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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2016-10-01

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

Glucose deprivation reversibly down-regulates tissue plasminogen activator via proteasomal degradation in rat primary astrocytes.

PubMed

Cho, Kyu Suk; Joo, So Hyun; Choi, Chang Soon; Kim, Ki Chan; Ko, Hyun Myung; Park, Jin Hee; Kim, Pitna; Hur, Jun; Lee, Sung Hoon; Bahn, Geon Ho; Ryu, Jong Hoon; Lee, Jongmin; Han, Seol-Heui; Kwon, Kyoung Ja; Shin, Chan Young

2013-05-20

Tissue plasminogen activator (tPA) is an essential neuromodulator whose involvement in multiple functions such as synaptic plasticity, cytokine-like immune function and regulation of cell survival mandates rapid and tight tPA regulation in the brain. We investigated the possibility that a transient metabolic challenge induced by glucose deprivation may affect tPA activity in rat primary astrocytes, the main cell type responsible for metabolic regulation in the CNS. Rat primary astrocytes were incubated in serum-free DMEM without glucose. Casein zymography was used to determine tPA activity, and tPA mRNA was measured by RT-PCR. The signaling pathways regulating tPA activity were identified by Western blotting. Glucose deprivation rapidly down-regulated the activity of tPA without affecting its mRNA level in rat primary astrocytes; this effect was mimicked by translational inhibitors. The down-regulation of tPA was accompanied by increased tPA degradation, which may be modulated by a proteasome-dependent degradation pathway. Glucose deprivation induced activation of PI3K-Akt-GSK3β, p38 and AMPK, and inhibition of these pathways using LY294002, SB203580 and compound C significantly inhibited glucose deprivation-induced tPA down-regulation, demonstrating the essential role of these pathways in tPA regulation in glucose-deprived astrocytes. Rapid and reversible regulation of tPA activity in rat primary astrocytes during metabolic crisis may minimize energy-requiring neurologic processes in stressed situations. This effect may thereby increase the opportunity to invest cellular resources in cell survival and may allow rapid re-establishment of normal cellular function after the crisis. Copyright © 2013 Elsevier Inc. All rights reserved.

OVEREXPRESSION OF SERUM RESPONSE FACTOR IN ASTROCYTES IMPROVES NEURONAL PLASTICITY IN A MODEL OF EARLY ALCOHOL EXPOSURE

PubMed Central

PAUL, ARCO P.; MEDINA, ALEXANDRE E.

2012-01-01

Neuronal plasticity deficits underlie many of the cognitive problems seen in Fetal Alcohol Spectrum Disorders (FASD). We have developed a ferret model showing that early alcohol exposure leads to a persistent disruption in ocular dominance (OD) plasticity. Recently, we showed that this deficit could be reversed by overexpression of serum response factor (SRF) in the primary visual cortex during the period of monocular deprivation (MD). Surprisingly, this restoration was observed throughout the extent of visual cortex and most of the cells transfected by the virus were positive for the astrocytic marker GFAP rather than the neuronal marker NeuN. Here we test whether overexpression of SRF exclusively in astrocytes is sufficient to restore OD plasticity in alcohol-exposed ferrets. To accomplish that, first we exposed cultured astrocytes to Sindbis viruses carrying either a constitutively active form of SRF (SRF+), a dominant negative (SRF−) or control GFP. After 24h, these astrocytes were implanted in the visual cortex of alcohol-exposed animals or saline controls one day before MD. Optical imaging of intrinsic signals showed that alcohol-exposed animals that were implanted with astrocytes expressing SRF, but not SRF− or GFP, showed robust restoration of OD plasticity in all visual cortex. These findings suggest that overexpression of SRF exclusively in astrocytes can improve neuronal plasticity in FASD. PMID:22742904

Assessment of mitochondrial electron transport chain function in a primary astrocyte cell model of hyperhomocystinaemia.

PubMed

Turkes, Fiona; Murphy, Elaine; Land, John; Demiray, Berna; Duberley, Kate; Briddon, Antony; Hargreaves, Iain

2013-07-01

Elevated plasma homocysteine (Hcy) has been detected in patients with various neurodegenerative conditions. Studies on neurones and cerebral tissue have revealed that hyperhomocystinaemia may inhibit mitochondrial electron transport chain (ETC) enzyme activity resulting in neuronal morbidity. As astrocytes convey a protective and supportive role towards neurones, we postulated that Hcy-induced astrocytic ETC inhibition may contribute to neurological dysfunction. In order to investigate this hypothesis, we established a cellular model of hyperhomocystinaemia using primary rat astrocytes. Which were incubated were incubated with 200 µM, 500 µM Hcy and the Hcy metabolite, thiolactone (10 µM). Following 96 h of incubation with 200 µM and 500 µM Hcy, an approximate two-fold (1.11 nmol/mg) and three-fold (1.45 nmol/mg) increase in mitochondrial levels of Hcy, respectively, were detected compared to control levels (0.54 nmol/mg). However, on exposure to Hcy (200 or 500 µM) and Hcy-thiolactone (10 µM), the activities of astrocytic ETC complex I, II-III and IV were found to be comparable to control levels. In addition, the extracellular lactate:pyruvate ratio and the intracellular glutathione status of primary rat astrocytes were not significantly different between Hcy (200 or 500 µM) treated and controls. In conclusion, the results of this study suggest that Hcy induced impairment of astrocytic ETC function may not contribute to the pathophysiology of hyperhomocystinaemia.

Methylglyoxal Induces Changes in the Glyoxalase System and Impairs Glutamate Uptake Activity in Primary Astrocytes

PubMed Central

Galland, Fabiana; Lirio, Franciane; de Souza, Daniela Fraga; Da Ré, Carollina; Pacheco, Rafaela Ferreira; Vizuete, Adriana Fernanda; Quincozes-Santos, André; Leite, Marina Concli; Gonçalves, Carlos-Alberto

2017-01-01

The impairment of astrocyte functions is associated with diabetes mellitus and other neurodegenerative diseases. Astrocytes have been proposed to be essential cells for neuroprotection against elevated levels of methylglyoxal (MG), a highly reactive aldehyde derived from the glycolytic pathway. MG exposure impairs primary astrocyte viability, as evaluated by different assays, and these cells respond to MG elevation by increasing glyoxalase 1 activity and glutathione levels, which improve cell viability and survival. However, C6 glioma cells have shown strong signs of resistance against MG, without significant changes in the glyoxalase system. Results for aminoguanidine coincubation support the idea that MG toxicity is mediated by glycation. We found a significant decrease in glutamate uptake by astrocytes, without changes in the expression of the major transporters. Carbenoxolone, a nonspecific inhibitor of gap junctions, prevented the cytotoxicity induced by MG in astrocyte cultures. Thus, our data reinforce the idea that astrocyte viability depends on gap junctions and that the impairment induced by MG involves glutamate excitotoxicity. The astrocyte susceptibility to MG emphasizes the importance of this compound in neurodegenerative diseases, where the neuronal damage induced by MG may be aggravated by the commitment of the cells charged with MG clearance. PMID:28685011

Methylglyoxal Induces Changes in the Glyoxalase System and Impairs Glutamate Uptake Activity in Primary Astrocytes.

PubMed

Hansen, Fernanda; Galland, Fabiana; Lirio, Franciane; de Souza, Daniela Fraga; Da Ré, Carollina; Pacheco, Rafaela Ferreira; Vizuete, Adriana Fernanda; Quincozes-Santos, André; Leite, Marina Concli; Gonçalves, Carlos-Alberto

2017-01-01

The impairment of astrocyte functions is associated with diabetes mellitus and other neurodegenerative diseases. Astrocytes have been proposed to be essential cells for neuroprotection against elevated levels of methylglyoxal (MG), a highly reactive aldehyde derived from the glycolytic pathway. MG exposure impairs primary astrocyte viability, as evaluated by different assays, and these cells respond to MG elevation by increasing glyoxalase 1 activity and glutathione levels, which improve cell viability and survival. However, C6 glioma cells have shown strong signs of resistance against MG, without significant changes in the glyoxalase system. Results for aminoguanidine coincubation support the idea that MG toxicity is mediated by glycation. We found a significant decrease in glutamate uptake by astrocytes, without changes in the expression of the major transporters. Carbenoxolone, a nonspecific inhibitor of gap junctions, prevented the cytotoxicity induced by MG in astrocyte cultures. Thus, our data reinforce the idea that astrocyte viability depends on gap junctions and that the impairment induced by MG involves glutamate excitotoxicity. The astrocyte susceptibility to MG emphasizes the importance of this compound in neurodegenerative diseases, where the neuronal damage induced by MG may be aggravated by the commitment of the cells charged with MG clearance.

Higher Vulnerability of Menadione-Exposed Cortical Astrocytes of Glutaryl-CoA Dehydrogenase Deficient Mice to Oxidative Stress, Mitochondrial Dysfunction, and Cell Death: Implications for the Neurodegeneration in Glutaric Aciduria Type I.

