Proliferation Of Human Astrocytes Research Articles

Regulation of proliferation rates in human astrocytes by modulating mitochondrial and cell metabolic activity.

Astrocytes contribute to the normal function and maintenance of the health of the central nervous system (CNS). The abnormal behavior of astrocytes can lead to a major alteration in neuronal function contributing to the pathogenesis of several neurological diseases, including Alzheimer's disease (AD). Dysfunctional astrocytes have been linked to the onset and development of overreacting inflammation in CNS tissue which is a major contributing factor to the onset and progression of AD. We hypothesize that by modulating mitochondrial activity and cell metabolism using antioxidants, key characteristics of astrogliosis could be regulated. Mainly, we believe that some antioxidants can help to regulate astrocyte response and promote their return back to a quiescent state. In this work, we explored the use of two antioxidant molecules (A1 and A2) as a way to regulate the metabolic activity, proliferation, and overreactive behavior of glial cells. Human astrocytes were cultured for five days in growth media supplemented with 0, 25, or 50 μM A1 or A2. Changes in DNA levels were used to calculate the doubling time of the cell population. DNA levels were quantified using PicoGreen dsDNA quantitation assay. Astrocytes were stained with JC-1 and fluorescence readings were taken to evaluate mitochondrial activity. Changes in metabolic activity were evaluated using the Vibrant Metabolic Assay and Adenosine Triphosphate (ATP) production. Quantitative assessments demonstrate that increasing levels of A1 significantly reduce human astrocyte proliferation while the addition of A2 at the chosen concentrations does not appear to have a significant effect on cell proliferation. The preliminary data for ATP production and metabolic activity illustrates that A1 appears to decrease cell metabolic activity which may lead to lower cell proliferation. In addition, quantitative analysis of the JC-1 mitochondrial staining suggests that the addition of A1 does not significantly impact mitochondrial membrane potential while increasing levels of A2 enhanced the polarization of the mitochondrial membrane. The modulation of mitochondrial and metabolic activity using antioxidant molecules may be used as a viable strategy to regulate the overreactive proliferative response of human astrocytes during astrogliosis.

The LncRNA H19/miR-1-3p/CCL2 axis modulates lipopolysaccharide (LPS) stimulation-induced normal human astrocyte proliferation and activation

Reactive astrocyte proliferation post SCI (spinal cord injury) leads to the formation of glial scars, thus hindering axon regeneration and SCI repair, during which the activation of astrocytes plays a central role. This study attempted to identify the lncRNA-miRNA-mRNA network which exerts a critical effect on normal human astrocyte (NHA) activation and proliferation during SCI inflammation. Herein, lncRNA H19 expression was increased by LPS in NHAs, and H19 was positively correlated with CCL2. H19 silencing in NHAs significantly attenuated the promoting effects of LPS stimulation on NHA proliferation and activation as manifested by inhibited cell viability and DNA synthesis capacity, reduced NHA activation markers, and reduced inflammatory factor concentrations (CCL2, IL-6, and TNF-α). miR-1-3p directly bound to H19 and the CCL2 3′UTR. miR-1-3p overexpression also attenuated the promoting effects of LPS stimulation on NHA proliferation and activation. H19 relieved miR-1-3p-induced inhibition of CCL2 expression by acting as a ceRNA. The inhibition of miR-1-3p could significantly reverse the effects of H19 silencing on NHA proliferation and activation, suggesting that the H19/miR-1-3p axis regulates the proliferation and activation of NHAs via CCL2. In conclusion, lncRNA H19, miR-1-3p, and CCL2 form a lncRNA-miRNA-mRNA axis that modulates NHA proliferation and activation in vitro.

Adenosine inhibits human astrocyte proliferation independently of adenosine receptor activation.

