12细胞折光性和光晕,而无促使其突起生长发育的作用.结论:PC12神经元细胞活力的提高与星形细胞分泌到条件培养液中的可溶性因子有关,而PC12神经元突起生长发育可能是和与星形细胞的直接接触以及二者的细胞数目比有关.%BACKGROUND: Although astrocytes are kown to provide structural andtrophic support to neurons and modulate synaptic activity, their role is farfrom being completely understood.OBJECTIVE: To investigate effects of cultured astrocytes fromSprague-Dawley rat cerebral cortex on the neurite development of PC12 cellsderived from rat pheochromocytoma.DESIGN: Completely randomized controlled trial.METHODS: PC12 cells were co-cultured with astrocyte according to dif-ferent astrocytes/neurons ratio(50: 1 -1: 1), or cultured with serum-freeconditioned medium of astrocytes (ACM).MAIN OUTCOME MEASURES: The vitality of PC12 cells was measuredby sensitive MTT method and their morphologic features were observed byOlympus light microscope.RFSULTS: When PC

  10. Neuron-glia signaling: Implications for astrocyte differentiation and synapse formation.

    Science.gov (United States)

    Stipursky, Joice; Romão, Luciana; Tortelli, Vanessa; Neto, Vivaldo Moura; Gomes, Flávia Carvalho Alcantara

    2011-10-10

    Glial cells are currently viewed as active partners of neurons in synapse formation. The close proximity of astrocytes to the synaptic cleft implicates that they strongly influence synapse function as well as suggests that these cells might be potential targets for neuronal-released molecules. In this review, we discuss the signaling pathways of astrocyte generation and the role of astrocyte-derived molecules in synapse formation in the central nervous system. Further, we discuss the role of the excitatory neurotransmitter, glutamate and transforming growth factor beta 1 (TGF-β1) pathway in astrocyte generation and differentiation. We provide evidence that astrocytes surrounding synapses are target of neuronal activity and shed light into the role of astroglial cells into neurological disorders associated with glutamate neurotoxicity.

  11. Neuroimmunological Implications of AQP4 in Astrocytes

    Science.gov (United States)

    Ikeshima-Kataoka, Hiroko

    2016-01-01

    The brain has high-order functions and is composed of several kinds of cells, such as neurons and glial cells. It is becoming clear that many kinds of neurodegenerative diseases are more-or-less influenced by astrocytes, which are a type of glial cell. Aquaporin-4 (AQP4), a membrane-bound protein that regulates water permeability is a member of the aquaporin family of water channel proteins that is expressed in the endfeet of astrocytes in the central nervous system (CNS). Recently, AQP4 has been shown to function, not only as a water channel protein, but also as an adhesion molecule that is involved in cell migration and neuroexcitation, synaptic plasticity, and learning/memory through mechanisms involved in long-term potentiation or long-term depression. The most extensively examined role of AQP4 is its ability to act as a neuroimmunological inducer. Previously, we showed that AQP4 plays an important role in neuroimmunological functions in injured mouse brain in concert with the proinflammatory inducer osteopontin (OPN). The aim of this review is to summarize the functional implication of AQP4, focusing especially on its neuroimmunological roles. This review is a good opportunity to compile recent knowledge and could contribute to the therapeutic treatment of autoimmune diseases through strategies targeting AQP4. Finally, the author would like to hypothesize on AQP4’s role in interaction between reactive astrocytes and reactive microglial cells, which might occur in neurodegenerative diseases. Furthermore, a therapeutic strategy for AQP4-related neurodegenerative diseases is proposed. PMID:27517922

  12. Comparative Metabolic Flux Profiling of Melanoma Cell Lines

    Science.gov (United States)

    Scott, David A.; Richardson, Adam D.; Filipp, Fabian V.; Knutzen, Christine A.; Chiang, Gary G.; Ronai, Ze'ev A.; Osterman, Andrei L.; Smith, Jeffrey W.

    2011-01-01

    Metabolic rewiring is an established hallmark of cancer, but the details of this rewiring at a systems level are not well characterized. Here we acquire this insight in a melanoma cell line panel by tracking metabolic flux using isotopically labeled nutrients. Metabolic profiling and flux balance analysis were used to compare normal melanocytes to melanoma cell lines in both normoxic and hypoxic conditions. All melanoma cells exhibited the Warburg phenomenon; they used more glucose and produced more lactate than melanocytes. Other changes were observed in melanoma cells that are not described by the Warburg phenomenon. Hypoxic conditions increased fermentation of glucose to lactate in both melanocytes and melanoma cells (the Pasteur effect). However, metabolism was not strictly glycolytic, as the tricarboxylic acid (TCA) cycle was functional in all melanoma lines, even under hypoxia. Furthermore, glutamine was also a key nutrient providing a substantial anaplerotic contribution to the TCA cycle. In the WM35 melanoma line glutamine was metabolized in the “reverse” (reductive) direction in the TCA cycle, particularly under hypoxia. This reverse flux allowed the melanoma cells to synthesize fatty acids from glutamine while glucose was primarily converted to lactate. Altogether, this study, which is the first comprehensive comparative analysis of metabolism in melanoma cells, provides a foundation for targeting metabolism for therapeutic benefit in melanoma. PMID:21998308

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

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    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.

  14. 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.

  15. Interlukin-18 Is a Pivot Regulatory Factor on Matrix Metalloproteinase-13 Expression and Brain Astrocytic Migration.

    Science.gov (United States)

    Chen, Jia-Hong; Tsai, Chon-Haw; Lin, Hsiao-Yun; Huang, Chien-Fang; Leung, Yuk-Man; Lai, Sheng-Wei; Tsai, Cheng-Fang; Chang, Pei-Chun; Lu, Dah-Yuu; Lin, Chingju

    2016-11-01

    The expression of matrix metalloproteinase-13 (MMP-13) has been shown to be elevated in some pathophysiological conditions and is involved in the degradation of extracellular matrix in astrocytes. In current study, the function of MMP-13 was further investigated. The conditioned medium (CM) collected from activated microglia increased interleukin (IL)-18 production and enhanced MMP-13 expression in astrocytes. Furthermore, treatment with recombinant IL-18 increased MMP-13 protein and mRNA levels in astrocytes. Recombinant IL-18 stimulation also increased the enzymatic activity of MMP-13 and the migratory activity of astrocytes, while administration of MMP-13 or pan-MMP inhibitors antagonized IL-18-induced migratory activity of astrocytes. In addition, administration of recombinant IL-18 to astrocytes led to the phosphorylation of JNK, Akt, or PKCδ, and treatment of astrocytes with JNK, PI3 kinase/Akt, or PKCδ inhibitors significantly decreased the IL-18-induced migratory activity. Taken together, the results suggest that IL-18-induced MMP-13 expression in astrocytes is regulated by JNK, PI3 kinase/Akt, and PKCδ signaling pathways. These findings also indicate that IL-18 is an important regulator leading to MMP-13 expression and cell migration in astrocytes.

  16. Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies

    OpenAIRE

    Krampe, Britta; Al-Rubeai, Mohamed

    2010-01-01

    Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members o...

  17. Peroxynitrite decomposition catalyst prevents apoptotic cell death in a human astrocytoma cell line incubated with supernatants of HIV-infected macrophages

    Directory of Open Access Journals (Sweden)

    Perno Carlo

    2002-09-01

    Full Text Available Abstract Background Oxidative stress has shown to contribute in the mechanisms underlying apoptotic cell death occuring in AIDS-dementia complex. Here we investigated the role of peroxynitrite in apoptosis occurring in astroglial cells incubated with supernatants of HIV-infected human primary macrophages (M/M. Results Flow cytometric analysis (FACS of human cultured astrocytes shortly incubated with HIV-1-infected M/M supernatants showed apoptotic cell death, an effect accompanied by pronounced staining for nitrotyrosine (footprint of peroxynitrite and by abnormal formation of malondialdehyde (MDA. Pretreatment of astrocytes with the peroxynitrite decomposition catalyst FeTMPS antagonized HIV-related astrocytic apoptosis, MDA formation and nitrotyrosine staining. Conclusions Taken together, our results suggest that inibition of peroxynitrite leads to protection against peroxidative stress accompanying HIV-related apoptosis of astrocytes. Overall results support the role of peroxynitrite in HIV-related programmed death of astrocytes and suggest the use of peroxynitrite decomposition catalyst to counteract HIV-1-related neurological disorders.

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Skin Biopsy and Patient-Specific Stem Cell Lines

    Science.gov (United States)

    Li, Yao; Nguyen, Huy V.; Tsang, Stephen H.

    2016-01-01

    The generation of patient-specific induced pluripotent stem (iPS) cells permits the development of next-generation patient-specific systems biology models reflecting personalized genomics profiles to better understand pathophysiology. In this chapter, we describe how to create a patient-specific iPS cell line. There are three major steps: (1) performing a skin biopsy procedure on the patient; (2) extracting human fibroblast cells from the skin biopsy tissue; and (3) reprogramming patient-specific fibroblast cells into the pluripotent stem cell stage. PMID:26141312

  1. The YUMM lines: a series of congenic mouse melanoma cell lines with defined genetic alterations.

    Science.gov (United States)

    Meeth, Katrina; Wang, Jake Xiao; Micevic, Goran; Damsky, William; Bosenberg, Marcus W

    2016-09-01

    The remarkable success of immune therapies emphasizes the need for immune-competent cancer models. Elegant genetically engineered mouse models of a variety of cancers have been established, but their effective use is limited by cost and difficulties in rapidly generating experimental data. Some mouse cancer cell lines are transplantable to immunocompetent host mice and have been utilized extensively to study cancer immunology. Here, we describe the Yale University Mouse Melanoma (YUMM) lines, a comprehensive system of mouse melanoma cell lines that are syngeneic to C57BL/6, have well-defined human-relevant driver mutations, and are genomically stable. This will be a useful tool for the study of tumor immunology and genotype-specific cancer biology.

  2. Intercellular synchronization of diffusively coupled astrocytes

    CERN Document Server

    Alam, Md Jahoor; Devi, Gurumayum Reenaroy; Singh, Heisnam Dinachandra; Singh, R K Brojen; Sharma, B Indrajit

    2010-01-01

    We examine the synchrony of the dynamics of localized [Ca^{2+}]_i oscillations in internal pool of astrocytes via diffusing coupling of a network of such cells in a certain topology where cytosolic Ca^{2+} and inositol 1,4,5-triphosphate (IP3) are coupling molecules; and possible long range interaction among the cells. Our numerical results claim that the cells exhibit fairly well coordinated behaviour through this coupling mechanism. It is also seen in the results that as the number of coupling molecular species is increased, the rate of synchrony is also increased correspondingly. Apart from the topology of the cells taken, as the number of coupled cells around any one of the cells in the system is increased, the cell process information faster.

  3. The analgesic effect on neuropathic pain of retrogradely transported botulinum neurotoxin A involves Schwann cells and astrocytes.

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    Sara Marinelli

    Full Text Available In recent years a growing debate is about whether botulinum neurotoxins are retrogradely transported from the site of injection. Immunodetection of cleaved SNAP-25 (cl-SNAP-25, the protein of the SNARE complex targeted by botulinum neurotoxin serotype A (BoNT/A, could represent an excellent approach to investigate the mechanism of action on the nociceptive pathways at peripheral and/or central level. After peripheral administration of BoNT/A, we analyzed the expression of cl-SNAP-25, from the hindpaw's nerve endings to the spinal cord, together with the behavioral effects on neuropathic pain. We used the chronic constriction injury of the sciatic nerve in CD1 mice as animal model of neuropathic pain. We evaluated immunostaining of cl-SNAP-25 in the peripheral nerve endings, along the sciatic nerve, in dorsal root ganglia and in spinal dorsal horns after intraplantar injection of saline or BoNT/A, alone or colocalized with either glial fibrillar acidic protein, GFAP, or complement receptor 3/cluster of differentiation 11b, CD11b, or neuronal nuclei, NeuN, depending on the area investigated. Immunofluorescence analysis shows the presence of the cl-SNAP-25 in all tissues examined, from the peripheral endings to the spinal cord, suggesting a retrograde transport of BoNT/A. Moreover, we performed in vitro experiments to ascertain if BoNT/A was able to interact with the proliferative state of Schwann cells (SC. We found that BoNT/A modulates the proliferation of SC and inhibits the acetylcholine release from SC, evidencing a new biological effect of the toxin and further supporting the retrograde transport of the toxin along the nerve and its ability to influence regenerative processes. The present results strongly sustain a combinatorial action at peripheral and central neural levels and encourage the use of BoNT/A for the pathological pain conditions difficult to treat in clinical practice and dramatically impairing patients' quality of life.