PubMed

Rodrigues, Marília Danyelle Nunes; Seminotti, Bianca; Zanatta, Ângela; de Mello Gonçalves, Aline; Bellaver, Bruna; Amaral, Alexandre Umpierrez; Quincozes-Santos, André; Goodman, Stephen Irwin; Woontner, Michael; Souza, Diogo Onofre; Wajner, Moacir

2017-08-01

Patients affected by glutaric aciduria type I (GA-I) show progressive cortical leukoencephalopathy whose pathogenesis is poorly known. In the present work, we exposed cortical astrocytes of wild-type (Gcdh +/+ ) and glutaryl-CoA dehydrogenase knockout (Gcdh -/- ) mice to the oxidative stress inducer menadione and measured mitochondrial bioenergetics, redox homeostasis, and cell viability. Mitochondrial function (MTT and JC1-mitochondrial membrane potential assays), redox homeostasis (DCFH oxidation, nitrate and nitrite production, GSH concentrations and activities of the antioxidant enzymes SOD and GPx), and cell death (propidium iodide incorporation) were evaluated in primary cortical astrocyte cultures of Gcdh +/+ and Gcdh -/- mice unstimulated and stimulated by menadione. We also measured the pro-inflammatory response (TNFα levels, IL1-β and NF-ƙB) in unstimulated astrocytes obtained from these mice. Gcdh -/- mice astrocytes were more vulnerable to menadione-induced oxidative stress (decreased GSH concentrations and altered activities of the antioxidant enzymes), mitochondrial dysfunction (decrease of MTT reduction and JC1 values), and cell death as compared with Gcdh +/+ astrocytes. A higher inflammatory response (TNFα, IL1-β and NF-ƙB) was also observed in Gcdh -/- mice astrocytes. These data indicate a higher susceptibility of Gcdh -/- cortical astrocytes to oxidative stress and mitochondrial dysfunction, probably leading to cell death. It is presumed that these pathomechanisms may contribute to the cortical leukodystrophy observed in GA-I patients.

Effects of dimethyl sulfoxide on the morphology and viability of primary cultured neurons and astrocytes.

PubMed

Zhang, Chen; Deng, Yuanying; Dai, Hongmei; Zhou, Wenjuan; Tian, Jing; Bing, Guoying; Zhao, Lingling

2017-01-01

Dimethyl sulfoxide (DMSO) is a widely used solvent and vehicle for in vivo and in vitro administration of test compounds. Effects of DMSO independent of the test compound, such as in studies examining morphological plasticity or neurotoxic responses, may lead to spurious results. To investigate effects of DMSO concentration ([DMSO]) on morphology and survival of primary cultured neurons and astrocytes. Primary cultured neurons and astrocytes were treated with 0.25%-10.00% [DMSO] for 12-48h. Viable cell number and morphology were compared to untreated cultures using the CCK-8 assay and phase-contrast microscopy. Expression levels of the neuronal marker NeuN and astrocyte marker glial fibrillary acidic protein (GFAP) were determined by immunofluorescence and western blotting. A [DMSO]≤0.50% had no effect on neuronal number or NeuN expression up to 24h, while ≥1.00% induced a progressive and dramatic loss of both viability and NeuN expression even after 12h. Brief (12h) exposure to ≤1.00% DMSO had no effect on astrocytes survival or GFAP expression, while ≥5.00% significantly reduced both at all exposure durations. In contrast to neurons, exposure to 0.50% and 1.00% DMSO for 24 or 48h enhanced astrocytes proliferation and GFAP expression. Astrocytic processes were maintained at 0.50% and 1.00% DMSO, while neurons exhibited marked neurite retraction at ≥0.50%. A [DMSO]≥0.5% markedly disrupts neuronal morphology and reduces viability, even after brief exposure. In astrocytes, 0.50% and 1.00% DMSO appear to induce reactive gliosis. For treatment of neural cells, [DMSO] should be ≤0.25% to obviate spurious vehicle effects. Copyright © 2016 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2016-01-01

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

Effects of Korean Red Ginseng extract on tissue plasminogen activator and plasminogen activator inhibitor-1 expression in cultured rat primary astrocytes

PubMed Central

Ko, Hyun Myung; Joo, So Hyun; Kim, Pitna; Park, Jin Hee; Kim, Hee Jin; Bahn, Geon Ho; Kim, Hahn Young; Lee, Jongmin; Han, Seol-Heui; Shin, Chan Young; Park, Seung Hwa

2013-01-01

Korean Red Ginseng (KRG) is an oriental herbal preparation obtained from Panax ginseng Meyer (Araliaceae). To expand our understanding of the action of KRG on central nervous system (CNS) function, we examined the effects of KRG on tissue plasminogen activator (tPA)/plasminogen activator inhibitor-1 (PAI-1) expression in rat primary astrocytes. KRG extract was treated in cultured rat primary astrocytes and neuron in a concentration range of 0.1 to 1.0 mg/mL and the expression of functional tPA/PAI-1 was examined by casein zymography, Western blot and reverse transcription-polymerase chain reaction. KRG extracts increased PAI-1 expression in rat primary astrocytes in a concentration dependent manner (0.1 to 1.0 mg/mL) without affecting the expression of tPA itself. Treatment of 1.0 mg/mL KRG increased PAI-1 protein expression in rat primary astrocytes to 319.3±65.9% as compared with control. The increased PAI-1 expression mediated the overall decrease in tPA activity in rat primary astrocytes. Due to the lack of PAI-1 expression in neuron, KRG did not affect tPA activity in neuron. KRG treatment induced a concentration dependent activation of PI3K, p38, ERK1/2, and JNK in rat primary astrocytes and treatment of PI3K or MAPK inhibitors such as LY294002, U0126, SB203580, and SP600125 (10 μM each), significantly inhibited 1.0 mg/mL KRG-induced expression of PAI- 1 and down-regulation of tPA activity in rat primary astrocytes. Furthermore, compound K but not other ginsenosides such as Rb1 and Rg1 induced PAI-1 expression. KRG-induced up-regulation of PAI-1 in astrocytes may play important role in the regulation of overall tPA activity in brain, which might underlie some of the beneficial effects of KRG on CNS such as neuroprotection in ischemia and brain damaging condition as well as prevention or recovery from addiction. PMID:24235858

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

PubMed Central

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

2012-01-01

Despite the widespread use of antiretroviral therapy that effectively limits viral replication, memory impairment remains a dilemma for HIV infected people. In the CNS, HIV infection of astrocytes leads to the production of the HIV-1 Nef protein without viral replication. Post mortem studies have found Nef expression in hippocampal astrocytes of people with HIV associated dementia suggesting that astrocytic Nef may contribute to HIV associated cognitive impairment even when viral replication is suppressed. To test whether astrocytic expression of Nef is sufficient to induce cognitive deficits, we examined the effect of implanting primary rat astrocytes expressing Nef into the hippocampus on spatial and recognition memory. Rats implanted unilaterally with astrocytes expressing Nef showed impaired novel location and novel object recognition in comparison with controls implanted with astrocytes expressing green fluorescent protein (GFP). This impairment was correlated with an increase in chemokine ligand 2 (CCL2) expression and the infiltration of peripheral macrophages into the hippocampus at the site of injection. Furthermore, the Nef exposed rats exhibited a bilateral loss of CA3 neurons. These results suggest that Nef protein expressed by the implanted astrocytes activates the immune system leading to neuronal damage and spatial and recognition memory deficits. Therefore, the continued expression of Nef by astrocytes in the absence of viral replication has the potential to contribute to HIV associated cognitive impairment. PMID:22926191

Neurotoxicity of Methylmercury in Isolated Astrocytes and Neurons: the Cytoskeleton as a Main Target.

PubMed

Pierozan, Paula; Biasibetti, Helena; Schmitz, Felipe; Ávila, Helena; Fernandes, Carolina Gonçalves; Pessoa-Pureur, Regina; Wyse, Angela T S

2017-10-01

In the present work, we focused on mechanisms of methylmercury (MeHg) toxicity in primary astrocytes and neurons of rats. Cortical astrocytes and neurons exposed to 0.5-5 μM MeHg present a link among morphological alterations, glutathione (GSH) depletion, glutamate dyshomeostasis, and cell death. Disrupted neuronal cytoskeleton was assessed by decreased neurite length and neurite/neuron ratio. Astrocytes presented reorganization of actin and glial fibrillary acidic protein (GFAP) networks and reduced cytoplasmic area. Glutamate uptake and Na + K + ATPase activity in MeHg-treated astrocytes were preserved; however, downregulated EAAC1-mediated glutamate uptake was associated with impaired Na + K + ATPase activity in neurons. Oxidative imbalance was found in astrocytes and neurons through increased 2'7'-dichlorofluorescein (DCF) production and misregulated superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GPX) activities. Glutathione (GSH) levels were downregulated in both astrocytes and neurons. MeHg reduced neuronal viability and induced caspase 3-dependent apoptosis together with downregulated PI3K/Akt pathway. In astrocytes, necrotic death was associated with increased TNF-α and JNK/MAPK activities. Cytoskeletal remodeling and cell death were fully prevented in astrocytes and neurons by GSH, but not melatonin or Trolox supplementation. These findings support a role for depleted GSH in the cytotoxicity of MeHg leading to disruption of the cytoskeleton and cell death. Moreover, in neurons, glutamate antagonists also prevented cytoskeletal disruption and neuronal death. We propose that cytoskeleton is an end point in MeHg cytotoxicity. Oxidative imbalance and glutamate mechanisms mediate MeHg cytoskeletal disruption and apoptosis in neurons. Otherwise, redox imbalance and glutamate-independent mechanisms disrupted the cytoskeleton and induced necrosis in MeHg-exposed astrocyte.