Brain adenosine concentrations can reach micromolar concentrations in stressful situations such as stroke, neurodegenerative diseases or hypoxic regions of brain tumours. Adenosine can act by receptor-independent mechanism by reversing the reaction catalysed by S-adenosylhomocysteine (SAH) hydrolase, leading to SAH accumulation and inhibition of S-adenosylmethionine (SAM)-dependent methyltransferases. Astrocytes are essential in maintaining brain homeostasis but their pathological activation and uncontrolled proliferation plays a role in neurodegeneration and glioma. Adenosine can affect cell proliferation, but the effect of increased adenosine concentration on proliferation of astrocytes is not clarified and was addressed in present work. Human astrocytes (HA) were treated for 3days with test drugs. Cell proliferation/viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay and by cell counting. Cell death was evaluated by assessing lactate dehydrogenase release and by western blot analysis of αII-Spectrin cleavage. 30µM-Adenosine caused a 40%±3% (p<.05, n=5) reduction in cell proliferation/viability, an effect reversed by 2U/ml-adenosine deaminase, but unchanged in the presence of antagonists of any of the adenosine receptors. Adenosine alone did not induce cell death. 100µM-Homocysteine alone caused 16%±3% (p<.05) decrease in HA proliferation. Combined action of adenosine and homocysteine decreased HA proliferation by 76%±4%, an effect higher (p<.05) than the sum of the effects of adenosine and homocysteine alone (56%±5%). The inhibitory effect of adenosine on HA proliferation/viability was mimicked by two adenosine kinase inhibitors and attenuated in the presence of folate (100µM) or SAM (50-100µM). The results suggest that adenosine reduces HA proliferation by a receptor-independent mechanism probably involving reversal of SAH hydrolase-catalysed reaction.

The inhibitory effect of adenosine on human astrocyte proliferation is absent in glioblastoma cell lines.

Event Abstract Back to Event The inhibitory effect of adenosine on human astrocyte proliferation is absent in glioblastoma cell lines. Helena Marcelino1, 2, Cecilia R. Santos2, Vanda S. Nogueira2, 3, 4, João F. Fonseca-Gomes3, 4, Joana Tomás2, Maria José Diógenes3, 4, Ana M. Sebastião3, 4 and José F. Cascalheira1, 2* 1 Department of Chemistry, Faculty of Sciences, University of Beira Interior, Portugal 2 Center for Research in Health Sciences, Faculty of Health Sciences, University of Beira Interior, Portugal 3 Institute of Pharmacology and Neurosciences, Faculty of Medicine, University of Lisbon, Portugal 4 Institute of Molecular Medicine João Lobo Antunes, Faculty of Medicine, University of Lisbon, Portugal Adenosine has been implicated in the proliferation and survival of various tumour cells, namely glioblastoma cells. Adenosine usually produces its cellular effects in the nervous system by activating G protein-coupled adenosine receptors. Increased adenosine concentrations are found in stressful conditions such as hypoxic regions in brain tumours. However it isn’t known if there any difference between the effects of increased adenosine concentration on cell proliferation in normal astrocytes when compared with malign transformed cells. This issue will be addressed in present work. Human astrocytes (HA), and human glioblastoma cell lines U-87MG, U-373 MG, SNB19, were treated for 3 days with 30µM-Adenosine, with or without the selective adenosine kinase inhibitor 5-idotubercidin (25µM). Cell proliferation was evaluated using MTT assay while cell death was assessed by measuring the released LDH activity and αII-Spectrin cleavage. 30µM-Adenosine produced a 40%±4% (P<0.005) reduction in HA cell proliferation but had no effect in any of the 3 glioblastoma cell lines (p<0.01, when compared with HA). Adenosine alone did not induce cell death, assessed by LDH release and αII-Spectrin cleavage. Curiously, incubation with 5-iodotubercidin+adenosine produced a strong and similar decrease (ranging from 86±5% for HA, to 75%±4% for U87, P<0.01) on cell proliferation in both HA and glioblastoma cell lines. The results show a strong attenuation of the antiproliferative effect of adenosine in glioblastoma cells compared to HA, probably resulting from an increased adenosine kinase activity in glioblastoma cells, suggesting an adaptation of the tumour cells to the antiproliferative effect of adenosine. Acknowledgements The present work was funded by a CENTRO 2020 and Lisboa 2020 (POCI-01-0145-FEDER-016822) and FCT – Fundação para a Ciência e Tecnologia, I.P. (PTDC/BIM¬ONC/7121/2014) research Grant. Keywords: Adenosine, Astrocytes, Cell Proliferation, Glioblastoma, Homocysteine Conference: XVI Meeting of the Portuguese Society for Neuroscience (SPN2019), Lisboa, Portugal, 30 May - 1 Jun, 2019. Presentation Type: Poster presentation Topic: Glia / Neuroinflammation Citation: Marcelino H, Santos CR, Nogueira VS, Fonseca-Gomes JF, Tomás J, Diógenes M, Sebastião AM and Cascalheira JF (2019). The inhibitory effect of adenosine on human astrocyte proliferation is absent in glioblastoma cell lines.. Front. Cell. Neurosci. Conference Abstract: XVI Meeting of the Portuguese Society for Neuroscience (SPN2019). doi: 10.3389/conf.fncel.2019.01.00004 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 16 Apr 2019; Published Online: 27 Sep 2019. * Correspondence: Prof. José F Cascalheira, Department of Chemistry, Faculty of Sciences, University of Beira Interior, Covilhã, Portugal, jfcascalheira@yahoo.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Helena Marcelino Cecilia R Santos Vanda S Nogueira João F Fonseca-Gomes Joana Tomás Maria José Diógenes Ana M Sebastião José F Cascalheira Google Helena Marcelino Cecilia R Santos Vanda S Nogueira João F Fonseca-Gomes Joana Tomás Maria José Diógenes Ana M Sebastião José F Cascalheira Google Scholar Helena Marcelino Cecilia R Santos Vanda S Nogueira João F Fonseca-Gomes Joana Tomás Maria José Diógenes Ana M Sebastião José F Cascalheira PubMed Helena Marcelino Cecilia R Santos Vanda S Nogueira João F Fonseca-Gomes Joana Tomás Maria José Diógenes Ana M Sebastião José F Cascalheira Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