  4. The Analgesic Effect on Neuropathic Pain of Retrogradely Transported botulinum Neurotoxin A Involves Schwann Cells and Astrocytes

    Science.gov (United States)

    Ricordy, Ruggero; Uggenti, Carolina; Tata, Ada Maria; Luvisetto, Siro; Pavone, Flaminia

    2012-01-01

    In recent years a growing debate is about whether botulinum neurotoxins are retrogradely transported from the site of injection. Immunodetection of cleaved SNAP-25 (cl-SNAP-25), the protein of the SNARE complex targeted by botulinum neurotoxin serotype A (BoNT/A), could represent an excellent approach to investigate the mechanism of action on the nociceptive pathways at peripheral and/or central level. After peripheral administration of BoNT/A, we analyzed the expression of cl-SNAP-25, from the hindpaw’s nerve endings to the spinal cord, together with the behavioral effects on neuropathic pain. We used the chronic constriction injury of the sciatic nerve in CD1 mice as animal model of neuropathic pain. We evaluated immunostaining of cl-SNAP-25 in the peripheral nerve endings, along the sciatic nerve, in dorsal root ganglia and in spinal dorsal horns after intraplantar injection of saline or BoNT/A, alone or colocalized with either glial fibrillar acidic protein, GFAP, or complement receptor 3/cluster of differentiation 11b, CD11b, or neuronal nuclei, NeuN, depending on the area investigated. Immunofluorescence analysis shows the presence of the cl-SNAP-25 in all tissues examined, from the peripheral endings to the spinal cord, suggesting a retrograde transport of BoNT/A. Moreover, we performed in vitro experiments to ascertain if BoNT/A was able to interact with the proliferative state of Schwann cells (SC). We found that BoNT/A modulates the proliferation of SC and inhibits the acetylcholine release from SC, evidencing a new biological effect of the toxin and further supporting the retrograde transport of the toxin along the nerve and its ability to influence regenerative processes. The present results strongly sustain a combinatorial action at peripheral and central neural levels and encourage the use of BoNT/A for the pathological pain conditions difficult to treat in clinical practice and dramatically impairing patients’ quality of life. PMID:23110146

  5. Human cell lines: A promising alternative for recombinant FIX production.

    Science.gov (United States)

    de Sousa Bomfim, Aline; Cristina Corrêa de Freitas, Marcela; Picanço-Castro, Virgínia; de Abreu Soares Neto, Mário; Swiech, Kamilla; Tadeu Covas, Dimas; Maria de Sousa Russo, Elisa

    2016-05-01

    Factor IX (FIX) is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the Hemophilia B treatment. We evaluated the potential of recombinant human FIX (rhFIX) expression in 293T and SK-Hep-1 human cell lines. SK-Hep-1-FIX cells produced higher levels of biologically active protein. The growth profile of 293T-FIX cells was not influenced by lentiviral integration number into the cellular genome. SK-Hep-1-FIX cells showed a significantly lower growth rate than SK-Hep-1 cells. γ-carboxylation process is significant to FIX biological activity, thus we performed a expression analysis of genes involved in this process. The 293T gene expression suggests that this cell line could efficiently carboxylate FIX, however only 28% of the total secreted protein is active. SK-Hep-1 cells did not express high amounts of VKORC1 and carboxylase, but this cell line secreted large amounts of active protein. Enrichment of culture medium with Ca(+2) and Mg(+2) ions did not affect positively rhFIX expression in SK-Hep-1 cells. In 293T cells, the addition of 0.5 mM Ca(+2) and 1 mM Mg(+2) resulted in higher rhFIX concentration. SK-Hep-1 cell line proved to be very effective in rhFIX production, and it can be used as a novel biotechnological platform for the production of recombinant proteins.

  6. Astrocyte Sodium Signalling and Panglial Spread of Sodium Signals in Brain White Matter.

    Science.gov (United States)

    Moshrefi-Ravasdjani, Behrouz; Hammel, Evelyn L; Kafitz, Karl W; Rose, Christine R

    2017-02-18

    In brain grey matter, excitatory synaptic transmission activates glutamate uptake into astrocytes, inducing sodium signals which propagate into neighboring astrocytes through gap junctions. These sodium signals have been suggested to serve an important role in neuro-metabolic coupling. So far, it is unknown if astrocytes in white matter-that is in brain regions devoid of synapses-are also able to undergo such intra- and intercellular sodium signalling. In the present study, we have addressed this question by performing quantitative sodium imaging in acute tissue slices of mouse corpus callosum. Focal application of glutamate induced sodium transients in SR101-positive astrocytes. These were largely unaltered in the presence of ionotropic glutamate receptors blockers, but strongly dampened upon pharmacological inhibition of glutamate uptake. Sodium signals induced in individual astrocytes readily spread into neighboring SR101-positive cells with peak amplitudes decaying monoexponentially with distance from the stimulated cell. In addition, spread of sodium was largely unaltered during pharmacological inhibition of purinergic and glutamate receptors, indicating gap junction-mediated, passive diffusion of sodium between astrocytes. Using cell-type-specific, transgenic reporter mice, we found that sodium signals also propagated, albeit less effectively, from astrocytes to neighboring oligodendrocytes and NG2 cells. Again, panglial spread was unaltered with purinergic and glutamate receptors blocked. Taken together, our results demonstrate that activation of sodium-dependent glutamate transporters induces sodium signals in white matter astrocytes, which spread within the astrocyte syncytium. In addition, we found a panglial passage of sodium signals from astrocytes to NG2 cells and oligodendrocytes, indicating functional coupling between these macroglial cells in white matter.

  7. Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

    Energy Technology Data Exchange (ETDEWEB)

    Medina, D.; Oborn, C.J. (Baylor College of Medicine, Houston, TX (USA)); Li, M.L.; Bissell, M.J. (Univ. of California, Berkeley (USA))

    1987-09-01

    The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.

  8. Analysis of LINE-1 expression in human pluripotent cells.

    Science.gov (United States)

    Muñoz-Lopez, Martin; Garcia-Cañadas, Marta; Macia, Angela; Morell, Santiago; Garcia-Perez, Jose L

    2012-01-01

    Half of the human genome is composed of repeated DNA, and some types are mobile within our genome (transposons and retrotransposons). Despite their abundance, only a small fraction of them are currently active in our genome (Long Interspersed Element-1 (LINE-1), Alu, and SVA elements). LINE-1 or L1 elements are a family of active non-LTR retrotransposons, the ongoing mobilization of which still impacts our genome. As selfish DNA elements, L1 activity is more prominent in early human development, where new insertions would be transmitted to the progeny. Here, we describe the conventional methods aimed to determine the expression level of LINE-1 elements in pluripotent human cells.

  9. 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.

  10. Establishment of cell suspension line of Populus tomentosa Carr

    Institute of Scientific and Technical Information of China (English)

    YAO Na; ZHANG Zhi-yi; AN Xin-min; YANG Kai

    2008-01-01

    Leaves of fine Populus tomentosa genotype TC152 were used as explants to establish cell suspension lines. The effects of plant growth regulators on callus induction and establishment of cell suspension lines were studied. The callus induction rate was the highest on a MS solid medium supplemented with 1.0 mg·L-1 2,4-D. A cell suspension line could be obtained by inoculating calli which were not subeultured into a MS liquid medium supplemented with 1.5 mg·L-1 2,4-D. The best subculture medium was MS+ 0.8 mg·L-1 2,4-D + 30 g·L-1 sucrose with a subculture cycle of seven days.

  11. Graphene Oxide Nanoribbons Induce Autophagic Vacuoles in Neuroblastoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Emanuela Mari

    2016-11-01

    Full Text Available Since graphene nanoparticles are attracting increasing interest in relation to medical applications, it is important to understand their potential effects on humans. In the present study, we prepared graphene oxide (GO nanoribbons by oxidative unzipping of single-wall carbon nanotubes (SWCNTs and analyzed their toxicity in two human neuroblastoma cell lines. Neuroblastoma is the most common solid neoplasia in children. The hallmark of these tumors is the high number of different clinical variables, ranging from highly metastatic, rapid progression and resistance to therapy to spontaneous regression or change into benign ganglioneuromas. Patients with neuroblastoma are grouped into different risk groups that are characterized by different prognosis and different clinical behavior. Relapse and mortality in high risk patients is very high in spite of new advances in chemotherapy. Cell lines, obtained from neuroblastomas have different genotypic and phenotypic features. The cell lines SK-N-BE(2 and SH-SY5Y have different genetic mutations and tumorigenicity. Cells were exposed to low doses of GO for different times in order to investigate whether GO was a good vehicle for biological molecules delivering individualized therapy. Cytotoxicity in both cell lines was studied by measuring cellular oxidative stress (ROS, mitochondria membrane potential, expression of lysosomial proteins and cell growth. GO uptake and cytoplasmic distribution of particles were studied by Transmission Electron Microscopy (TEM for up to 72 h. The results show that GO at low concentrations increased ROS production and induced autophagy in both neuroblastoma cell lines within a few hours of exposure, events that, however, are not followed by growth arrest or death. For this reason, we suggest that the GO nanoparticle can be used for therapeutic delivery to the brain tissue with minimal effects on healthy cells.

  12. Dipeptidyl peptidase IV in two human glioma cell lines

    Directory of Open Access Journals (Sweden)

    A Sedo

    2009-12-01

    Full Text Available There is growing evidence that dipeptidyl peptidase IV [DPP-IV, EC 3.4.14.5] takes part in the metabolism of biologically active peptides participating in the regulation of growth and transformation of glial cells. However, the knowledge on the DPP-IV expression in human glial and glioma cells is still very limited. In this study, using histochemical and biochemical techniques, the DPP-IV activity was demonstrated in two commercially available human glioma cell lines of different transformation degree, as represented by U373 astrocytoma (Grade III and U87 glioblastoma multiforme (Grade IV lines. Higher total activity of the enzyme, as well as its preferential localisation in the plasma membrane, was observed in U87 cells. Compared to U373 population, U87 cells were morphologically more pleiomorphic, they were cycling at lower rate and expressing less Glial Fibrillary Acidic Protein. The data revealed positive correlation between the degree of transformation of cells and activity of DPP-IV. Great difference in expression of this enzyme, together with the phenotypic differences of cells, makes these lines a suitable standard model for further 57 studies of function of this enzyme in human glioma cells.