Cortical astrocytes exposed to tributyltin undergo morphological changes in vitro.

PubMed

Mizuhashi, S; Ikegaya, Y; Nishiyama, N; Matsuki, N

2000-11-01

We investigated the effect of tributyltin (TBT), an endocrine-disrupting chemical, on the morphology and viability of cultured rat cortical astrocytes. Cultured astrocytes exhibited smooth and planiform morphology under normal conditions. Following exposure to TBT, however, they showed rapid morphological changes that are characterized by asteriated cell bodies and process formation in a time- and concentration-dependent manner. Higher concentrations of TBT produced progressive cell death of the astrocytes. In serum-free medium, TBT at a concentration as low as 200 nM induced the stellation. Pharmacological studies revealed that the morphological changes were alleviated by application of diverse free radical scavengers or antioxidants such as catalase, superoxide dismutase, Trolox, ascorbic acid and N-acetyl-L-cysteine, suggesting that TBT-induced stellation is caused by oxidative stress involving free radicals, particularly reactive oxygen species. Furthermore, we found that the astrocyte stellation was abolished by treatment with inhibitors of phospholipase C, mitogen-activated protein kinase kinase or tyrosine phosphatase. The data suggest that TBT causes the stellation through intracellular signaling cascades rather than its non-specific toxicity. These findings provide an important insight for reconciling the problems in assumed aversive actions of this environmental pollutant for mammals.

Huperzine A, but not tacrine, stimulates S100B secretion in astrocyte cultures.

PubMed

Lunardi, Paula; Nardin, Patrícia; Guerra, Maria Cristina; Abib, Renata; Leite, Marina Concli; Gonçalves, Carlos-Alberto

2013-04-09

The loss of cholinergic function in the central nervous system contributes significantly to the cognitive decline associated with advanced age and dementias. Huperzine A (HupA) is a selective inhibitor of acetylcholinesterase (AChE) and has been shown to significantly reduce cognitive impairment in animal models of dementia. Based on the importance of astrocytes in physiological and pathological brain activities, we investigated the effect of HupA and tacrine on S100B secretion in primary astrocyte cultures. S100B is an astrocyte-derived protein that has been proposed to be a marker of brain injury. Primary astrocyte cultures were exposed to HupA, tacrine, cholinergic agonists, and S100B secretion was measured by enzyme-linked immunosorbent assay (ELISA) at 1 and 24h. HupA, but not tacrine, at 100μM significantly increased S100B secretion in astrocyte cultures. Nicotine (at 100 and 1000μM) was able to stimulate S100B secretion in astrocyte cultures. Our data reinforce the idea that AChE inhibitors, particularly HupA, do not act exclusively on the acetylcholine balance. This effect of HupA could contribute to improve the cognitive deficit observed in patients, which are attributed to cholinergic dysfunction. In addition, for the first time, to our knowledge, these data indicate that S100B secretion can be modulated by nicotinic receptors, in addition to glutamate, dopamine and serotonin receptors. Copyright © 2013 Elsevier Inc. All rights reserved.

Leptin inhibits amyloid β-protein degradation through decrease of neprilysin expression in primary cultured astrocytes.

PubMed

Yamamoto, Naoki; Tanida, Mamoru; Ono, Yoko; Kasahara, Rika; Fujii, Yuko; Ohora, Kentaro; Suzuki, Kenji; Sobue, Kazuya

2014-02-28

Pathogenesis of Alzheimer's disease (AD) is characterized by accumulation of extracellular deposits of amyloid β-protein (Aβ) in the brain. The steady state level of Aβ in the brain is determined by the balance between its production and removal; the latter occurring through egress across blood and CSF barriers as well as Aβ degradation. The major Aβ-degrading enzymes in the brain are neprilysin (NEP) and insulin-degrading enzyme (IDE), which may promote Aβ deposition in patients with sporadic late-onset AD. Epidemiological studies have suggested an inverse relationship between the adipocytokine leptin levels and the onset of AD. However, the mechanisms underlying the relationship remain uncertain. We investigated whether leptin is associated with Aβ degradation by inducing NEP and IDE expression within primary cultured astrocytes. Leptin significantly decreased the expression of NEP but not IDE in a concentration- and time-dependent manner through the activation of extracellular signal-regulated kinase (ERK) in cultured rat astrocytes. Furthermore, leptin inhibited the degradation of exogenous Aβ in primary cultured astrocytes. These results suggest that leptin suppresses Aβ degradation by NEP through activation of ERK. Copyright © 2014 Elsevier Inc. All rights reserved.

Furosemide- and bumetanide-sensitive ion transport and volume control in primary astrocyte cultures from rat brain.

PubMed

Kimelberg, H K; Frangakis, M V

1985-12-30

K+ and Cl- transport using 42K+ and 36Cl- was studied in primary astrocyte cultures prepared from neonatal rat brains. A component of 42K+ uptake was sensitive to both furosemide and bumetanide with maximum inhibition being obtained at 1 and 0.01 mM concentrations of the inhibitors, respectively. Furosemide and bumetanide also markedly inhibited uptake of 36Cl-. 42K+ uptake in the presence of ouabain was also sensitive to the omission of medium Na+ and Cl-. These results suggest the existence of a K+ + Na+ + Cl- cotransport system in astrocyte cultures which in many cells has been shown to be involved in volume regulation. We studied volume changes using uptake of [14C]3-O- methyl-D-glucose ([14C]3-OMG), and also ion transport, in attached cells in response to exposure to hyper- or hypotonic medium. Exposure to medium made hypertonic with mannitol resulted in shrinkage of the [14C]3-OMG space of the cells, but did not affect 36Cl- content, expressed as nmol/mg protein. Exposure to hypotonic medium led to a marked increase in the [14C]3-OMG space, rapidly followed by a decrease towards control values. After the cells were then exposed to isotonic medium there was an immediate decrease followed by a slower increase in the [14C]3-OMG space. The increase in the [14C]3-OMG space was partially inhibited by 1 mM furosemide.(ABSTRACT TRUNCATED AT 250 WORDS)

3D Electrospun scaffolds promote a cytotrophic phenotype of cultured primary astrocytes.

PubMed

Lau, Chew L; Kovacevic, Michelle; Tingleff, Tine S; Forsythe, John S; Cate, Holly S; Merlo, Daniel; Cederfur, Cecilia; Maclean, Francesca L; Parish, Clare L; Horne, Malcolm K; Nisbet, David R; Beart, Philip M

2014-07-01

Astrocytes are a target for regenerative neurobiology because in brain injury their phenotype arbitrates brain integrity, neuronal death and subsequent repair and reconstruction. We explored the ability of 3D scaffolds to direct astrocytes into phenotypes with the potential to support neuronal survival. Poly-ε-caprolactone scaffolds were electrospun with random and aligned fibre orientations on which murine astrocytes were sub-cultured and analysed at 4 and 12 DIV. Astrocytes survived, proliferated and migrated into scaffolds adopting 3D morphologies, mimicking in vivo stellated phenotypes. Cells on random poly-ε-caprolactone scaffolds grew as circular colonies extending processes deep within sub-micron fibres, whereas astrocytes on aligned scaffolds exhibited rectangular colonies with processes following not only the direction of fibre alignment but also penetrating the scaffold. Cell viability was maintained over 12 DIV, and cytochemistry for F-/G-actin showed fewer stress fibres on bioscaffolds relative to 2D astrocytes. Reduced cytoskeletal stress was confirmed by the decreased expression of glial fibrillary acidic protein. PCR demonstrated up-regulation of genes (excitatory amino acid transporter 2, brain-derived neurotrophic factor and anti-oxidant) reflecting healthy biologies of mature astrocytes in our extended culture protocol. This study illustrates the therapeutic potential of bioengineering strategies using 3D electrospun scaffolds which direct astrocytes into phenotypes supporting brain repair. Astrocytes exist in phenotypes with pro-survival and destructive components, and their biology can be modulated by changing phenotype. Our findings demonstrate murine astrocytes adopt a healthy phenotype when cultured in 3D. Astrocytes proliferate and extend into poly-ε-caprolactone scaffolds displaying 3D stellated morphologies with reduced GFAP expression and actin stress fibres, plus a cytotrophic gene profile. Bioengineered 3D scaffolds have potential

Cortical Astrocytes Acutely Exposed to the Monomethylarsonous Acid (MMAIII) Show Increased Pro-inflammatory Cytokines Gene Expression that is Consistent with APP and BACE-1: Over-expression.