MiR-140/BDNF axis regulates normal human astrocyte proliferation and LPS-induced IL-6 and TNF-α secretion.

Reactive astrocyte proliferation after spinal cord injury (SCI) contributes to glial scar formation that impedes axonal regeneration. The mechanisms underlying astrocyte proliferation upon injury remain partially understood. MicroRNAs (miRNAs) function as a major class of post-transcriptional gene expression regulators that participate in many biological processes. In this study, we focused on the functional role of miR-140 in normal human astrocyte (NHA) cell proliferation. Ectopic miR-140 expression significantly inhibited NHA cell viability and proliferation; miR-140 inhibition exerted the opposite function. Commonly, miRNAs exert functions through targeting downstream genes to inhibit their expression. In the present study, brain-derived neurotrophic factor (BDNF), a regulator of astrocyte proliferation and differentiation, confirmed as a direct target of miR-140 in NHA. Through binding to the 3'UTR of BDNF, miR-140 inhibited BDNF expression. BDNF overexpression significantly promoted NHA cell viability and proliferation; the regulatory effect of miR-140/BDNF on NHA proliferation was mediated by PI3K/AKT pathway. Moreover, we evaluated the functional role of miR-140 in Lipopolysaccharide (LPS)-induced in vitro injury model of astroglial cultures; a significantly up-regulated BDNF, interleukin (IL)-6 and tumor necrosis factor (TNF)-α expression in response to LPS stimulation was observed. After ectopic miR-140 expression, the promotive effect of LPS on BDNF, IL-6 and TGF-α expression was partially restored. Taken together, miR-140/BDNF axis regulates NHA proliferation through PI3K/AKT pathway; miR-140 could inhibit BDNF, IL-6 and TGF-α expression in LPS-induced in vitro injury model. MiR-140/BDNF might serve as a promising target in strategy against reactive astrocyte proliferation after SCI.

Cocaine promotes primary human astrocyte proliferation via JNK-dependent up-regulation of cyclin A2.