  13. Oral bioavailability of glyphosate: studies using two intestinal cell lines.

    Science.gov (United States)

    Vasiluk, Luba; Pinto, Linda J; Moore, Margo M

    2005-01-01

    Glyphosate is a commonly used nonselective herbicide that inhibits plant growth through interference with the production of essential aromatic amino acids. In vivo studies in mammals with radiolabeled glyphosate have shown that 34% of radioactivity was associated with intestinal tissue 2 h after oral administration. The aim of our research was to investigate the transport, binding, and toxicity of glyphosate to the cultured human intestinal epithelial cell line, Caco-2, and the rat small intestinal crypt-derived cell line, ileum epithelial cells-18 (IEC-18). An in vitro analysis of the transport kinetics of [14C]-glyphosate showed that 4 h after exposure, approximately 8% of radiolabeled glyphosate moved through the Caco-2 monolayer in a dose-dependent manner. Binding of glyphosate to cells was saturable and approximately 4 x 10(11) binding sites/cell were estimated from bound [14C]. Exposure of Caco-2 cells to > or =10 mg/ml glyphosate reduced transmembrane electrical resistance (TEER) by 82 to 96% and increased permeability to [3H]-mannitol, indicating that paracellular permeability increased in glyphosate-treated cells. At 10-mg/ml glyphosate, both IEC-18 and Caco-2 cells showed disruption in the actin cytoskeleton. In Caco-2 cells, significant lactate dehydrogenase leakage was observed when cells were exposed to 15 mg/ml of glyphosate. These data indicate that at doses >10 mg/ml, glyphosate significantly disrupts the barrier properties of cultured intestinal cells.

  14. Derivation of a Homozygous Human Androgenetic Embryonic Stem Cell Line.

    Science.gov (United States)

    Ding, Chenhui; Huang, Sunxing; Qi, Quan; Fu, Rui; Zhu, Wanwan; Cai, Bing; Hong, Pingping; Liu, Zhengxin; Gu, Tiantian; Zeng, Yanhong; Wang, Jing; Xu, Yanwen; Zhao, Xiaoyang; Zhou, Qi; Zhou, Canquan

    2015-10-01

    Human embryonic stem cells (hESCs) have long been considered as a promising source for cell replacement therapy. However, one major obstacle for the use of these cells is immune compatibility. Histocompatible human parthenogenetic ESCs have been reported as a new method for generating human leukocyte antigen (HLA)-matched hESCs. To further investigate the possibility of obtaining histocompatible stem cells from uniparental embryos, we tried to produce androgenetic haploid human embryos by injecting a single spermatozoon into enucleated human oocyte, and establish human androgenetic embryonic stem (hAGES) cell lines from androgenetic embryos. In the present study, a diploid hAGES cell line has been established, which exhibits typical features of human ESCs, including the expression of pluripotency markers, having differentiation potential in vitro and in vivo, and stable propagation in an undifferentiated state (>P40). Bisulfite sequencing of the H19, Snrpn, Meg3, and Kv imprinting control regions suggested that hAGES cells maintained to a certain extent a sperm methylation pattern. Genome-wide single nucleotide polymorphism, short tandem repeat, and HLA analyses revealed that the hAGES cell genome was highly homozygous. These results suggest that hAGES cells from spermatozoon could serve as a useful tool for studying the mechanisms underlying genomic imprinting in humans. It might also be used as a potential resource for cell replacement therapy as parthenogenetic stem cells.

  15. The effects of trypsin on rat brain astrocyte activation.

    Directory of Open Access Journals (Sweden)

    Masoud Fereidoni

    2013-12-01

    Full Text Available Astrocytes are cells within the central nervous system which are activated in a wide spectrum of infections, and autoimmune and neurodegenerative diseases. In pathologic states, they produce inflammatory cytokines, chemokines, and nitric oxide (NO, and sometimes they induce apoptosis. Their protease-activated receptors (PARs can be activated by proteases, e.g. thrombin and trypsin, which are important in brain inflammation. The current study aimed to investigate the effects of different concentrations of trypsin (1 to 100U/ml on cultured astrocytes.In the present study, two-day rat infants' brains were isolated and homogenized after meninges removal, then cultivated in DMEM + 10% FBS medium. 10 days later, astrocytes were harvested and recultivated for more purification (up to 95%, using Immunocytochemistry method, in order to be employed for tests. They were affected by different concentrations of trypsin (1, 5, 10, 15, 20, 40, 60, 80, and 100 U/ml. To reveal the inflammation progress, NO concentrations (the Griess test were assessed after 24 and 48 hours.The results showed that trypsin concentration up to 20 U/ml caused a significant increase in NO, in a dose-dependent manner, on cultured astrocytes (P < 0.001. Trypsin 20 U/ml increased NO production fivefold the control group (P < 0.001. At higher concentrations than 20 U/ml, NO production diminished (P < 0.001. At 100 U/ml, NO production was less than the control group (P < 0.001.Inflammatory effects of trypsin 5-20 U/ml are probably due to the stimulation of astrocytes' PAR-2 receptors and the increasing of the activation of NF-κB, PKC, MAPKs. Stimulation of astrocytes' PAR-2 receptors causes an increase in iNOS activation which in turn leads to NO production. However, higher trypsin concentration possibly made astrocyte apoptosis; therefore, NO production diminished. These assumptions need to be further investigated.

  16. Antiproliferative effect of Tualang honey on oral squamous cell carcinoma and osteosarcoma cell lines

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    Ismail Noorliza M

    2010-09-01

    Full Text Available Abstract Background The treatment of oral squamous cell carcinomas (OSCC and human osteosarcoma (HOS includes surgery and/or radiotherapy which often lead to reduced quality of life. This study was aimed to study the antiproliferative activity of local honey (Tualang on OSCC and HOS cell lines. Methods Several concentrations of Tualang honey (1% - 20% were applied on OSCC and HOS cell lines for 3, 6, 12, 24, 48 and 72 hours. Morphological characteristics were observed under light and fluorescent microscope. Cell viability was assessed using MTT assay and the optical density for absorbance values in each experiment was measured at 570 nm by an ELISA reader. Detection of cellular apoptosis was done using the Annexin V-FITC Apoptosis Detection Kit. Results Morphological appearance showed apoptotic cellular changes like becoming rounded, reduction in cell number, blebbed membrane and apoptotic nuclear changes like nuclear shrinkage, chromatin condensation and fragmented nucleus on OSCC and HOS cell lines. Cell viability assay showed a time and dose-dependent inhibitory effect of honey on both cell lines. The 50% inhibitory concentration (IC50 for OSCC and HOS cell lines was found to be 4% and 3.5% respectively. The maximum inhibition of cell growth of ≥80% was obtained at 15% for both cell lines. Early apoptosis was evident by flow cytometry where percentage of early apoptotic cells increased in dose and time dependent manner. Conclusion Tualang honey showed antiproliferative effect on OSCC and HOS cell lines by inducing early apoptosis.

  17. Identification and Characterization of Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma Cell Lines

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    Valentina Pozzi

    2015-05-01

    Full Text Available Background/Aims: Head and neck squamous cell carcinoma (HNSCC ranks sixth worldwide for tumor-related mortality. A subpopulation of tumor cells, termed cancer stem cells (CSCs, has the ability to support cancer growth. Therefore, profiling CSC-enriched populations could be a reliable tool to study cancer biology. Methods: We performed phenotypic characterization of 7 HNSCC cell lines and evaluated the presence of CSCs. CSCs from Hep-2 cell line and HNSCC primary cultures were enriched through sphere formation and sphere-forming cells have been characterized both in vitro and in vivo. In addition, we investigated the expression levels of Nicotinamide N-methyltransferase (NNMT, an enzyme overexpressed in several malignancies. Results: CSC markers were markedly expressed in Hep-2 cell line, which was found to be highly tumorigenic. CSC-enriched populations displayed increased expression of CSC markers and a strong capability to form tumors in vivo. We also found an overexpression of CSC markers in tumor formed by CSC-enriched populations. Interestingly, NNMT levels were significantly higher in CSC-enriched populations compared with parental cells. Conclusion: Our study provides an useful procedure for CSC identification and enrichment in HNSCC. Moreover, results obtained seem to suggest that CSCs may represent a promising target for an anticancer therapy.

  18. Boldine: a potential new antiproliferative drug against glioma cell lines.

    Science.gov (United States)

    Gerhardt, Daniéli; Horn, Ana Paula; Gaelzer, Mariana Maier; Frozza, Rudimar Luiz; Delgado-Cañedo, Andrés; Pelegrini, Alessandra Luiza; Henriques, Amélia T; Lenz, Guido; Salbego, Christianne

    2009-12-01

    Malignant gliomas are the most common and devastating primary tumors of the central nervous system. Currently no efficient treatment is available. This study evaluated the effect and underlying mechanisms of boldine, an aporphine alkaloid of Peumus boldus, on glioma proliferation and cell death. Boldine decreased the cell number of U138-MG, U87-MG and C6 glioma lines at concentrations of 80, 250 and 500 muM. We observed that cell death caused by boldine was cell-type specific and dose-dependent. Exposure to boldine for 24 h did not activate key mediators of apoptosis. However, it induced alterations in the cell cycle suggesting a G(2)/M arrest in U138-MG cells. Boldine had no toxic effect on non-tumor cells when used at the same concentrations as those used on tumor cells. Based on these results, we speculate that boldine may be a promising compound for evaluation as an anti-cancer agent.

  19. Effect of 8-bromo-cAMP and dexamethasone on glutamate metabolism in rat astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Zielke, H.R.; Tildon, J.T.; Landry, M.E.; Max, S.R. (Univ. of Maryland School of Medicine, Baltimore (USA))

    1990-11-01

    Glutamine synthetase (GS) activity in cultured rat astrocytes was measured in extracts and compared to the intracellular rate of glutamine synthesis by intact control astrocytes or astrocytes exposed to 1 mM 8-bromo-cAMP (8Br-cAMP) + 1 microM dexamethasone (DEX) for 4 days. GS activity in extracts of astrocytes treated with 8Br-cAMP + DEX was 7.5 times greater than the activity in extracts of control astrocytes. In contrast, the intracellular rate of glutamine synthesis by intact cells increased only 2-fold, suggesting that additional intracellular effectors regulate the expression of GS activity inside the intact cell. The rate of glutamine synthesis by astrocytes was 4.3 times greater in MEM than in HEPES buffered Hank's salts. Synthesis of glutamine by intact astrocytes cultured in MEM was independent of the external glutamine or ammonia concentrations but was increased by higher extracellular glutamate concentrations. In studies with intact astrocytes 80% of the original (U-{sup 14}C)glutamate was recovered in the medium as radioactive glutamine, 2-3% as aspartate, and 7% as glutamate after 2 hours for both control and treated astrocytes. The results suggest: (1) astrocytes are highly efficient in the conversion of glutamate to glutamine; (2) induction of GS activity increases the rate of glutamate conversion to glutamine by astrocytes and the rate of glutamine release into the medium; (3) endogenous intracellular regulators of GS activity control the flux of glutamate through this enzymatic reaction; and (4) the composition of the medium alters the rate of glutamine synthesis from external glutamate.

  20. Characterization of cloned cells from an immortalized fetal pulmonary type II cell line

    Energy Technology Data Exchange (ETDEWEB)

    Henderson, R.F.; Waide, J.J.; Lechner, J.F.

    1995-12-01

    A cultured cell line that maintained expression of pulmonary type II cell markers of differentiation would be advantageous to generate a large number of homogenous cells in which to study the biochemical functions of type II cells. Type II epithelial cells are the source of pulmonary surfactant and a cell of origin for pulmonary adenomas. Last year our laboratory reported the induction of expression of two phenotypic markers of pulmonary type II cells (alkaline phosphatase activity and surfactant lipid synthesis) in cultured fetal rat lung epithelial (FRLE) cells, a spontaneously immortalized cell line of fetal rat lung type II cell origin. Subsequently, the induction of the ability to synthesize surfactant lipid became difficult to repeat. We hypothesized that the cell line was heterogenuous and some cells were more like type II cells than others. The purpose of this study was to test this hypothesis and to obtain a cultured cell line with type II cell phenotypic markers by cloning several FRLE cells and characterizing them for phenotypic markers of type II cells (alkaline phosphatase activity and presence of surfactant lipids). Thirty cloned cell lines were analyzed for induced alkaline phosphatase activity (on x-axis) and for percent of phospholipids that were disaturated (i.e., surfactant).