PubMed

Escudero-Lourdes, C; Uresti-Rivera, E E; Oliva-González, C; Torres-Ramos, M A; Aguirre-Bañuelos, P; Gandolfi, A J

2016-10-01

Long-term exposure to inorganic arsenic (iAs) through drinking water has been associated with cognitive impairment in children and adults; however, the related pathogenic mechanisms have not been completely described. Increased or chronic inflammation in the brain is linked to impaired cognition and neurodegeneration; iAs induces strong inflammatory responses in several cells, but this effect has been poorly evaluated in central nervous system (CNS) cells. Because astrocytes are the most abundant cells in the CNS and play a critical role in brain homeostasis, including regulation of the inflammatory response, any functional impairment in them can be deleterious for the brain. We propose that iAs could induce cognitive impairment through inflammatory response activation in astrocytes. In the present work, rat cortical astrocytes were acutely exposed in vitro to the monomethylated metabolite of iAs (MMA III ), which accumulates in glial cells without compromising cell viability. MMA III LD 50 in astrocytes was 10.52 μM, however, exposure to sub-toxic MMA III concentrations (50-1000 nM) significantly increased IL-1β, IL-6, TNF-α, COX-2, and MIF-1 gene expression. These effects were consistent with amyloid precursor protein (APP) and β-secretase (BACE-1) increased gene expression, mainly for those MMA III concentrations that also induced TNF-α over-expression. Other effects of MMA III on cortical astrocytes included increased proliferative and metabolic activity. All tested MMA III concentrations led to an inhibition of intracellular lactate dehydrogenase (LDH) activity. Results suggest that MMA III induces important metabolic and functional changes in astrocytes that may affect brain homeostasis and that inflammation may play a major role in cognitive impairment-related pathogenicity in As-exposed populations.

Neurotensin and neuromedin N undergo distinct catabolic processes in murine astrocytes and primary cultured neurons.

PubMed

Vincent, B; Vincent, J P; Checler, F

1994-04-01

We examined the occurrence of various endopeptidases and exopeptidases and their subcellular partition within soluble and membrane-associated compartments of 15-day-old astrocytes and 4-day-old primary cultured neurons. Peptidases were monitored with chromogenic or fluorimetric substrates and identified by means of specific inhibitors. We assessed the contribution of these peptidases in the catabolism of two related neuropeptides, neurotensin and neuromedin N. Metabolites were separated by HPLC and the identity of the proteolytic activities involved in their formation was established using specific inhibitors. Neuromedin N and neurotensin undergo both quantitative and qualitative differential proteolysis. Initial maximal rates of neuromedin N degradation were higher than those of neurotensin in both cell types. Furthermore, the two peptides were inactivated much more rapidly by the soluble than by the membrane-associated fractions prepared from both cell cultures. Neuromedin N was rapidly broken down by an aminopeptidase M/leucine aminopeptidase attack, leading to the functionally silent Des-Lys1-neuromedin N metabolite. In the astrocytic membrane-associated fraction, neuromedin N underwent an additional minor endoproteolytic cleavage at the Pro3-Tyr4 bond elicited by endopeptidase 24.11, as suggested by the protective effect of its blocking agent phosphoramidon. Unlike neuromedin N, neurotensin totally resisted hydrolysis by aminopeptidases. Primary inactivating cleavages detected in both cell types appeared mainly located at the Arg8-Arg9 and Pro10-Tyr11 bonds, leading to the formations of neurotensin-(1-8) and neurotensin-(1-10) as the major biologically inactive neurotensin catabolites. Endopeptidase 24.15 appeared mainly responsible for neurotensin-(1-8) formation by the soluble fraction of neurons and astrocytes. In contrast, endopeptidase 24.16 was involved in neurotensin-(1-10) formation by both soluble and membrane-associated fractions of the two cell

Obesity induces functional astrocytic leptin receptors in hypothalamus

PubMed Central

Hsuchou, Hung; He, Yi; Kastin, Abba J.; Tu, Hong; Markadakis, Emily N.; Rogers, Richard C.; Fossier, Paul B.

2009-01-01

The possible role of astrocytes in the regulation of feeding has been overlooked. It is well-established that the endothelial cells constituting the blood–brain barrier transport leptin from blood to brain and that hypothalamic neurons respond to leptin to induce anorexic signaling. However, few studies have addressed the role of astrocytes in either leptin transport or cellular activation. We recently showed that the obese agouti viable yellow mouse has prominent astrocytic expression of the leptin receptor. In this study, we test the hypothesis that diet-induced obesity increases astrocytic leptin receptor expression and function in the hypothalamus. Double-labelling immunohistochemistry and confocal microscopic analysis showed that all astrocytes in the hypothalamus express leptin receptors. In adult obese mice, 2 months after being placed on a high-fat diet, there was a striking increase of leptin receptor (+) astrocytes, most prominent in the dorsomedial hypothalamus and arcuate nucleus. Agouti viable yellow mice with their adult-onset obesity showed similar changes, but the increase of leptin receptor (+) astrocytes was barely seen in ob/ob or db/db mice with their early-onset obesity and defective leptin systems. The marked leptin receptor protein expression in the astrocytes, shown with several antibodies against different receptor epitopes, was supported by RT–PCR detection of leptin receptor-a and -b mRNAs in primary hypothalamic astrocytes. Unexpectedly, the protein expression of GFAP, a marker of astrocytes, was also increased in adult-onset obesity. Real-time confocal imaging showed that leptin caused a robust increase of calcium signalling in primary astrocytes from the hypothalamus, confirming their functionality. The results indicate that metabolic changes in obese mice can rapidly alter leptin receptor expression and astrocytic activity, and that leptin receptor is responsible for leptin-induced calcium signalling in astrocytes. This novel and

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

PubMed Central

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

2015-01-01

Astrocytes outnumber neurons and serve many metabolic and trophic functions in the mammalian brain. Preserving astrocytes is critical for normal brain function as well as for protecting the brain against various insults. Our previous studies have indicated that methylene blue (MB) functions as an alternative electron carrier and enhances brain metabolism. In addition, MB has been shown to be protective against neurodegeneration and brain injury. In the current study, we investigated the protective role of MB in astrocytes. Cell viability assays showed that MB treatment significantly protected primary astrocytes from oxygen-glucose deprivation (OGD) & reoxygenation induced cell death. We also studied the effect of MB on cellular oxygen and glucose metabolism in primary astrocytes following OGD-reoxygenation injury. MB treatment significantly increased cellular oxygen consumption, glucose uptake and ATP production in primary astrocytes. In conclusion our study demonstrated that MB protects astrocytes against OGD-reoxygenation injury by improving astrocyte cellular respiration. PMID:25848957

The antidiabetic drug metformin decreases mitochondrial respiration and tricarboxylic acid cycle activity in cultured primary rat astrocytes.

PubMed

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

2017-11-01

Metformin is an antidiabetic drug that is used daily by millions of patients worldwide. Metformin is able to cross the blood-brain barrier and has recently been shown to increase glucose consumption and lactate release in cultured astrocytes. However, potential effects of metformin on mitochondrial tricarboxylic acid (TCA) cycle metabolism in astrocytes are unknown. We investigated this by mapping 13 C labeling in TCA cycle intermediates and corresponding amino acids after incubation of primary rat astrocytes with [U- 13 C]glucose. The presence of metformin did not compromise the viability of cultured astrocytes during 4 hr of incubation, but almost doubled cellular glucose consumption and lactate release. Compared with control cells, the presence of metformin dramatically lowered the molecular 13 C carbon labeling (MCL) of the cellular TCA cycle intermediates citrate, α-ketoglutarate, succinate, fumarate, and malate, as well as the MCL of the TCA cycle intermediate-derived amino acids glutamate, glutamine, and aspartate. In addition to the total molecular 13 C labeling, analysis of the individual isotopomers of TCA cycle intermediates confirmed a severe decline in labeling and a significant lowering in TCA cycling ratio in metformin-treated astrocytes. Finally, the oxygen consumption of mitochondria isolated from metformin-treated astrocytes was drastically reduced in the presence of complex I substrates, but not of complex II substrates. These data demonstrate that exposure to metformin strongly impairs complex I-mediated mitochondrial respiration in astrocytes, which is likely to cause the observed decrease in labeling of mitochondrial TCA cycle intermediates and the stimulation of glycolytic lactate production. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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

PubMed

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

2013-11-01

An increasing amount of evidence has emerged to suggest that neuroinflammatory process is involved in the pathogenesis of Parkinson's disease (PD). Activated microglia and astrocytes are found in the substantia nigra (SN) of Parkinson's disease brains as well as in animal models of Parkinson's disease. Although reactive astrocytes are involved in the progression of PD, the role of reactive astrocytes in neuroinflammation of PD has received limited attention to date. Recently, Glycogen synthase kinase-3β (GSK-3β) was identified as a crucial regulator of the inflammatory response. The purpose of this study was to explore the mechanism by which 6-hydroxydopamine (6-OHDA) induces inflammatory response in astrocytes and observe the anti-inflammatory effect of lithium chloride (LiCl) on 6-OHDA-treated astrocytes. In the present study, we found that glial fibrillary acidic protein (GFAP) was markedly upregulated in the presence of 6-OHDA. Moreover, our results revealed that proinflammatory molecules including inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase-2(COX-2), prostaglandins E2 (PGE2), and tumor necrosis factor-α (TNF-α) were obviously increased in astrocytes exposed to 6-OHDA. Western blot analysis revealed that 6-OHDA significantly increased dephosphorylation/activation of GSK-3β as well as the nuclear translocation of nuclear factor-κB (NF-κB) p65. Besides, GSK-3β inhibitor LiCl and SB415286 inhibited the GSK-3β/NF-κB signaling pathway, leading to the reduction of proinflammatory molecules in 6-OHDA-activated astrocytes. These results confirmed that GSK-3β inhibitor LiCl and SB415286 provide protection against neuroinflammation in 6-OHDA-treated astrocytes. Therefore, GSK-3β may be a potential therapeutic target for the treatment of PD. Copyright © 2013. Published by Elsevier Ltd.