Astrocytes perform a plethora of important functions in the central nervous system (CNS) and are involved in cocaine-evoked synaptic plasticity. Previously, we showed that while cocaine decreased cyclin A2 expression in primary human neural progenitor cells, it increased cyclin A2 expression in human astrocytes. Since cyclin A2 is an essential regulator of the cell cycle, the aim of the present study is to clarify the effect of cocaine on proliferation of human astrocytes and elucidate the underlying molecular mechanisms. Primary human astrocytes were treated with either 1, 10, or 100 μM cocaine for 48 hr, and cell proliferation was measured using the CyQUANT cell proliferation assay. To elucidate the molecular mechanisms through which cocaine affects the proliferation of astrocytes, we analyzed gene expression profiles in cocaine-treated primary human astrocytes using a human focused cDNA array. Gene ontology/pathway enrichment analysis, STRING protein-protein interaction analysis, RT-qPCR, and western blotting were used to identify signal transduction pathways that are involved in cocaine-induced astrocyte dysfunction. Cocaine at 10 and 100 μM significantly increased human astrocyte proliferation. Gene expression profiling revealed the JNK MAP kinase pathway as a driver of cell proliferation affected by cocaine in human astrocytes. Further experiments showed that cocaine-induced JNK activation induced up-regulation of cyclin A2, leading to enhanced proliferation of human astrocytes. Cocaine-induced abnormal increases in the number of astrocytes may cause disruption in neuron-glia signaling and contribute to synaptic impairment in the CNS. Understanding the mechanisms of cocaine's effects on human astrocytes may help to reveal the involvement of glial cells in addictive behaviors.

Negative Regulation of Human Astrocytes by Interferon (IFN) α in Relation to Growth Inhibition and Impaired Glucose Utilization

The present study assessed the direct effects of IFNs on human astrocytes. Human astrocytes were exposed to human recombinant IFNs, and the proliferation of cells was measured. Type I IFN receptor mRNA and protein expression, the phosphoprotein levels of signaling molecules including JNK, ERK1/2, IκB, p38MAPK, Stat3, and the expression of cytokines were determined respectively. In addition, cellular glucose consumption was measured as well as Glut-1 protein and activation of GSK-3β/mTOR signal were determined. The expression of Type I IFN receptor was detected in cultured human astrocytes. 2IU/ml IFNα2a and IFNα2b significantly decreased the proliferation of human astrocytes respectively, compared to control. IFNβ had no significant effect on the proliferation of the cells. The phosphorylation of JNK stimulated by all IFNs detected was more pronounced and sustained than ERK1/2 and IκB. No effects were observed on the activation of p38MAPK and Stat3. Moreover, Treatment with IFNα, especially with IFNα2b, decreased glucose consumption and stimulated phosphorylation of GSK-3β and mTOR, but decreased the expression of Glut-1. In contrast, IFNβ had no significant effect on either glucose consumption or activation of GSK-3β/mTOR signals. INFα2b significantly decreased the levels of IL-8 whereas the levels of GM-CSF were increased. The present study demonstrates direct inhibitory effects of IFNα on cell proliferation, cell signaling and glucose utilization in human astrocytes.

Transformation by the (R)-enantiomer of 2-hydroxyglutarate linked to EGLN activation

The identification of succinate dehydrogenase (SDH), fumarate hydratase (FH), and isocitrate dehydrogenase (IDH) mutations in human cancers has rekindled the idea that altered cellular metabolism can transform cells. Inactivating SDH and FH mutations cause the accumulation of succinate and fumarate, respectively, which can inhibit 2-oxoglutarate (2-OG)-dependent enzymes, including the EglN prolyl 4-hydroxylases that mark the HIF transcription factor for polyubiquitylation and proteasomal degradation 1. Inappropriate HIF activation is suspected of contributing to the pathogenesis of SDH-defective and FH-defective tumors but can suppress tumor growth in some other contexts. IDH1 and IDH2, which catalyze the interconversion of isocitrate and 2-OG, are frequently mutated in human brain tumors and leukemias. The resulting mutants display the neomorphic ability to convert 2-OG to the R-enantiomer of 2-hydroxyglutarate (R-2HG) 2, 3. Here we show that R-2HG, but not S-2HG, stimulates EglN activity leading to diminished HIF levels, which enhances the proliferation and soft agar growth of human astrocytes.