  1. LINEing germ and embryonic stem cells' silencing of retrotransposons.

    Science.gov (United States)

    Ishiuchi, Takashi; Torres-Padilla, Maria-Elena

    2014-07-01

    Almost half of our genome is occupied by transposable elements. Although most of them are inactive, one type of non-long terminal repeat (LTR) retrotransposon, long interspersed nuclear element 1 (LINE1), is capable of retrotransposition. Two studies in this issue, Pezic and colleagues (pp. 1410-1428) and Castro-Diaz and colleagues (pp. 1397-1409), provide novel insight into the regulation of LINE1s in human embryonic stem cells and mouse germ cells and shed new light on the conservation of complex mechanisms to ensure silencing of transposable elements in mammals.

  2. Metallothionein-3 regulates lysosomal function in cultured astrocytes under both normal and oxidative conditions.

    Science.gov (United States)

    Lee, Sook-Jeong; Park, Mi-Ha; Kim, Hyun-Jae; Koh, Jae-Young

    2010-08-01

    Cellular zinc plays a key role in lysosomal change and cell death in neurons and astrocytes under oxidative stress. Here, using astrocytes lacking metallothionein-3 (MT3), a potential source of labile zinc in the brain, we studied the role of MT3 in oxidative stress responses. H(2)O(2) induced a large increase in labile zinc in wild-type (WT) astrocytes, but stimulated only a modest rise in MT3-null astrocytes. In addition, H(2)O(2)-induced lysosomal membrane permeabilization (LMP) and cell death were comparably attenuated in MT3-null astrocytes. Expression and glycosylation of Lamp1 (lysosome-associated membrane protein 1) and Lamp2 were increased in MT3-null astrocytes, and the activities of several lysosomal enzymes were significantly reduced, indicating an effect of MT3 on lysosomal components. Consistent with lysosomal dysfunction in MT3-null cells, the level of LC3-II (microtubule-associated protein 1 light chain 3), a marker of early autophagy, was increased by oxidative stress in WT astrocytes, but not in MT3-null cells. Similar changes in Lamp1, LC3, and cathepsin-D were induced by the lysosomal inhibitors bafilomycin A1, chloroquine, and monensin, indicating that lysosomal dysfunction may lie upstream of changes observed in MT3-null astrocytes. Consistent with this idea, lysosomal accumulation of cholesterol and lipofuscin were augmented in MT3-null astrocytes. Similar to the results seen in MT3-null cells, MT3 knockdown by siRNA inhibited oxidative stress-induced increases in zinc and LMP. These results indicate that MT3 may play a key role in normal lysosomal function in cultured astrocytes.

  3. Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies.

    Science.gov (United States)

    Krampe, Britta; Al-Rubeai, Mohamed

    2010-07-01

    Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members of the anti-apoptosis Bcl-2 family genes in several cell types has extended the knowledge of their molecular function and interaction with other proteins, and their regulation of cell death. In this review, we describe the various modes of cell death and their death pathways at molecular and organelle level and discuss the relevance of the growing knowledge of anti-apoptotic engineering strategies to inhibit cell death and increase productivity in mammalian cell culture.

  4. 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.

  5. Cellular and Phenotypic Characterization of Canine Osteosarcoma Cell Lines

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    Marie E. Legare, Jamie Bush, Amanda K. Ashley, Taka Kato, William H. Hanneman

    2011-01-01

    Full Text Available Canine and human osteosarcoma (OSA have many similarities, with the majority of reported cases occurring in the appendicular skeleton, gender predominance noted, high rate of metastasis at the time of presentation, and a lack of known etiology for this devastating disease. Due to poor understanding of the molecular mechanisms underlying OSA, we have characterized seven different OSA canine cell lines: Abrams, D17, Grey, Hughes, Ingles, Jarques, and Marisco and compared them to U2, a human OSA cell line, for the following parameters: morphology, growth, contact inhibition, migrational tendencies, alkaline phosphatase staining, heterologous tumor growth, double-strand DNA breaks, and oxidative damage. All results demonstrated the positive characteristics of the Abrams cell line for use in future studies of OSA. Of particular interest, the robust growth of a subcutaneous tumor and rapid pulmonary metastasis of the Abrams cell line in an immunocompromised mouse shows incredible potential for the future use of Abrams as a canine OSA model. Further investigations utilizing a canine cell model of OSA, such as Abrams, will be invaluable to understanding the molecular events underlying OSA, pharmaceutical inhibition of metastasis, and eventual prevention of this devastating disease.

  6. Translation of the prion protein mRNA is robust in astrocytes but does not amplify during reactive astrocytosis in the mouse brain.

    Science.gov (United States)

    Jackson, Walker S; Krost, Clemens; Borkowski, Andrew W; Kaczmarczyk, Lech

    2014-01-01

    Prion diseases induce neurodegeneration in specific brain areas for undetermined reasons. A thorough understanding of the localization of the disease-causing molecule, the prion protein (PrP), could inform on this issue but previous studies have generated conflicting conclusions. One of the more intriguing disagreements is whether PrP is synthesized by astrocytes. We developed a knock-in reporter mouse line in which the coding sequence of the PrP expressing gene (Prnp), was replaced with that for green fluorescent protein (GFP). Native GFP fluorescence intensity varied between and within brain regions. GFP was present in astrocytes but did not increase during reactive gliosis induced by scrapie prion infection. Therefore, reactive gliosis associated with prion diseases does not cause an acceleration of local PrP production. In addition to aiding in Prnp gene activity studies, this reporter mouse line will likely prove useful for analysis of chimeric animals produced by stem cell and tissue transplantation experiments.

  7. Interleukin-1β and anaphylatoxins exert a synergistic effect on NGF expression by astrocytes

    Directory of Open Access Journals (Sweden)

    Patte Christine

    2006-04-01

    Full Text Available Abstract C3a and C5a anaphylatoxins are proinflammatory polypeptides released during complement activation. They exert their biological activities through interaction with two G protein-coupled receptors named C3aR and C5aR, respectively. In the brain, these receptors are expressed on glial cells, and some recent data have suggested that anaphylatoxins could mediate neuroprotection. In this study, we used RT-PCR and ribonuclease protection assays (RPA to investigate the role of anaphylatoxins on neurotrophin expression by the human glioblastoma cell line T98G and by rat astrocytes. Our data show that for both cell types, anaphylatoxins upregulate expression of NGF mRNA. This response depended on a G protein-coupled pathway since pre-treatment of cells with pertussis toxin (PTX completely blocked NGF mRNA increases. This effect was anaphylatoxin-specific since pre-incubation with anti-C3a or anti-C5aR antibodies abolished the effects of C3a and C5a, respectively. The regulation of NGF mRNA by anaphylatoxins was not accompanied by translation into protein expression, but there was a significant synergic effect of anaphylatoxins/IL-1b costimulation. Our demonstration of involvement of anaphylatoxins in the NGF release process by astrocytes suggests that C3a and C5a could modulate neuronal survival in the CNS.

  8. 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.

  9. EFFECTS OF CULTURED ASTROCYTES FROM RAT CEREBRAL CORTEX ON THE DEVELOPMENT OF PC12 CELLS%星形神经胶质细胞对PC12细胞生长发育的影响

    Institute of Scientific and Technical Information of China (English)

    莫永炎; 陈瑗; 周玫; 张宝

    2000-01-01

    在神经系统的生长发育过程中,星形胶质细胞对神经元生长发育的作用是一项重要的研究课题。本文以体外培养的SD大鼠大脑皮质星形胶质细胞与PC12神经元按不同细胞数目比例(50:1~1:1)共同培养,并用其制备的条件培养液培养PC12细胞,用快速灵敏的MTT比色法测定PC12神经元的细胞活力,用光学相差显微镜观察PC12细胞形态学变化。结果显示,星形胶质细胞条件培养液可增强PC12细胞活力(MTT测定的 OD值由0.255±0.012提高到0.510±0.036,P<0.001,且细胞折光性较对照组强),却不能促使PC12神经元突起的生出。将星形胶质细胞与PC12细胞按30:1~1:1的比例共同培养时,既可提高PC12细胞折光性和光晕又可促使其突起的生长;如按50:1~40:1的比例共同培养时,只观察到提高PC12细胞折光性和光晕,而无促使其突起生长发育的作用。本文结果提示,PC12神经元细胞活力的提高与星形胶质细胞分泌到条件培养液中的可溶性因子有关,而PC12神经元突起生长发育可能是和与星形胶质细胞的直接接触以及二者的细胞数且比有关。%To investigate effects of cultured astrocytes from Sprague Dawley rat cerebral cortex on the development of PC12 cellsderived from rat pheochromocytoma, PC12 cells were cocultured with astrocyte according to different astrocytes/neurons ratio(50:1~1:1) , or with serum-free conditioned medium of astrocytes(ACM). The vitality of PC12 cells was measured by sensi-tive MTT method and their morphologic features were observed by Olympus light microscope. The results showed: (1) WhenPC12 cells were cultured with ACM, compared with the control group, the vitality of PC12 cells was increased significantly (0.255+0. 012 vs 0. 510±0. 036, P<0. 001) and the morphological changes were not obvious in the experimental group. (2) WhenPC12 cells were cocultured with astrocyte in the ratio of 30: 1

  10. Gain-of-function defects of astrocytic Kir4.1 channels in children with autism spectrum disorders and epilepsy

    Science.gov (United States)

    Sicca, Federico; Ambrosini, Elena; Marchese, Maria; Sforna, Luigi; Servettini, Ilenio; Valvo, Giulia; Brignone, Maria Stefania; Lanciotti, Angela; Moro, Francesca; Grottesi, Alessandro; Catacuzzeno, Luigi; Baldini, Sara; Hasan, Sonia; D’Adamo, Maria Cristina; Franciolini, Fabio; Molinari, Paola; Santorelli, Filippo M.; Pessia, Mauro

    2016-01-01

    Dysfunction of the inwardly-rectifying potassium channels Kir4.1 (KCNJ10) represents a pathogenic mechanism contributing to Autism-Epilepsy comorbidity. To define the role of Kir4.1 variants in the disorder, we sequenced KCNJ10 in a sample of affected individuals, and performed genotype-phenotype correlations. The effects of mutations on channel activity, protein trafficking, and astrocyte function were investigated in Xenopus laevis oocytes, and in human astrocytoma cell lines. An in vivo model of the disorder was also explored through generation of kcnj10a morphant zebrafish overexpressing the mutated human KCNJ10. We detected germline heterozygous KCNJ10 variants in 19/175 affected children. Epileptic spasms with dysregulated sensory processing represented the main disease phenotype. When investigated on astrocyte-like cells, the p.R18Q mutation exerted a gain-of-function effect by enhancing Kir4.1 membrane expression and current density. Similarly, the p.R348H variant led to gain of channel function through hindrance of pH-dependent current inhibition. The frequent polymorphism p.R271C seemed, instead, to have no obvious functional effects. Our results confirm that variants in KCNJ10 deserve attention in autism-epilepsy, and provide insight into the molecular mechanisms of autism and seizures. Similar to neurons, astrocyte dysfunction may result in abnormal synaptic transmission and electrical discharge, and should be regarded as a possible pharmacological target in autism-epilepsy. PMID:27677466

  11. Osmotic stress affects functional properties of human melanoma cell lines

    CERN Document Server

    La Porta, Caterina A M; Pasini, Maria; Laurson, Lasse; Alava, Mikko J; Zapperi, Stefano; Amar, Martine Ben

    2015-01-01

    Understanding the role of microenvironment in cancer growth and metastasis is a key issue for cancer research. Here, we study the effect of osmotic pressure on the functional properties of primary and metastatic melanoma cell lines. In particular, we experimentally quantify individual cell motility and transmigration capability. We then perform a circular scratch assay to study how a cancer cell front invades an empty space. Our results show that primary melanoma cells are sensitive to a low osmotic pressure, while metastatic cells are less. To better understand the experimental results, we introduce and study a continuous model for the dynamics of a cell layer and a stochastic discrete model for cell proliferation and diffusion. The two models capture essential features of the experimental results and allow to make predictions for a wide range of experimentally measurable parameters.