Toll-Like Receptor 4 (TLR4) and Triggering Receptor Expressed on Myeloid Cells-2 (TREM-2) Activation Balance Astrocyte Polarization into a Proinflammatory Phenotype.

PubMed

Rosciszewski, Gerardo; Cadena, Vanesa; Murta, Veronica; Lukin, Jeronimo; Villarreal, Alejandro; Roger, Thierry; Ramos, Alberto Javier

2018-05-01

Astrocytes react to brain injury with a generic response known as reactive gliosis, which involves activation of multiple intracellular pathways including several that may be beneficial for neuronal survival. However, by unknown mechanisms, reactive astrocytes can polarize into a proinflammatory phenotype that induces neurodegeneration. In order to study reactive gliosis and astroglial polarization into a proinflammatory phenotype, we used cortical devascularization-induced brain ischemia in Wistar rats and primary astroglial cell cultures exposed to oxygen-glucose deprivation (OGD). We analyzed the profile of TLR4 expression and the consequences of its activation by gain- and loss-of-function studies, and the effects produced by the activation of triggering receptor expressed on myeloid cells-2 (TREM-2), a negative regulator of TLR4 signaling. Both OGD exposure on primary astroglial cell cultures and cortical devascularization brain ischemia in rats induced TLR4 expression in astrocytes. In vivo, astroglial TLR4 expression was specifically observed in the ischemic penumbra surrounding necrotic core. Functional studies showed that OGD increased the astroglial response to the TLR4 agonist lipopolysaccharide (LPS), and conversely, TLR4 knockout primary astrocytes had impaired nuclear factor kappa-B (NF-κB) activation when exposed to LPS. In gain-of-function studies, plasmid-mediated TLR4 over-expression exacerbated astroglial response to LPS as shown by sustained NF-κB activation and increased expression of proinflammatory cytokines IL-1β and TNFα. TREM-2 expression, although present in naïve primary astrocytes, was induced by OGD, LPS, or high-mobility group box 1 protein (HMGB-1) exposure. TREM-2 activation by antibody cross-linking or the overexpression of TREM-2 intracellular adaptor, DAP12, partially suppressed LPS-induced NF-κB activation in purified astrocytic cultures. In vivo, TREM-2 expression was observed in macrophages and astrocytes located in the

Wildtype motoneurons, ALS-Linked SOD1 mutation and glutamate profoundly modify astrocyte metabolism and lactate shuttling.

PubMed

Madji Hounoum, Blandine; Mavel, Sylvie; Coque, Emmanuelle; Patin, Franck; Vourc'h, Patrick; Marouillat, Sylviane; Nadal-Desbarats, Lydie; Emond, Patrick; Corcia, Philippe; Andres, Christian R; Raoul, Cédric; Blasco, Hélène

2017-04-01

The selective degeneration of motoneuron that typifies amyotrophic lateral sclerosis (ALS) implicates non-cell-autonomous effects of astrocytes. However, mechanisms underlying astrocyte-mediated neurotoxicity remain largely unknown. According to the determinant role of astrocyte metabolism in supporting neuronal function, we propose to explore the metabolic status of astrocytes exposed to ALS-associated conditions. We found a significant metabolic dysregulation including purine, pyrimidine, lysine, and glycerophospholipid metabolism pathways in astrocytes expressing an ALS-causing mutated superoxide dismutase-1 (SOD1) when co-cultured with motoneurons. SOD1 astrocytes exposed to glutamate revealed a significant modification of the astrocyte metabolic fingerprint. More importantly, we observed that SOD1 mutation and glutamate impact the cellular shuttling of lactate between astrocytes and motoneurons with a decreased in extra- and intra-cellular lactate levels in astrocytes. Based on the emergent strategy of metabolomics, this work provides novel insight for understanding metabolic dysfunction of astrocytes in ALS conditions and opens the perspective of therapeutics targets through focusing on these metabolic pathways. GLIA 2017 GLIA 2017;65:592-605. © 2017 Wiley Periodicals, Inc.

The Synaptic Cell Adhesion Molecule, SynCAM1, Mediates Astrocyte-to-Astrocyte and Astrocyte-to-GnRH Neuron Adhesiveness in the Mouse Hypothalamus

PubMed Central

Sandau, Ursula S.; Mungenast, Alison E.; McCarthy, Jack; Biederer, Thomas; Corfas, Gabriel

2011-01-01

We previously identified synaptic cell adhesion molecule 1 (SynCAM1) as a component of a genetic network involved in the hypothalamic control of female puberty. Although it is well established that SynCAM1 is a synaptic adhesion molecule, its contribution to hypothalamic function is unknown. Here we show that, in addition to the expected neuronal localization illustrated by its presence in GnRH neurons, SynCAM1 is expressed in hypothalamic astrocytes. Cell adhesion assays indicated that SynCAM is recognized by both GnRH neurons and astrocytes as an adhesive partner and promotes cell-cell adhesiveness via homophilic, extracellular domain-mediated interactions. Alternative splicing of the SynCAM1 primary mRNA transcript yields four mRNAs encoding membrane-spanning SynCAM1 isoforms. Variants 1 and 4 are predicted to be both N and O glycosylated. Hypothalamic astrocytes and GnRH-producing GT1-7 cells express mainly isoform 4 mRNA, and sequential N- and O-deglycosylation of proteins extracted from these cells yields progressively smaller SynCAM1 species, indicating that isoform 4 is the predominant SynCAM1 variant expressed in astrocytes and GT1-7 cells. Neither cell type expresses the products of two other SynCAM genes (SynCAM2 and SynCAM3), suggesting that SynCAM-mediated astrocyte-astrocyte and astrocyte-GnRH neuron adhesiveness is mostly mediated by SynCAM1 homophilic interactions. When erbB4 receptor function is disrupted in astrocytes, via transgenic expression of a dominant-negative erbB4 receptor form, SynCAM1-mediated adhesiveness is severely compromised. Conversely, SynCAM1 adhesive behavior is rapidly, but transiently, enhanced in astrocytes by ligand-dependent activation of erbB4 receptors, suggesting that erbB4-mediated events affecting SynCAM1 function contribute to regulate astrocyte adhesive communication. PMID:21486931

1H NMR-based metabolic profiling reveals the effects of fluoxetine on lipid and amino acid metabolism in astrocytes.

PubMed

Bai, Shunjie; Zhou, Chanjuan; Cheng, Pengfei; Fu, Yuying; Fang, Liang; Huang, Wen; Yu, Jia; Shao, Weihua; Wang, Xinfa; Liu, Meiling; Zhou, Jingjing; Xie, Peng

2015-04-15

Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), is a prescribed and effective antidepressant and generally used for the treatment of depression. Previous studies have revealed that the antidepressant mechanism of fluoxetine was related to astrocytes. However, the therapeutic mechanism underlying its mode of action in astrocytes remains largely unclear. In this study, primary astrocytes were exposed to 10 µM fluoxetine; 24 h post-treatment, a high-resolution proton nuclear magnetic resonance (1H NMR)-based metabolomic approach coupled with multivariate statistical analysis was used to characterize the metabolic variations of intracellular metabolites. The orthogonal partial least-squares discriminant analysis (OPLS-DA) score plots of the spectra demonstrated that the fluoxetine-treated astrocytes were significantly distinguished from the untreated controls. In total, 17 differential metabolites were identified to discriminate the two groups. These key metabolites were mainly involved in lipids, lipid metabolism-related molecules and amino acids. This is the first study to indicate that fluoxetine may exert antidepressant action by regulating the astrocyte's lipid and amino acid metabolism. These findings should aid our understanding of the biological mechanisms underlying fluoxetine therapy.