Nuclear accumulation of FGF-2 is associated with proliferation of human astrocytes and glioma cells.

FGF-2 has been implicated in the neoplastic transformation of glioma cells and in the transition of normal quiescent astrocytes to a proliferating, reactive state. In the present study we have observed that in human glial cells, levels and subcellular localization of FGF-2 are different in quiescent and proliferating cells. FGF-2 was detected in the cytoplasm of non-reactive astrocytes in human brain sections. In contrast FGF-2 was located within the cytoplasm and nuclei of reactive astrocytes in gliotic brain tissue and in neoplastic cells of glioma tumors. In vitro, FGF-2 was found predominantly in the nucleus of subconfluent proliferating astrocytes, but was detected only in the cytoplasm of density arrested quiescent astrocytes. Our results suggest that reduced cell contact stimulates nuclear accumulation of FGF-2, accompanying mitotic activation of reactive human astrocytes. FGF-2 was constitutively localized to the nucleus of continuously proliferating glioma cells independent of cell density. A role for intracellular FGF-2 was further suggested by the observation that glioma cells that are not stimulated to proliferate by extracellular FGF-2 proliferated faster when transfected with FGF-2 expressing vectors. This increased proliferation correlated with nuclear accumulation of FGF-2. Cell proliferation was attenuated by 5'-deoxy-5'-methylthioadenosine, a FGF-2 receptor tyrosine kinase inhibitor that acts within the cell, but was unaffected by myo-inositol hexakis [dihydrogen phosphate] that disrupts FGF-2 binding to plasma membrane receptors. Our results indicate that FGF-2 serves as a nuclear regulator of proliferation in astrocytic cells. In glioma cells, the constitutive presence of FGF-2 in the nucleus may promote proliferation that is insensitive to cell contact inhibition.

Counteracting effect of IFN-beta on IFN-gamma-induced proliferation of human astrocytes in culture.

Recent clinical trials have shown that interferon beta (IFN-beta) is effective in reducing exacerbations in relapsing-remitting MS, while interferon gamma (IFN-gamma) precipitates the relapses. To investigate mechanisms underlying the beneficial effects of IFN-beta and the detrimental effects of IFN-gamma in MS, cell growth-regulatory effects of IFNs were examined in astrocyte-enriched cultures isolated from fetal brains of 12-20 weeks' gestation. Treatment with IFN-gamma (50 or 500 IU ml-1) stimulated significantly the proliferation of astrocytes in 6 out of 9 culture series examined, while IFN-beta (50 or 500 IU ml-1) inhibited the astrocytic proliferation in 3 out of 9 cultures, and IFN-alpha (50 or 500 IU ml-1) did not affect the proliferation IFN-beta and to a lesser degree IFN-alpha reduced the astrocytic proliferation induced by IFN-gamma-treatment in 8 out of 9 culture series. The counteracting effect of IFN-alpha/IFN-beta against IFN-gamma-induced astrocytic proliferation was verified by the DNA content distribution analysis of propidium iodide-labeled cells. The antagonistic effect of IFN-alpha/IFN-beta on the growth-promoting activity of IFN-gamma in cultured human astrocytes suggests that interferons serve as growth regulators of astrocytes at sites of reactive gliosis lesions of MS.

A new double labelling immunofluorescence technique for the determination of proliferation of human astrocytes in culture

We describe a method that can positively identify proliferating human astrocytes in culture. The procedure is an indirect double immunofluorescence staining technique that uses bromodeoxyuridine together with specific antibodies directed against it and glial fibrillary acidic protein. The method is simple, rapid, reproducible, and promises to decrease many of the limitations inherent in other previously used methods. Using this technique, we observed that mitosis occurred in 57 and 11% of human astrocytes that have been in culture for 11 days and 16 weeks, respectively.