  12. Proteomic analysis of astrocytic secretion that regulates neurogenesis using quantitative amine-specific isobaric tagging

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Hu; Zhou, Wenhao [Children' s Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102 (China); Wei, Liming; Zhong, Fan [Institutes of Biomedical Sciences, Fudan University, 138 Yixueyuan Roda, Shanghai 200032 (China); Yang, Yi, E-mail: yyang@shmu.edu.cn [Children' s Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102 (China)

    2010-01-08

    Astrocytes are essential components of neurogenic niches that affect neurogenesis through membrane association and/or the release of soluble factors. To identify factors released from astrocytes that could regulate neural stem cell differentiation and proliferation, we used mild oxygen-glucose deprivation (OGD) to inhibit the secretory capacity of astrocytes. Using the Transwell co-culture system, we found that OGD-treated astrocytes could not promote neural stem cell differentiation and proliferation. Next, isobaric tagging for the relative and absolute quantitation (iTRAQ) proteomics techniques was performed to identify the proteins in the supernatants of astrocytes (with or without OGD). Through a multi-step analysis and gene ontology classification, 130 extracellular proteins were identified, most of which were involved in neuronal development, the inflammatory response, extracellular matrix composition and supportive functions. Of these proteins, 44 had never been reported to be produced by astrocytes. Using ProteinPilot software analysis, we found that 60 extracellular proteins were significantly altered (27 upregulated and 33 downregulated) in the supernatant of OGD-treated astrocytes. Among these proteins, 7 have been reported to be able to regulate neurogenesis, while others may have the potential to regulate neurogenesis. This study profiles the major proteins released by astrocytes, which play important roles in the modulation of neurogenesis.

  13. IFN-γ signaling to astrocytes protects from autoimmune mediated neurological disability.

    Directory of Open Access Journals (Sweden)

    Claudia Hindinger

    Full Text Available Demyelination and axonal degeneration are determinants of progressive neurological disability in patients with multiple sclerosis (MS. Cells resident within the central nervous system (CNS are active participants in development, progression and subsequent control of autoimmune disease; however, their individual contributions are not well understood. Astrocytes, the most abundant CNS cell type, are highly sensitive to environmental cues and are implicated in both detrimental and protective outcomes during autoimmune demyelination. Experimental autoimmune encephalomyelitis (EAE was induced in transgenic mice expressing signaling defective dominant-negative interferon gamma (IFN-γ receptors on astrocytes to determine the influence of inflammation on astrocyte activity. Inhibition of IFN-γ signaling to astrocytes did not influence disease incidence, onset, initial progression of symptoms, blood brain barrier (BBB integrity or the composition of the acute CNS inflammatory response. Nevertheless, increased demyelination at peak acute disease in the absence of IFN-γ signaling to astrocytes correlated with sustained clinical symptoms. Following peak disease, diminished clinical remission, increased mortality and sustained astrocyte activation within the gray matter demonstrate a critical role of IFN-γ signaling to astrocytes in neuroprotection. Diminished disease remission was associated with escalating demyelination, axonal degeneration and sustained inflammation. The CNS infiltrating leukocyte composition was not altered; however, decreased IL-10 and IL-27 correlated with sustained disease. These data indicate that astrocytes play a critical role in limiting CNS autoimmune disease dependent upon a neuroprotective signaling pathway mediated by engagement of IFN-γ receptors.

  14. Concurrent loss and proliferation of astrocytes following lateral fluid percussion brain injury in the adult rat.

    Science.gov (United States)

    Hill-Felberg, S J; McIntosh, T K; Oliver, D L; Raghupathi, R; Barbarese, E

    1999-07-15

    Astrocyte populations were analyzed over a period of 1 month in the hippocampus following lateral fluid percussion (FP) brain injury. Rats (n = 23) were subjected either to a brain injury of moderate severity, or to anesthesia and surgery without injury (n = 7). At 3 days, 1, 2, or 4 weeks postinjury, subgroups of animals were sacrificed and the brains removed and sectioned for histochemical analysis. The density of astrocytes, identified with gold sublimate staining, decreased significantly in the ipsilateral hippocampus of injured rats 3 days following injury, eventually falling to 64% of the total astrocyte population present in uninjured animals by 1 week postinjury. One month postinjury, the density of hippocampal astrocytes had returned to 85% of the total number of astrocytes observed in the hippocampus of uninjured animals. In order to characterize the post-traumatic formation of new astrocytes, immunohistochemistry was performed using antibodies to proliferating cell nuclear antigen (PCNA) and to glial fibriallary acidic protein (GFAP). Positive immunolabeling for both PCNA and GFAP was most abundant at 3 days following FP brain injury in regions where the blood brain barrier was compromised, and was not detectable by 1 month postinjury. These results indicate that astrocyte proliferation after injury may be evoked by mitogens released from vascular sources, and may be an attempt to compensate for some of the astrocytic cell loss observed after injury.

  15. 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.

  16. Optimized protocol for derivation of human embryonic stem cell lines.

    Science.gov (United States)

    Camarasa, María Vicenta; Galvez, Víctor Miguel; Brison, Daniel Roy; Bachiller, Daniel

    2012-09-01

    For the past 12 years, the biology and applications of human embryonic stem cells (hESCs) have received great attention from the scientific community. Derivatives of the first hESC line obtained by J. Thomson's group (Science 282(5391):1145-1147, 1998) have been used in clinical trials in patients with spinal cord injury, and other hESC lines have now been used to generate cells for use in treating blindness (Lancet 379(9817):713-720, 2012). In addition to the classical protocol based on mouse or human feeder layers using open culture methods (In Vitro Cellular & Developmental Biology - Animal 46(3-4):386-394, 2010; Stem Cells 23(9):1221-1227, 2005; Nature Biotechnology 24(2):185-187, 2006; Human Reproduction 21(2):503-511, 2006; Human Reproduction 20(8):2201-2206, 2005; Fertility and Sterility 83(5):1517-1529, 2005), novel hESC lines have been derived xeno-free (without using animal derived reagents) (PLoS One 5 (4):1024-1026, 2010), feeder-free (without supporting cell monolayers) (Lancet 365(9471):1601-1603, 2005), in microdrops under oil (In Vitro Cellular & Developmental Biology - Animal 46(3-4):236-41, 2010) and in suspension with ROCK inhibitor (Nature Biotechnology 28(4):361-4, 2010). Regardless of the culture system, successful hESC derivation usually requires optimization of embryo culture, the careful and timely isolation of its inner cell mass (ICM), and precise culture conditions up to the establishment of pluripotent cell growth during hESC line derivation. Herein we address the crucial steps of the hESC line derivation protocol, and provide tips to apply quality control to each step of the procedure.

  17. Cysteine modified polyaniline films improve biocompatibility for two cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Yslas, Edith I., E-mail: eyslas@exa.unrc.edu.ar [Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina); Cavallo, Pablo; Acevedo, Diego F.; Barbero, César A. [Departamento de Química, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina); Rivarola, Viviana A. [Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina)

    2015-06-01

    This work focuses on one of the most exciting application areas of conjugated conducting polymers, which is cell culture and tissue engineering. To improve the biocompatibility of conducting polymers we present an easy method that involves the modification of the polymer backbone using L-cysteine. In this publication, we show the synthesis of polyaniline (PANI) films supported onto Polyethylene terephthalate (PET) films, and modified using cysteine (PANI-Cys) in order to generate a biocompatible substrate for cell culture. The PANI-Cys films are characterized by Fourier Transform infrared and UV–visible spectroscopy. The changes in the hydrophilicity of the polymer films after and before the modification were tested using contact angle measurements. After modification the contact angle changes from 86° ± 1 to 90° ± 1, suggesting a more hydrophylic surface. The adhesion properties of LM2 and HaCaT cell lines on the surface of PANI-Cys films in comparison with tissue culture plastic (TCP) are studied. The PANI-Cys film shows better biocompatibility than PANI film for both cell lines. The cell morphologies on the TCP and PANI-Cys film were examined by florescence and Atomic Force Microscopy (AFM). Microscopic observations show normal cellular behavior when PANI-Cys is used as a substrate of both cell lines (HaCaT and LM2) as when they are cultured on TCP. The ability of these PANI-Cys films to support cell attachment and growth indicates their potential use as biocompatible surfaces and in tissue engineering. - Highlights: • A new surface PANI-Cys was produced on films of polyethylene terephthalate. • The relationship between surface characteristics and biocompatibility is analyzed. • The PANI-Cys film presents good biocompatibility for two cell lines.

  18. Astrocytes in oligodendrocyte lineage development and white matter pathology

    Directory of Open Access Journals (Sweden)

    Jiasi eLi

    2016-05-01

    Full Text Available White matter is primarily composed of myelin and myelinated axons. Structural and functional completeness of myelin is critical for the reliable and efficient transmission of information. White matter injury has been associated with the development of many demyelinating diseases. Despite a variety of scientific advances aimed at promoting re-myelination, their benefit has proven at best to be marginal. Research suggests that the failure of the re-myelination process may be the result of an unfavorable microenvironment. Astrocytes, are the most ample and diverse type of glial cells in central nervous system which display multiple functions for the cells of the oligodendrocytes lineage. As such, much attention has recently been drawn to astrocyte function in terms of white matter myelin repair. They are different in white matter from those in grey matter in specific regards to development, morphology, location, protein expression and other supportive functions. During the process of demyelination and re-myelination, the functions of astrocytes are dynamic in that they are able to change functions in accordance to different time points, triggers or reactive pathways resulting in vastly different biologic effects. They have pivotal effects on oligodendrocytes and other cell types in the oligodendrocyte lineage by serving as an energy supplier, a participant of immunological and inflammatory functions, a source of trophic factors and iron and a sustainer of homeostasis. Astrocytic impairment has been shown to be directly linked to the development of neuromyelities optica. In addition, astroctyes have also been implicated in other white matter conditions such as psychiatric disorders and neurodegenerative diseases such as Alzheimer’s disease, multiple sclerosis and amyotrophic lateral sclerosis. Inhibiting specifically detrimental signaling pathways in astrocytes while preserving their beneficial functions may be a promising approach for

  19. Astrocytes in Oligodendrocyte Lineage Development and White Matter Pathology

    Science.gov (United States)

    Li, Jiasi; Zhang, Lei; Chu, Yongxin; Namaka, Michael; Deng, Benqiang; Kong, Jiming; Bi, Xiaoying

    2016-01-01

    White matter is primarily composed of myelin and myelinated axons. Structural and functional completeness of myelin is critical for the reliable and efficient transmission of information. White matter injury has been associated with the development of many demyelinating diseases. Despite a variety of scientific advances aimed at promoting re-myelination, their benefit has proven at best to be marginal. Research suggests that the failure of the re-myelination process may be the result of an unfavorable microenvironment. Astrocytes, are the most ample and diverse type of glial cells in central nervous system (CNS) which display multiple functions for the cells of the oligodendrocytes lineage. As such, much attention has recently been drawn to astrocyte function in terms of white matter myelin repair. They are different in white matter from those in gray matter in specific regards to development, morphology, location, protein expression and other supportive functions. During the process of demyelination and re-myelination, the functions of astrocytes are dynamic in that they are able to change functions in accordance to different time points, triggers or reactive pathways resulting in vastly different biologic effects. They have pivotal effects on oligodendrocytes and other cell types in the oligodendrocyte lineage by serving as an energy supplier, a participant of immunological and inflammatory functions, a source of trophic factors and iron and a sustainer of homeostasis. Astrocytic impairment has been shown to be directly linked to the development of neuromyelities optica (NMO). In addition, astroctyes have also been implicated in other white matter conditions such as psychiatric disorders and neurodegenerative diseases such as Alzheimer’s disease (AD), multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Inhibiting specifically detrimental signaling pathways in astrocytes while preserving their beneficial functions may be a promising approach for

  20. Astrocytes mediate the neuroprotective effects of Tibolone following brain injury

    Directory of Open Access Journals (Sweden)

    Luis Miguel Garcia-Segura

    2015-04-01

    Full Text Available Recently, astrocytes have become a key central player in mediating important functions in the brain. These physiological processes include neurotransmitter recycling, energy management, metabolic shuttle, immune sensing, K+ buffer, antioxidant supply and release of neurotrophic factors and gliotransmitters. These astrocytic roles are somehow altered upon brain injury, therefore strategies aimed at better protecting astrocytes are an essential asset to maintain brain homeostasis. In this context, estrogenic compounds, such as Tibolone, have attracted attention for their beneficial effects in acute and chronic degenerative diseases. Tibolone may act through binding to estrogen, androgen or progesterone receptors and exert protective effects by reducing astrocytes cell death and oxidative stress signaling mechanisms. Although Tibolone has a multifactorial effect in the brain, its mechanisms of action are not completely understood. In this work, we highlight the role of Tibolone in brain protection upon damage, how astrocytes might mediate part of its neuroprotective actions and discuss the effects of Tibolone in diminishing the harmful consequences of a metabolic insult and energy failure in the setting of a pathological event.