Astrocytes expressing ALS‐linked mutant FUS induce motor neuron death through release of tumor necrosis factor‐alpha

PubMed Central

Kia, Azadeh; McAvoy, Kevin; Krishnamurthy, Karthik; Trotti, Davide

2018-01-01

Mutations in fused in sarcoma (FUS) are linked to amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease affecting both upper and lower motor neurons. While it is established that astrocytes contribute to the death of motor neurons in ALS, the specific contribution of mutant FUS (mutFUS) through astrocytes has not yet been studied. Here, we used primary astrocytes expressing a N‐terminally GFP tagged R521G mutant or wild‐type FUS (WTFUS) and show that mutFUS‐expressing astrocytes undergo astrogliosis, damage co‐cultured motor neurons via activation of an inflammatory response and produce conditioned medium (ACM) that is toxic to motor neurons in isolation. Time lapse imaging shows that motor neuron cultures exposed to mutFUS ACM, but not WTFUS ACM, undergo significant cell loss, which is preceded by progressive degeneration of neurites. We found that Tumor Necrosis Factor‐Alpha (TNFα) is secreted into ACM of mutFUS‐expressing astrocytes. Accordingly, mutFUS astrocyte‐mediated motor neuron toxicity is blocked by targeting soluble TNFα with neutralizing antibodies. We also found that mutant astrocytes trigger changes to motor neuron AMPA receptors (AMPAR) that render them susceptible to excitotoxicity and AMPAR‐mediated cell death. Our data provide the first evidence of astrocytic involvement in FUS‐ALS, identify TNFα as a mediator of this toxicity, and provide several potential therapeutic targets to protect motor neurons in FUS‐linked ALS. PMID:29380416

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

PubMed

Oyanagi, Koshi; Tashiro, Tomoko; Negishi, Takayuki

2015-08-01

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

Aniracetam attenuates apoptosis of astrocytes subjected to simulated ischemia in vitro.

PubMed

Gabryel, Bozena; Adamczyk, Jakub; Huzarska, Małgorzata; Pudełko, Anna; Trzeciak, Henryk I

2002-09-01

The aim of the present study was to establish whether aniracetam is capable of protecting cultured rat astrocytes against ischemic injury. Treatment of the cultures with aniracetam (1, 10 and 100 mM) during 24 h ischemia simulated in vitro significantly decreased the number of apoptotic cells. The antiapoptotic effects of the drug were confirmed by the increase of intracellular ATP and phosphocreatine (PCr) levels and the inhibition of the caspase-3 activity. Aniracetam also attenuated cellular oxidative stress by decreased production of reactive oxygen species (ROS). These effects were associated with the decrease in levels of c-fos and c-jun mRNA in primary astrocyte cultures exposed to 24 h ischemia. When cultured astrocytes were incubated during 24 h simulated ischemia with wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor or PD98059, a mitogen-activated protein (MAP)/extracellular signal regulated kinase (ERK) (MEK) inhibitor the cell apoptosis was accelerated. This effect was antagonized by adding 100 mM aniracetam to the culture medium. These findings suggest that the protective effect of aniracetam is mediated by PI 3-kinase and MEK pathways in the downstream mechanisms.

HIV-1-associated inflammation and antiretroviral therapy regulate astrocyte endoplasmic reticulum stress responses.

PubMed

Nooka, Shruthi; Ghorpade, Anuja

2017-01-01

Antiretroviral (ARV) therapy (ART) has effectively suppressed the incidence of human immunodeficiency virus (HIV)-associated dementia in HIV-1 positive individuals. However, the prevalence of more subtle forms of neurocognitive dysfunction continues to escalate. Recently, endoplasmic reticulum (ER) stress has been linked to many neurological diseases; yet, its role in HIV/neuroAIDS remains largely unexplored. Furthermore, upregulation of astrocyte elevated gene-1 ( AEG-1 ), a novel HIV-1 inducible gene, along with ER stress markers in a Huntington's disease model, suggests a possible role in HIV-associated ER stress. The current study is focused on unfolded protein responses (UPRs) and AEG-1 regulation in primary human astrocytes exposed to HIV-associated neurocognitive disorders (HAND)-relevant stimuli (HIV-1 virions, inflammation and ARV drugs). Interleukin (IL)-1 β and the nucleoside reverse transcriptase inhibitor abacavir upregulated expression of ER stress markers in human astrocytes, including binding immunoglobulin protein (BiP), C/EBP homologous protein (CHOP), and calnexin. In addition, IL-1 β activated all three well-known UPR pathways: protein kinase RNA-like ER kinase (PERK); activating transcription factor 6 (ATF-6); and inositol-requiring enzyme 1 α (IRE1 α ). AEG-1 upregulation correlated to ER stress and demonstrated astrocyte AEG-1 interaction with the calcium-binding chaperone, calnexin. IL-1 β and abacavir enhanced intracellular calcium signaling in astrocytes in the absence of extracellular calcium, illustrating ER-associated calcium release. Alternatively, calcium evoked in response to HAND-relevant stimuli led to mitochondrial permeability transition pore (mPTP) opening in human astrocytes. Importantly, IL-1 β - and abacavir-induced UPR and mPTP opening were inhibited by the intracellular calcium chelation, indicating the critical role of calcium signaling in HAND-relevant ER stress in astrocytes. In summary, our study highlights that

Human central nervous system astrocytes support survival and activation of B cells: implications for MS pathogenesis.

PubMed

Touil, Hanane; Kobert, Antonia; Lebeurrier, Nathalie; Rieger, Aja; Saikali, Philippe; Lambert, Caroline; Fawaz, Lama; Moore, Craig S; Prat, Alexandre; Gommerman, Jennifer; Antel, Jack P; Itoyama, Yasuto; Nakashima, Ichiro; Bar-Or, Amit

2018-04-19

The success of clinical trials of selective B cell depletion in patients with relapsing multiple sclerosis (MS) indicates B cells are important contributors to peripheral immune responses involved in the development of new relapses. Such B cell contribution to peripheral inflammation likely involves antibody-independent mechanisms. Of growing interest is the potential that B cells, within the MS central nervous system (CNS), may also contribute to the propagation of CNS-compartmentalized inflammation in progressive (non-relapsing) disease. B cells are known to persist in the inflamed MS CNS and are more recently described as concentrated in meningeal immune-cell aggregates, adjacent to the subpial cortical injury which has been associated with progressive disease. How B cells are fostered within the MS CNS and how they may contribute locally to the propagation of CNS-compartmentalized inflammation remain to be elucidated. We considered whether activated human astrocytes might contribute to B cell survival and function through soluble factors. B cells from healthy controls (HC) and untreated MS patients were exposed to primary human astrocytes that were either maintained under basal culture conditions (non-activated) or pre-activated with standard inflammatory signals. B cell exposure to astrocytes included direct co-culture, co-culture in transwells, or exposure to astrocyte-conditioned medium. Following the different exposures, B cell survival and expression of T cell co-stimulatory molecules were assessed by flow cytometry, as was the ability of differentially exposed B cells to induce activation of allogeneic T cells. Secreted factors from both non-activated and activated human astrocytes robustly supported human B cell survival. Soluble products of pre-activated astrocytes also induced B cell upregulation of antigen-presenting cell machinery, and these B cells, in turn, were more efficient activators of T cells. Astrocyte-soluble factors could support survival

Rat astrocytes are more supportive for mouse OPC self-renewal than mouse astrocytes in culture.

PubMed

Cheng, Xuejun; Xie, Binghua; Qi, Jiajun; Zhao, Xiaofeng; Zhang, Zunyi; Qiu, Mengsheng; Yang, Junlin

2017-09-01

Mouse primary oligodendrocyte precursor cells (OPCs) are increasingly used to study the molecular mechanisms underlying the phenotype changes in oligodendrocyte differentiation and axonal myelination observed in transgenic or mutant mouse models. However, mouse OPCs are much more difficult to be isolated by the simple dissociation culture of brain tissues than their rat counterparts. To date, the mechanisms underlying the species difference in OPC preparation remain obscure. In this study, we showed that astrocytes from rats have a stronger effect than those from mouse in promoting OPC proliferation and survival in vitro. Mouse astrocytes displayed significantly weaker viability in culture and reduced potential in maintaining OPC self-renewal, as confirmed by culturing OPCs with conditioned media from rat or mouse astrocytes. These results explained the reason for why stratified cultures of OPCs and astrocytes are difficult to be achieved in mouse CNS tissues. Based on these findings, we adopted inactivated rat astrocytes as feeder cells to support the self-renewal of mouse cortical OPCs and preparation of high-purity mouse OPCs. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 907-916, 2017. © 2016 Wiley Periodicals, Inc.

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2015-01-01

Silver nanoparticles (Ag-NPs) can enter the brain and induce neurotoxicity. However, the toxicity of Ag-NPs on the blood-brain barrier (BBB) and the underlying mechanism(s) of action on the BBB and the brain are not well understood. To investigate Ag-NP suspension (Ag-NPS)-induced toxicity, a triple coculture BBB model of rat brain microvascular endothelial cells, pericytes, and astrocytes was established. The BBB permeability and tight junction protein expression in response to Ag-NPS, NP-released Ag ions, and polystyrene-NP exposure were investigated. Ultrastructural changes of the microvascular endothelial cells, pericytes, and astrocytes were observed using transmission electron microscopy (TEM). Global gene expression of astrocytes was measured using a DNA microarray. A triple coculture BBB model of primary rat brain microvascular endothelial cells, pericytes, and astrocytes was established, with the transendothelial electrical resistance values >200 Ω·cm(2). After Ag-NPS exposure for 24 hours, the BBB permeability was significantly increased and expression of the tight junction (TJ) protein ZO-1 was decreased. Discontinuous TJs were also observed between microvascular endothelial cells. After Ag-NPS exposure, severe mitochondrial shrinkage, vacuolations, endoplasmic reticulum expansion, and Ag-NPs were observed in astrocytes by TEM. Global gene expression analysis showed that three genes were upregulated and 20 genes were downregulated in astrocytes treated with Ag-NPS. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the 23 genes were associated with metabolic processes, biosynthetic processes, response to stimuli, cell death, the MAPK pathway, and so on. No GO term and KEGG pathways were changed in the released-ion or polystyrene-NP groups. Ag-NPS inhibited the antioxidant defense of the astrocytes by increasing thioredoxin interacting protein, which inhibits the Trx system, and decreasing Nr4a1 and Dusp1

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

PubMed Central

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

2015-01-01

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

GPR30 Regulates Glutamate Transporter GLT-1 Expression in Rat Primary Astrocytes*

PubMed Central

Lee, Eunsook; Sidoryk-Wêgrzynowicz, Marta; Wang, Ning; Webb, Anton; Son, Deok-Soo; Lee, Kyuwon; Aschner, Michael

2012-01-01

The G protein-coupled estrogen receptor GPR30 contributes to the neuroprotective effects of 17β-estradiol (E2); however, the mechanisms associated with this protection have yet to be elucidated. Given that E2 increases astrocytic expression of glutamate transporter-1 (GLT-1), which would prevent excitotoxic-induced neuronal death, we proposed that GPR30 mediates E2 action on GLT-1 expression. To investigate this hypothesis, we examined the effects of G1, a selective agonist of GPR30, and GPR30 siRNA on astrocytic GLT-1 expression, as well as glutamate uptake in rat primary astrocytes, and explored potential signaling pathways linking GPR30 to GLT-1. G1 increased GLT-1 protein and mRNA levels, subject to regulation by both MAPK and PI3K signaling. Inhibition of TGF-α receptor suppressed the G1-induced increase in GLT-1 expression. Silencing GPR30 reduced the expression of both GLT-1 and TGF-α and abrogated the G1-induced increase in GLT-1 expression. Moreover, the G1-induced increase in GLT-1 protein expression was abolished by a protein kinase A inhibitor and an NF-κB inhibitor. G1 also enhanced cAMP response element-binding protein (CREB), as well as both NF-κB p50 and NF-κB p65 binding to the GLT-1 promoter. Finally, to model dysfunction of glutamate transporters, manganese was used, and G1 was found to attenuate manganese-induced impairment in GLT-1 protein expression and glutamate uptake. Taken together, the present data demonstrate that activation of GPR30 increases GLT-1 expression via multiple pathways, suggesting that GPR30 is worthwhile as a potential target to be explored for developing therapeutics of excitotoxic neuronal injury. PMID:22645130

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

NASA Astrophysics Data System (ADS)

Salazar-García, Samuel; Silva-Ramírez, Ana Sonia; Ramirez-Lee, Manuel A.; Rosas-Hernandez, Hector; Rangel-López, Edgar; Castillo, Claudia G.; Santamaría, Abel; Martinez-Castañon, Gabriel A.; Gonzalez, Carmen

2015-11-01

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 (AgNO3) 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.

The MicroRNA and MessengerRNA Profile of the RNA-Induced Silencing Complex in Human Primary Astrocyte and Astrocytoma Cells

PubMed Central

Moser, Joanna J.; Fritzler, Marvin J.

2010-01-01

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 possibly in

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

PubMed

Moser, Joanna J; Fritzler, Marvin J

2010-10-18

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. 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. 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 possibly in other malignancies.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

2014-05-16

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

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

PubMed

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

2016-05-13

Nicotinamide adenine dinucleotide (NAD(+)) participates in redox reactions and NAD(+)-dependent signaling pathways. Although the redox reactions are critical for efficient mitochondrial metabolism, they are not accompanied by any net consumption of the nucleotide. On the contrary, NAD(+)-dependent signaling processes lead to its degradation. Three distinct families of enzymes consume NAD(+) as substrate: poly(ADP-ribose) polymerases, ADP-ribosyl cyclases (CD38 and CD157), and sirtuins (SIRT1-7). Because all of the above enzymes generate nicotinamide as a byproduct, mammalian cells have evolved an NAD(+) salvage pathway capable of resynthesizing NAD(+) from nicotinamide. Overexpression of the rate-limiting enzyme in this pathway, nicotinamide phosphoribosyltransferase, increases total and mitochondrial NAD(+) levels in astrocytes. Moreover, targeting nicotinamide phosphoribosyltransferase to the mitochondria also enhances NAD(+) salvage pathway in astrocytes. Supplementation with the NAD(+) precursors nicotinamide mononucleotide and nicotinamide riboside also increases NAD(+) levels in astrocytes. Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Superoxide dismutase 1 (SOD1) mutations account for up to 20% of familial ALS and 1-2% of apparently sporadic ALS cases. Primary astrocytes isolated from mutant human superoxide dismutase 1-overexpressing mice as well as human post-mortem ALS spinal cord-derived astrocytes induce motor neuron death in co-culture. Increasing total and mitochondrial NAD(+) content in ALS astrocytes increases oxidative stress resistance and reverts their toxicity toward co-cultured motor neurons. Taken together, our results suggest that enhancing the NAD(+) salvage pathway in astrocytes could be a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS. © 2016 by The American Society for Biochemistry and Molecular

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

PubMed

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

2016-01-01

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

Temporal Differences in MicroRNA Expression Patterns in Astrocytes and Neurons after Ischemic Injury

PubMed Central

Ziu, Mateo; Fletcher, Lauren; Rana, Shushan; Jimenez, David F.; Digicaylioglu, Murat

2011-01-01

MicroRNAs (miRNAs) are small, non-protein-coding RNA molecules that modulate gene translation. Their expression is altered in many central nervous system (CNS) injuries suggesting a role in the cellular response to stress. Current studies in brain tissue have not yet described the cell-specific temporal miRNA expression patterns following ischemic injury. In this study, we analyzed the expression alterations of a set of miRNAs in neurons and astrocytes subjected to 60 minutes of ischemia and collected at different time-points following this injury. To mimic ischemic conditions and reperfusion in vitro, cortical primary neuronal and astrocytic cultures prepared from fetal rats were first placed in oxygen and glucose deprived (OGD) medium for 60 minutes, followed by their transfer into normoxic pre-conditioned medium. Total RNA was extracted at different time-points after the termination of the ischemic insult and the expression levels of miRNAs were measured. In neurons exposed to OGD, expression of miR-29b was upregulated 2-fold within 6 h and up to 4-fold at 24 h post-OGD, whereas induction of miR-21 was upregulated 2-fold after 24 h when compared to expression in neurons under normoxic conditions. In contrast, in astrocytes, miR-29b and miR-21 were upregulated only after 12 h. MiR-30b, 107, and 137 showed expression alteration in astrocytes, but not in neurons. Furthermore, we show that expression of miR-29b was significantly decreased in neurons exposed to Insulin-Like Growth Factor I (IGF-I), a well documented neuroprotectant in ischemic models. Our study indicates that miRNAs expression is altered in neurons and astrocytes after ischemic injury. Furthermore, we found that following OGD, specific miRNAs have unique cell-specific temporal expression patterns in CNS. Therefore the specific role of each miRNA in different intracellular processes in ischemic brain and the relevance of their temporal and spatial expression patterns warrant further investigation that

Inhibition or ablation of transglutaminase 2 impairs astrocyte migration.

PubMed

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

2017-01-22

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

Electrospun fiber surface nanotopography influences astrocyte-mediated neurite outgrowth.

PubMed

Johnson, Christopher D; D'Amato, Anthony R; Puhl, Devan L; Wich, Douglas M; Vespermann, Amanda; Gilbert, Ryan J

2018-05-15

Aligned, electrospun fiber scaffolds provide topographical guidance for regenerating neurons and glia after central nervous system injury. To date, no study has explored how fiber surface nanotopography affects astrocyte response to fibrous scaffolds. Astrocytes play important roles in the glial scar, the blood brain barrier, and in maintaining homeostasis in the central nervous system. In this study, electrospun poly L-lactic acid fibers were engineered with smooth, pitted, or divoted surface nanotopography. Cortical or spinal cord primary rat astrocytes were cultured on the surfaces for either 1 or 3 days to examine the astrocyte response over time. The results showed that cortical astrocytes were significantly shorter and broader on the pitted and divoted fibers compared to those on smooth fibers. However, spinal cord astrocyte morphology was not significantly altered by the surface features. These findings indicate that astrocytes from unique anatomical locations respond differently to the presence of nanotopography. Western Blot results show that the differences in morphology were not associated with significant changes in GFAP or vinculin in either astrocyte population, suggesting that surface pits and divots do not induce a reactive phenotype in either cortical or spinal cord astrocytes. Finally, astrocytes were co-cultured with dorsal root ganglia to determine how the surfaces affected astrocyte-mediated neurite outgrowth. Astrocytes cultured on the fibers for shorter periods of time (1 day) generally supported longer neurite outgrowth. Pitted and divoted fibers restricted spinal cord astrocyte-mediated neurite outgrowth, while smooth fibers increased 3 day spinal cord astrocyte-mediated neurite outgrowth. In total, fiber surface nanotopography can influence astrocyte elongation and influence the capability of astrocytes to direct neurites. Therefore, fiber surface characteristics should be carefully controlled to optimize astrocyte-mediated axonal

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

PubMed

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

2003-06-01

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

Quantification of a thermal damage threshold for astrocytes using infrared laser generated heat gradients.