  1. UOK 268 Cell Line for Hereditary Leiomyomatosis and Renal Cell Carcinoma | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Cancer Institute’s Urologic Oncology Branch seeks parties to co-develop the UOK 262 immortalized cell line as research tool to study aggressive hereditary leiomyomatosis and renal cell carcinoma (HLRCC)-associated recurring kidney cancer.

  2. Third-line chemotherapy for small cell lung cancer

    NARCIS (Netherlands)

    de Jong, WK; ten Hacken, NHT; Groen, HJM

    2006-01-01

    Efficacy of third-line chemotherapy treatment for small cell lung cancer (SCLC) is unknown. We present our experience with third-tine chemotherapy for recurrent SCLC. Between January 1996 and July 2004 all. consecutive patients treated for SCLC were retrospectively studied. We recorded patient chara

  3. UV light blocks EGFR signalling in human cancer cell lines

    DEFF Research Database (Denmark)

    Olsen, BB; Neves-Petersen, M T; Klitgaard, S

    2007-01-01

    UV light excites aromatic residues, causing these to disrupt nearby disulphide bridges. The EGF receptor is rich in aromatic residues near the disulphide bridges. Herein we show that laser-pulsed UV illumination of two different skin-derived cancer cell lines i.e. Cal-39 and A431, which both...

  4. Silicon Carbide Tiles for Sidewall Lining in Aluminium Electrolysis Cells

    Institute of Scientific and Technical Information of China (English)

    RUANBo; ZHAOJunguo; 等

    1999-01-01

    The paper introduces the nitride bonded silicon carbide used for sidewall lining in aluminium eletrolysis cells ,including technical process,main properties and application results.Comparison tests on various physical properties of silicon carbide products made by LIRR and other producers worldwide have also been conducted in an independent laboratory.

  5. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project

    Science.gov (United States)

    2011-03-24

    ...; Identification of Human Cell Lines Project AGENCY: National Institute of Standards and Technology (NIST...) profiling up to 1500 human cell line samples as part of the Identification of Human Cell Lines Project. All... for Biotechnology Information (NCBI) and will be used to differentiate among cell lines, as...

  6. Identification and pharmacological characterization of the histamine H3 receptor in cultured rat astrocytes.

    Science.gov (United States)

    Mele, Tina; Jurič, Damijana Mojca

    2013-11-15

    Recently we reported that cultured rat cortical astrocytes express histamine H3 receptor that is functionally coupled to Gi/o proteins and participates to the stimulatory effect of histamine. Due to the lack of data on the distribution of histamine H3 receptors on glial cells we further investigated their presence in cultured astrocytes from different brain regions. Real-time PCR was performed to examine the expression of native histamine H3 receptor in cultured rat astrocytes from cortex,cerebellum, hippocampus and striatum.Double-antigen immunofluorescence staining and[3H]N-α-methylhistamine([3H]NαMH) binding studies were utilized to specifically identify and characterize receptor binding sites in astrocytes. Histamine H3 receptor mRNA was detected in rat astrocytes from all the regions under investigation with the highest levels in striatal astrocytes followed by hippocampal astrocytes and approximately equal levels in cerebellar and cortical astrocytes.Double-antigen immunofluorescence confirmed the presence of histamine H3 receptors on the membrane of all examined astroglial populations.[3H]NαMH bound with high affinity and specificity to an apparently single class of saturable sites on cortical astrocytic membranes(KD¼4.5570.46 nM; Bmax¼5.6370.21 fmol/mg protein)and competition assays with selective agonists and antagonists were consistent with labeling of histamine H3 receptor(range of pKi values 7.50–8.87). Our study confirmed the ability of cultured astrocytes from different rat brain regions to express histamine H3 receptors.The observed diverse distribution of the receptors within various astrocytic populations possibly mirrors their heterogeneity in the brain and indicates their active involvement in histamine-mediated effects.

  7. 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.

  8. Glioactive ATP controls BDNF recycling in cortical astrocytes

    Science.gov (United States)

    Vignoli, Beatrice; Canossa, Marco

    2017-01-01

    ABSTRACT We have recently reported that long-term memory retention requires synaptic glia for proBDNF uptake and recycling. Through the recycling course, glial cells release endocytic BDNF, a mechanism that is activated in response to glutamate via AMPA and mGluRI/II receptors. Cortical astrocytes express receptors for many different transmitters suggesting for a complex signaling controlling endocytic BDNF secretion. Here, we demonstrated that the extracellular nucleotide ATP, activating P2X and P2Y receptors, regulates endocytic BDNF secretion in cultured astrocytes. Our data indicate that distinct glioactive molecules can participate in BDNF glial recycling and suggest that cortical astrocytes contributing to neuronal plasticity can be influenced by neurotransmitters in tune with synaptic needs.

  9. The indispensable roles of microglia and astrocytes during brain development

    Directory of Open Access Journals (Sweden)

    Kitty Reemst

    2016-11-01

    Full Text Available Glia are essential for brain functioning during development and in the adult brain. Here, we discuss the various roles of both microglia and astrocytes, and their interactions during brain development. Although both cells are fundamentally different in origin and function, they often affect the same developmental processes such as neuro-/gliogenesis, angiogenesis, axonal outgrowth, synaptogenesis and synaptic pruning. Due to their important instructive roles in these processes, dysfunction of microglia or astrocytes during brain development could contribute to neurodevelopmental disorders and potentially even late-onset neuropathology. A better understanding of the origin, differentiation process and developmental functions of microglia and astrocytes will help to fully appreciate their role both in the developing as well as in the adult brain, in health and disease.

  10. Crude subcellular fractionation of cultured mammalian cell lines

    Directory of Open Access Journals (Sweden)

    Holden Paul

    2009-12-01

    Full Text Available Abstract Background The expression and study of recombinant proteins in mammalian culture systems can be complicated during the cell lysis procedure by contaminating proteins from cellular compartments distinct from those within which the protein of interest resides and also by solubility issues that may arise from the use of a single lysis buffer. Partial subcellular fractionation using buffers of increasing stringency, rather than whole cell lysis is one way in which to avoid or reduce this contamination and ensure complete recovery of the target protein. Currently published protocols involve time consuming centrifugation steps which may require expensive equipment and commercially available kits can be prohibitively expensive when handling large or multiple samples. Findings We have established a protocol to sequentially extract proteins from cultured mammalian cells in fractions enriched for cytosolic, membrane bound organellar, nuclear and insoluble proteins. All of the buffers used can be made inexpensively and easily and the protocol requires no costly equipment. While the method was optimized for a specific cell type, we demonstrate that the protocol can be applied to a variety of commonly used cell lines and anticipate that it can be applied to any cell line via simple optimization of the primary extraction step. Conclusion We describe a protocol for the crude subcellular fractionation of cultured mammalian cells that is both straightforward and cost effective and may facilitate the more accurate study of recombinant proteins and the generation of purer preparations of said proteins from cell extracts.

  11. Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

    Directory of Open Access Journals (Sweden)

    Chen Lei

    2011-06-01

    Full Text Available Abstract Background Cancer stem cells (CSCs are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. Methods Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs. Results The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44. Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability. Conclusions Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.

  12. THP-1 cell line: an in vitro cell model for immune-modulation approach : Review

    NARCIS (Netherlands)

    Chanput, W.; Mes, J.J.; Wichers, H.J.

    2014-01-01

    THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review a

  13. Pre-Conditioning Induces the Precocious Differentiation of Neonatal Astrocytes to Enhance Their Neuroprotective Properties

    Directory of Open Access Journals (Sweden)

    Ellora Sen

    2011-07-01

    Full Text Available Hypoxic preconditioning reprogrammes the brain's response to subsequent H/I (hypoxia-ischaemia injury by enhancing neuroprotective mechanisms. Given that astrocytes normally support neuronal survival and function, the purpose of the present study was to test the hypothesis that a hypoxic preconditioning stimulus would activate an adaptive astrocytic response. We analysed several functional parameters 24 h after exposing rat pups to 3 h of systemic hypoxia (8% O2. Hypoxia increased neocortical astrocyte maturation as evidenced by the loss of GFAP (glial fibrillary acidic proteinpositive cells with radial morphologies and the acquisition of multipolar GFAP-positive cells. Interestingly, many of these astrocytes had nuclear S100B. Accompanying their differentiation, there was increased expression of GFAP, GS (glutamine synthetase, EAAT-1 (excitatory amino acid transporter-1; also known as GLAST, MCT-1 (monocarboxylate transporter-1 and ceruloplasmin. A subsequent H/I insult did not result in any further astrocyte activation. Some responses were cell autonomous, as levels of GS and MCT-1 increased subsequent to hypoxia in cultured forebrain astrocytes. In contrast, the expression of GFAP, GLAST and ceruloplasmin remained unaltered. Additional experiments utilized astrocytes exposed to exogenous dbcAMP (dibutyryl-cAMP, which mimicked several aspects of the preconditioning response, to determine whether activated astrocytes could protect neurons from subsequent excitotoxic injury. dbcAMP treatment increased GS and glutamate transporter expression and function, and as hypothesized, protected neurons from glutamate excitotoxicity. Taken altogether, these results indicate that a preconditioning stimulus causes the precocious differentiation of astrocytes and increases the acquisition of multiple astrocytic functions that will contribute to the neuroprotection conferred by a sublethal preconditioning stress.

  14. Pre-conditioning induces the precocious differentiation of neonatal astrocytes to enhance their neuroprotective properties

    Directory of Open Access Journals (Sweden)

    Sandra J Hewett

    2011-07-01

    Full Text Available Hypoxic preconditioning reprogrammes the brain's response to subsequent H/I (hypoxia–ischaemia injury by enhancing neuroprotective mechanisms. Given that astrocytes normally support neuronal survival and function, the purpose of the present study was to test the hypothesis that a hypoxic preconditioning stimulus would activate an adaptive astrocytic response. We analysed several functional parameters 24 h after exposing rat pups to 3 h of systemic hypoxia (8% O2. Hypoxia increased neocortical astrocyte maturation as evidenced by the loss of GFAP (glial fibrillary acidic protein-positive cells with radial morphologies and the acquisition of multipolar GFAP-positive cells. Interestingly, many of these astrocytes had nuclear S100B. Accompanying their differentiation, there was increased expression of GFAP, GS (glutamine synthetase, EAAT-1 (excitatory amino acid transporter-1; also known as GLAST, MCT-1 (monocarboxylate transporter-1 and ceruloplasmin. A subsequent H/I insult did not result in any further astrocyte activation. Some responses were cell autonomous, as levels of GS and MCT-1 increased subsequent to hypoxia in cultured forebrain astrocytes. In contrast, the expression of GFAP, GLAST and ceruloplasmin remained unaltered. Additional experiments utilized astrocytes exposed to exogenous dbcAMP (dibutyryl-cAMP, which mimicked several aspects of the preconditioning response, to determine whether activated astrocytes could protect neurons from subsequent excitotoxic injury. dbcAMP treatment increased GS and glutamate transporter expression and function, and as hypothesized, protected neurons from glutamate excitotoxicity. Taken altogether, these results indicate that a preconditioning stimulus causes the precocious differentiation of astrocytes and increases the acquisition of multiple astrocytic functions that will contribute to the neuroprotection conferred by a sublethal preconditioning stress.

  15. Human Embryonic St me Cell Lines fromthe Chinese Population and Differentiation to Liver and Muscle Cell Types

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    We have established 6 hES cell lines from IVF surplus blastocysts. Characterization of these lines have shown that 4 of the 6 lines meet all of the criterion (Science) for hES cell lines and 2 of them display most characteristics of hES cells but do not form teratoma. In order to produce hES cell lines without using mouse feeders, we have produced a hES cell line using feeders derived from hES cells themselves, and showed that hES-derived feeders are capable of supporting the derivation of new hES cell line...

  16. Cytotoxicity and genotoxicity of phenazine in two human cell lines.

    Science.gov (United States)

    McGuigan, Claire F; Li, Xing-Fang

    2014-06-01

    Phenazine was recently identified as a drinking water disinfection byproduct (DBP), but little is known of its toxic effects. We examined in vitro cytotoxicity and genotoxicity of phenazine (1.9-123 μM) in HepG2 and T24 cell lines. Cytotoxicity was determined by an impedance-based real-time cell analysis instrument. The BrdU (5-bromo-2'-deoxyuridine) proliferation and MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assays were used to examine mechanisms of cytotoxicity. Genotoxicity was determined using the alkaline comet assay. Concentration-dependent cytotoxicity was observed in HepG2 cells, primarily due to an antiproliferative effect (BrdU 24 h IC50: 11 μM; 48 h IC50: 7.8 μM) observed as low as 1.9 μM. T24 cells experienced a minor antiproliferative effect (BrdU 24 h IC50: 47 μM; 48 h IC50: 17 μM). IC50 values for HepG2 proliferation and viability were 54-77% lower compared to T24 cells. In both cell lines, IC50 values for proliferation were 66-90% lower than those for viability. At phenazine concentrations producing equivalent cytotoxicity, HepG2 cells (1.9-30.8 μM) experienced no significant genotoxic effects, while T24 cells (7.7-123 μM) experienced significant genotoxicity at ⩾61.5 μM. While these effects were seen at phenazine concentrations above those found in disinfected water, the persistence of the antiproliferative effect and the differential toxicity in each cell line deserves further study.

  17. Effect of cell confluence on production of cloned mice using an inbred embryonic stem cell line.

    Science.gov (United States)

    Gao, Shaorong; McGarry, Michelle; Ferrier, Tricia; Pallante, Benedetta; Priddle, Helen; Gasparrini, Bianca; Fletcher, Judy; Harkness, Linda; De Sousa, Paul; McWhir, Jim; Wilmut, Ian

    2003-02-01

    Mice have been successfully cloned from both somatic cells and hybrid embryonic stem (ES) cells. Heterozygosity of the donor ES cell genome has been suggested as a crucial factor for long-term survival of cloned mice. In the present study, an inbred ES cell line, HM-1 (129/Ola), and a well-tested ES cell line, R1 (129/Sv x 129/Sv-CP), were used as donor cells to evaluate the developmental potential of nuclear transfer embryos. We found that ES cell confluence dramatically affects the developmental potential of reconstructed embryos. With the ES cell line HM-1 and 80-90% confluence, 49% of reconstructed embryos developed to the morula/blastocyst stage, 9% of these embryos developed to live pups when transferred to the surrogate mothers, and 5 of 18 live pups survived to adulthood. By contrast, at 60-70% confluence, only 22% of embryos developed to the morula/blastocyst stage, and after transfer, only a single fetus reached term. Consistent with previous reports, the nuclei of R1 ES cells were also shown to direct development to term, but no live pups were derived from cells at later passages (>20). Our results show that the developmental potential of reconstructed embryos is determined by both cell confluence and cell passage. These results also demonstrate that the inbred ES cell line, HM-1, can be used to produce viable cloned mice, although less efficiently than most heterozygous ES cell lines.

  18. Relation of cell proliferation to expression of peripheral benzodiazepine receptors in human breast cancer cell lines.

    Science.gov (United States)

    Beinlich, A; Strohmeier, R; Kaufmann, M; Kuhl, H

    2000-08-01

    Peripheral benzodiazepine receptor (PBR) agonist [(3)H]Ro5-4864 has been shown to bind with high affinity to the human breast cancer cell line BT-20. Therefore, we investigated different human breast cancer cell lines with regard to binding to [(3)H]Ro5-4864 and staining with the PBR-specific monoclonal antibody 8D7. Results were correlated with cell proliferation characteristics. In flow cytometric analysis, the estrogen receptor (ER)-negative breast cancer cell lines BT-20, MDA-MB-435-S, and SK-BR-3 showed significantly higher PBR expression (relative fluorescence intensity) than the ER-positive cells T47-D, MCF-7 and BT-474 (Pdiazepam-binding inhibitor are possibly involved in the regulation of cell proliferation of human breast cancer cell lines.

  19. Mitochondrial DNA sequence analysis of two mouse hepatocarcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Ji-Gang Dai; Xia Lei; Jia-Xin Min; Guo-Qiang Zhang; Hong Wei

    2005-01-01

    AIM: To study genetic difference of mitochondrial DNA (mtDNA)between two hepatocarcinoma cell lines (Hca-F and Hca-P)with diverse metastatic characteristics and the relationship between mtDNA changes in cancer cells and their oncogenic phenotype.METHODS: Mitochondrial DNA D-loop, tRNAMet+Glu+Ile and ND3gene fragments from the hepatocarcinoma cell lines with 1100, 1126 and 534 bp in length respectively were analysed by PCR amplification and restriction fragment length polymorphism techniques. The D-loop 3' end sequence of the hepatocarcinoma cell lines was determined by sequencing.RESULTS: No amplification fragment length polymorphism and restriction fragment length polymorphism were observed in tRNAMet+Glu+Ile,ND3 and D-loop of mitochondrial DNA of the hepatocarcinoma cells. Sequence differences between Hca-F and Hca-P were found in mtDNA D-loop.CONCLUSION: Deletion mutations of mitochondrial DNA restriction fragment may not play a significant role in carcinogenesis. Genetic difference of mtDNA D-loop between Hca-F and Hca-P, which may reflect the environmental and genetic influences during tumor progression, could be linked to their tumorigenic phenotypes.

  20. Centrosomal dysregulation in human metastatic melanoma cell lines.

    Science.gov (United States)

    Charters, Geoffrey A; Stones, Clare J; Shelling, Andrew N; Baguley, Bruce C; Finlay, Graeme J

    2011-09-01

    Correct partitioning of the replicated genome during mitosis is orchestrated by centrosomes, and chromosomal instability is a commonly reported feature of human cancer. Melanomas are notorious for their genetic instability and rapid clonal evolution that may be manifested as aggressive growth and facile generation of therapy-resistant variants. We characterized the centrosomal status, ploidy, and gene status (TP53, CDKN2A/B, BRAF, and NRAS) of 15 human metastatic melanoma cell lines. Cells were labelled for pericentrin (a centrosomal marker), DNA and α-tubulin, and scored for centrosome morphology, supernumerary centrosomes, and mitotic symmetry. The incidence of supernumerary centrosomes correlated with that of gross centrosomal abnormalities (r = 0.90), mitotic asymmetry (r = 0.90), and, surprisingly, increased content of G/M cells (r = 0.79). Centrosomal numerical dysregulation, observed in all cell lines, was found not to be specifically related to the status of any of the characterized gene mutations that were found in 13/15 cell lines. We conclude that centrosomal dysregulation may arise from multiple mechanisms and may drive the generation of genetic and phenotypic diversity in melanoma.

  1. BRITER: a BMP responsive osteoblast reporter cell line.

    Directory of Open Access Journals (Sweden)

    Prem Swaroop Yadav

    Full Text Available BACKGROUND: BMP signaling pathway is critical for vertebrate development and tissue homeostasis. High-throughput molecular genetic screening may reveal novel players regulating BMP signaling response while chemical genetic screening of BMP signaling modifiers may have clinical significance. It is therefore important to generate a cell-based tool to execute such screens. METHODOLOGY/PRINCIPAL FINDINGS: We have established a BMP responsive reporter cell line by stably integrating a BMP responsive dual luciferase reporter construct in the immortalized calvarial osteoblast cells isolated from tamoxifen inducible Bmp2; Bmp4 double conditional knockout mouse strain. This cell line, named BRITER (BMP Responsive Immortalized Reporter cell line, responds robustly, promptly and specifically to exogenously added BMP2 protein. The sensitivity to added BMP may be further increased by depleting the endogenous BMP2 and BMP4 proteins. CONCLUSION: As the dynamic range of the assay (for BMP responsiveness is very high for BRITER and as it responds specifically and promptly to exogenously added BMP2 protein, BRITER may be used effectively for chemical or molecular genetic screening for BMP signaling modifiers. Identification of novel molecular players capable of influencing BMP signaling pathway may have clinical significance.

  2. Differences in radiosensitivity between three HER2 overexpressing cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Steffen, Ann-Charlott; Tolmachev, Vladimir; Stenerloew, Bo [Uppsala University, Unit of Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala (Sweden); Goestring, Lovisa [Affibody AB, Bromma (Sweden); Palm, Stig [Sahlgrenska Academy at Goeteborg University, Department of Radiation Physics, Goeteborg (Sweden); Carlsson, Joergen [Uppsala University, Unit of Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala (Sweden); Rudbeck Laboratory, Biomedical Radiation Sciences, Uppsala (Sweden)

    2008-06-15

    HER2 is a potential target for radionuclide therapy, especially when HER2 overexpressing breast cancer cells are resistant to Herceptin {sup registered} treatment. Therefore, it is of interest to analyse whether HER2 overexpressing tumour cells have different inherent radiosensitivity. The radiosensitivity of three often used HER2 overexpressing cell lines, SKOV-3, SKBR-3 and BT-474, was analysed. The cells were exposed to conventional photon irradiation, low linear energy transfer (LET), to characterise their inherent radiosensitivity. The analysis was made with clonogenic survival and growth extrapolation assays. The cells were also exposed to alpha particles, high LET, from {sup 211}At decays using the HER2-binding affibody molecule {sup 211}At-(Z{sub HER2:4}){sub 2} as targeting agent. Assays for studies of internalisation of the affibody molecule were applied. SKOV-3 cells were most radioresistant, SKBR-3 cells were intermediate and BT-474 cells were most sensitive as measured with the clonogenic and growth extrapolation assays after photon irradiation. The HER2 dependent cellular uptake of {sup 211}At was qualitatively similar for all three cell lines. However, the sensitivity to the alpha particles from {sup 211}At differed; SKOV-3 was most resistant, SKBR-3 intermediate and BT-474 most sensitive. These differences were unexpected because it is assumed that all types of cells should have similar sensitivity to high-LET radiation. The sensitivity to alpha particle exposure correlated with internalisation of the affibody molecule and with size of the cell nucleus. There can be differences in radiosensitivity, which, if they also exist between patient breast cancer cells, are important to consider for both conventional radiotherapy and for HER2-targeted radionuclide therapy. (orig.)

  3. Effect of 8-Chloroadenosine on Undifferentiatied HL-60 Cell Line

    Institute of Scientific and Technical Information of China (English)

    CUIJing-rong; HUIYu; XIANGYou-qing; ZHANGLi-he

    2003-01-01

    Aim To study the effect of 8-chloroadenosine (8-CA)on undifferentiatied HL-60 cell line. Methods The IC50 of cancer cell proliferation was determined using a microculture plate reader at 570 nm (MTT) and 540 nm (SRB).Morphology of HL-60 cells was observed under a scanning electron microscope and a transmission electron microscope. The differentiation of HL-60 cells was examined by nitro blue tetrazolium reduction (NBT) and acid phosphatase assay. The cycle of HL-60 cells was analyzed by flow cytometry. Results 8-CA inhibited proliferation of eight human cancer cell lines.The IC50 ranked in the following order; KB (0.05 μmol·L-1 ) < HL-60 (0.25 μmol·L-1) < Bel-7402 (0.56μmol·L-1 )< MCF-7 (0.65μmol·L-1) < HCT (0.79 μmol·L-1) < HeLa (0.89μmol·L-1) < BGC-823 ( 1.149μmol·L-1) cell surface shortened, and the shape of HL-60 cells nuclei changed to kidney-shaped, horse shoe-shaped and bilob ated after treatment with 8-CA. Meanwhile, 8-CA promoted NBT reduction and increased activity of acid phosphatase in HL-60 ceils in a time and concentration-dependent manner. Flow cytometry analysis indicated that 8-CA induced an appreciable increase of the cell population in G1 phase with a marked reduction in S phase. Conclusion 8-CA can induce differentiation of HL-60 cells and block the cells at G1 phase, thus inhibiting proliferation of HL-60 cells.

  4. Plasmids and packaging cell lines for use in phage display

    Science.gov (United States)

    Bradbury, Andrew M.

    2012-07-24

    The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, in fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.

  5. 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.

  6. The indispensable roles of microglia and astrocytes during brain development

    NARCIS (Netherlands)

    Reemst, Kitty; Noctor, Stephen C.; Lucassen, Paul J.; Hol, Elly M.

    2016-01-01

    Glia are essential for brain functioning during development and in the adult brain. Here, we discuss the various roles of both microglia and astrocytes, and their interactions during brain development. Although both cells are fundamentally different in origin and function, they often affect the same

  7. 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.

  8. Loss of ATRX, associated with DNA methylation pattern of chromosome end, impacted biological behaviors of astrocytic tumors

    OpenAIRE

    CAI, JINQUAN; Chen, Jing; Zhang, Wei; Yang, Pei; Zhang, Chuanbao; Li, Mingyang; Yao, Kun; Wang, Hongjun; Li, Qingbin; Jiang, Chuanlu; Jiang., Tao

    2015-01-01

    Loss of ATRX leads to epigenetic alterations, including abnormal levels of DNA methylation at repetitive elements such as telomeres in murine cells. We conducted an extensive DNA methylation and mRNA expression profile study on a cohort of 82 patients with astrocytic tumors to study whether ATRX expression was associated with DNA methylation level in astrocytic tumors and in which cellular functions it participated. We observed that astrocytic tumors with lower ATRX expression harbored higher...

  9. Survey of Differentially Methylated Promoters in Prostate Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Yipeng Wang

    2005-08-01

    Full Text Available DNA methylation, copy number in the genomes of three immortalized prostate epithelial, five cancer cell lines (LNCaP, PC3, PC3M, PC3M-Pro4, PC3MLN4 were compared using a microarray-based technique. Genomic DNA is cut with a methylation-sensitive enzyme Hpall, followed by linker ligation, polymerase chain reaction (PCR amplification, labeling, hybridization to an array of promoter sequences. Only those parts of the genomic DNA that have unmethylated restriction sites within a few hundred base pairs generate PCR products detectable on an array. Of 2732 promoter sequences on a test array, 504 (18.5% showed differential hybridization between immortalized prostate epithelial, cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, there were eight (CD44, CDKN1A, ESR1, PLAU, RARB, SFN, TNFRSF6, TSPY previously observed in prostate cancer, 13 previously known methylation targets in other cancers (ARHI, bcl-2, BRCA1, CDKN2C, GADD45A, MTAP, PGR, SLC26A4, SPARC, SYK, TJP2, UCHL1, WIT-1. The majority of genes that appear to be both differentially methylated, differentially regulated between prostate epithelial, cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, GG2-1, representing a rich new source of candidate genes used to study the role of DNA methylation in prostate tumors.

  10. Highly efficient site-specific transgenesis in cancer cell lines

    Directory of Open Access Journals (Sweden)

    Michael Iacovos P

    2012-12-01

    Full Text Available Abstract Background Transgenes introduced into cancer cell lines serve as powerful tools for identification of genes involved in cancer. However, the random nature of genomic integration site of a transgene highly influences the fidelity, reliability and level of its expression. In order to alleviate this bottleneck, we characterized the potential utility of a novel PhiC31 integrase-mediated site-specific insertion system (PhiC31-IMSI for introduction of transgenes into a pre-inserted docking site in the genome of cancer cells. Methods According to this system, a “docking-site” was first randomly inserted into human cancer cell lines and clones with a single copy were selected. Subsequently, an “incoming” vector containing the gene of interest was specifically inserted in the docking-site using PhiC31. Results Using the Pc-3 and SKOV-3 cancer cell lines, we showed that transgene insertion is reproducible and reliable. Furthermore, the selection system ensured that all surviving stable transgenic lines harbored the correct integration site. We demonstrated that the expression levels of reporter genes, such as green fluorescent protein and luciferase, from the same locus were comparable among sister, isogenic clones. Using in vivo xenograft studies, we showed that the genetically altered cancer cell lines retain the properties of the parental line. To achieve temporal control of transgene expression, we coupled our insertion strategy with the doxycycline inducible system and demonstrated tight regulation of the expression of the antiangiogenic molecule sFlt-1-Fc in Pc-3 cells. Furthermore, we introduced the luciferase gene into the insertion cassette allowing for possible live imaging of cancer cells in transplantation assays. We also generated a series of Gateway cloning-compatible intermediate cassettes ready for high-throughput cloning of transgenes and demonstrated that PhiC31-IMSI can be achieved in a high throughput 96-well plate

  11. Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

    Science.gov (United States)

    Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

    2016-02-03

    After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts.

  12. Understanding spatial and temporal patterning of astrocyte calcium transients via interactions between network transport and extracellular diffusion

    Science.gov (United States)

    Shtrahman, E.; Maruyama, D.; Olariu, E.; Fink, C. G.; Zochowski, M.

    2017-02-01

    Astrocytes form interconnected networks in the brain and communicate via calcium signaling. We investigate how modes of coupling between astrocytes influence the spatio-temporal patterns of calcium signaling within astrocyte networks and specifically how these network interactions promote coordination within this group of cells. To investigate these complex phenomena, we study reduced cultured networks of astrocytes and neurons. We image the spatial temporal patterns of astrocyte calcium activity and quantify how perturbing the coupling between astrocytes influences astrocyte activity patterns. To gain insight into the pattern formation observed in these cultured networks, we compare the experimentally observed calcium activity patterns to the patterns produced by a reduced computational model, where we represent astrocytes as simple units that integrate input through two mechanisms: gap junction coupling (network transport) and chemical release (extracellular diffusion). We examine the activity patterns in the simulated astrocyte network and their dependence upon these two coupling mechanisms. We find that gap junctions and extracellular chemical release interact in astrocyte networks to modulate the spatiotemporal patterns of their calcium dynamics. We show agreement between the computational and experimental findings, which suggests that the complex global patterns can be understood as a result of simple local coupling mechanisms.

  13. Modulation of Pineal Melatonin Synthesis by Glutamate Involves Paracrine Interactions between Pinealocytes and Astrocytes through NF-κB Activation

    Directory of Open Access Journals (Sweden)

    Darine Villela

    2013-01-01

    Full Text Available The glutamatergic modulation of melatonin synthesis is well known, along with the importance of astrocytes in mediating glutamatergic signaling in the central nervous system. Pinealocytes and astrocytes are the main cell types in the pineal gland. The objective of this work was to investigate the interactions between astrocytes and pinealocytes as a part of the glutamate inhibitory effect on melatonin synthesis. Rat pinealocytes isolated or in coculture with astrocytes were incubated with glutamate in the presence of norepinephrine, and the melatonin content, was quantified. The expression of glutamate receptors, the intracellular calcium content and the NF-κB activation were analyzed in astrocytes and pinealocytes. TNF-α's possible mediation of the effect of glutamate was also investigated. The results showed that glutamate's inhibitory effect on melatonin synthesis involves interactions between astrocytes and pinealocytes, possibly through the release of TNF-α. Moreover, the activation of the astrocytic NF-κB seems to be a necessary step. In astrocytes and pinealocytes, AMPA, NMDA, and group I metabotropic glutamate receptors were observed, as well as the intracellular calcium elevation. In conclusion, there is evidence that the modulation of melatonin synthesis by glutamate involves paracrine interactions between pinealocytes and astrocytes through the activation of the astrocytic NF-κB transcription factor and possibly by subsequent TNF-α release.

  14. Correlation between Twist expression and multidrug resistance of breast cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    Yue-Xi Wang; Xiao-Mei Chen; Jun Yan; Zhi-Ping Li

    2016-01-01

    Objective:To study the correlation between Twist expression and multidrug resistance of breast cancer cell lines. Methods:Human breast cancer cell lines MCF-7, cisplatin-resistant human breast cancer cell lines MCF-7/DDP, doxorubicin-resistant human breast cancer cell lines MCF-7/Adr and taxol-resistant human breast cancer cell lines MCF/PTX were cultured, Twist in human breast cancer cell lines MCF-7 was overexpressed and treated with doxorubicin, and then cell viability and expression levels of EMT marker molecules and related signaling pathway molecules were detected. Results:mRNA contents and protein contents of Twist in drug-resistant breast cancer cell lines MCF-7/DDP, MCF-7/Adr and MCF/PTX were higher than those in MCF-7 cell lines;after doxorubicin treatment, inhibitory rates of cell viability in MCF-7 cell lines were higher than those in MCF-7/Adr and MCF-7/Twist cell lines;E-cadherin expression levels in MCF-7/Adr cell lines and MCF-7/Twist cell lines were lower than those in MCF-7 cell lines, and mRNA contents and protein contents of N-cadherin, Vimentin, TGF-β, Smad, Wnt,β-catenin, TNF-αand NF-kB were higher than those in MCF-7 cell lines. Conclusion:Increased expression of Twist is associated with the occurrence of drug resistance in breast cancer cells.

  15. Neuron to astrocyte communication via cannabinoid receptors is necessary for sustained epileptiform activity in rat hippocampus.

    Directory of Open Access Journals (Sweden)

    Guyllaume Coiret

    Full Text Available Astrocytes are integral functional components of synapses, regulating transmission and plasticity. They have also been implicated in the pathogenesis of epilepsy, although their precise roles have not been comprehensively characterized. Astrocytes integrate activity from neighboring synapses by responding to neuronally released neurotransmitters such as glutamate and ATP. Strong activation of astrocytes mediated by these neurotransmitters can promote seizure-like activity by initiating a positive feedback loop that induces excessive neuronal discharge. Recent work has demonstrated that astrocytes express cannabinoid 1 (CB1 receptors, which are sensitive to endocannabinoids released by nearby pyramidal cells. In this study, we tested whether this mechanism also contributes to epileptiform activity. In a model of 4-aminopyridine induced epileptic-like activity in hippocampal slice cultures, we show that pharmacological blockade of astrocyte CB1 receptors did not modify the initiation, but significantly