PubMed

Liljemalm, Rickard; Nyberg, Tobias

2014-04-01

The response of cells and tissues to elevated temperatures is highly important in several research areas, especially in the area of infrared neural stimulation. So far, only the heat response of neurons has been considered. In this study, primary rat astrocytes were exposed to infrared laser pulses of various pulse lengths and the resulting cell morphology changes and cell migration was studied using light microscopy. By using a finite element model of the experimental setup the temperature distribution was simulated and the temperatures and times to induce morphological changes and migration were extracted. These threshold temperatures were used in the commonly used first-order reaction model according to Arrhenius to extract the kinetic parameters, i.e., the activation energy, E a, and the frequency factor, A c, for the system. A damage signal ratio threshold was defined and calculated to be 6% for the astrocytes to change morphology and start migrating.

AQP5 is differentially regulated in astrocytes during metabolic and traumatic injuries.

PubMed

Chai, Rui Chao; Jiang, Jiao Hua; Wong, Ann Yuen Kwan; Jiang, Feng; Gao, Kai; Vatcher, Greg; Hoi Yu, Albert Cheung

2013-10-01

Water movement plays vital roles in both physiological and pathological conditions in the brain. Astrocytes are responsible for regulating this water movement and are the major contributors to brain edema in pathological conditions. Aquaporins (AQPs) in astrocytes play critical roles in the regulation of water movement in the brain. AQP1, 3, 4, 5, 8, and 9 have been reported in the brain. Compared with AQP1, 4, and 9, AQP3, 5, and 8 are less studied. Among the lesser known AQPs, AQP5, which has multiple functions identified outside the central nervous system, is also indicated to be involved in hypoxia injury in astrocytes. In our study, AQP5 expression could be detected both in primary cultures of astrocytes and neurons, and AQP5 expression in astrocytes was confirmed in 1- to 4-week old primary cultures of astrocytes. AQP5 was localized on the cytoplasmic membrane and in the cytoplasm of astrocytes. AQP5 expression was downregulated during ischemia treatment and upregulated after scratch-wound injury, which was also confirmed in a middle cerebral artery occlusion model and a stab-wound injury model in vivo. The AQP5 increased after scratch injury was polarized to the migrating processes and cytoplasmic membrane of astrocytes in the leading edge of the scratch-wound, and AQP5 over-expression facilitated astrocyte process elongation after scratch injury. Taken together, these results indicate that AQP5 might be an important water channel in astrocytes that is differentially expressed during various brain injuries. Copyright © 2013 Wiley Periodicals, Inc.

1H NMR-Based Metabolic Profiling Reveals the Effects of Fluoxetine on Lipid and Amino Acid Metabolism in Astrocytes

PubMed Central

Bai, Shunjie; Zhou, Chanjuan; Cheng, Pengfei; Fu, Yuying; Fang, Liang; Huang, Wen; Yu, Jia; Shao, Weihua; Wang, Xinfa; Liu, Meiling; Zhou, Jingjing; Xie, Peng

2015-01-01

Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), is a prescribed and effective antidepressant and generally used for the treatment of depression. Previous studies have revealed that the antidepressant mechanism of fluoxetine was related to astrocytes. However, the therapeutic mechanism underlying its mode of action in astrocytes remains largely unclear. In this study, primary astrocytes were exposed to 10 µM fluoxetine; 24 h post-treatment, a high-resolution proton nuclear magnetic resonance (1H NMR)-based metabolomic approach coupled with multivariate statistical analysis was used to characterize the metabolic variations of intracellular metabolites. The orthogonal partial least-squares discriminant analysis (OPLS-DA) score plots of the spectra demonstrated that the fluoxetine-treated astrocytes were significantly distinguished from the untreated controls. In total, 17 differential metabolites were identified to discriminate the two groups. These key metabolites were mainly involved in lipids, lipid metabolism-related molecules and amino acids. This is the first study to indicate that fluoxetine may exert antidepressant action by regulating the astrocyte’s lipid and amino acid metabolism. These findings should aid our understanding of the biological mechanisms underlying fluoxetine therapy. PMID:25884334

An Efficient Platform for Astrocyte Differentiation from Human Induced Pluripotent Stem Cells.

PubMed

Tcw, Julia; Wang, Minghui; Pimenova, Anna A; Bowles, Kathryn R; Hartley, Brigham J; Lacin, Emre; Machlovi, Saima I; Abdelaal, Rawan; Karch, Celeste M; Phatnani, Hemali; Slesinger, Paul A; Zhang, Bin; Goate, Alison M; Brennand, Kristen J

2017-08-08

Growing evidence implicates the importance of glia, particularly astrocytes, in neurological and psychiatric diseases. Here, we describe a rapid and robust method for the differentiation of highly pure populations of replicative astrocytes from human induced pluripotent stem cells (hiPSCs), via a neural progenitor cell (NPC) intermediate. We evaluated this protocol across 42 NPC lines (derived from 30 individuals). Transcriptomic analysis demonstrated that hiPSC-astrocytes from four individuals are highly similar to primary human fetal astrocytes and characteristic of a non-reactive state. hiPSC-astrocytes respond to inflammatory stimulants, display phagocytic capacity, and enhance microglial phagocytosis. hiPSC-astrocytes also possess spontaneous calcium transient activity. Our protocol is a reproducible, straightforward (single medium), and rapid (<30 days) method to generate populations of hiPSC-astrocytes that can be used for neuron-astrocyte and microglia-astrocyte co-cultures for the study of neuropsychiatric disorders. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Phagocytic response of astrocytes to damaged neighboring cells

PubMed Central

Cruz, Gladys Mae S.; Ro, Clarissa C.; Moncada, Emmanuel G.; Khatibzadeh, Nima; Flanagan, Lisa A.; Berns, Michael W.

2018-01-01

This study aims to understand the phagocytic response of astrocytes to the injury of neurons or other astrocytes at the single cell level. Laser nanosurgery was used to damage individual cells in both primary mouse cortical astrocytes and an established astrocyte cell line. In both cases, the release of material/substances from laser-irradiated astrocytes or neurons induced a phagocytic response in near-by astrocytes. Propidium iodide stained DNA originating from irradiated cells was visible in vesicles of neighboring cells, confirming phagocytosis of material from damaged cortical cells. In the presence of an intracellular pH indicator dye, newly formed vesicles correspond to acidic pH fluorescence, thus suggesting lysosome bound degradation of cellular debris. Cells with shared membrane connections prior to laser damage had a significantly higher frequency of induced phagocytosis compared to isolated cells with no shared membrane. The increase in phagocytic response of cells with a shared membrane occurred regardless of the extent of shared membrane (a thin filopodial connection vs. a cell cluster with significant shared membrane). In addition to the presence (or lack) of a membrane connection, variation in phagocytic ability was also observed with differences in injury location within the cell and distance separating isolated astrocytes. These results demonstrate the ability of an astrocyte to respond to the damage of a single cell, be it another astrocyte, or a neuron. This single-cell level of analysis results in a better understanding of the role of astrocytes to maintain homeostasis in the CNS, particularly in the sensing and removal of debris in damaged or pathologic nervous tissue. PMID:29708987

Differentiation of Inflammation-Responsive Astrocytes from Glial Progenitors Generated from Human Induced Pluripotent Stem Cells.

PubMed

Santos, Renata; Vadodaria, Krishna C; Jaeger, Baptiste N; Mei, Arianna; Lefcochilos-Fogelquist, Sabrina; Mendes, Ana P D; Erikson, Galina; Shokhirev, Maxim; Randolph-Moore, Lynne; Fredlender, Callie; Dave, Sonia; Oefner, Ruth; Fitzpatrick, Conor; Pena, Monique; Barron, Jerika J; Ku, Manching; Denli, Ahmet M; Kerman, Bilal E; Charnay, Patrick; Kelsoe, John R; Marchetto, Maria C; Gage, Fred H

2017-06-06

Astrocyte dysfunction and neuroinflammation are detrimental features in multiple pathologies of the CNS. Therefore, the development of methods that produce functional human astrocytes represents an advance in the study of neurological diseases. Here we report an efficient method for inflammation-responsive astrocyte generation from induced pluripotent stem cells (iPSCs) and embryonic stem cells. This protocol uses an intermediate glial progenitor stage and generates functional astrocytes that show levels of glutamate uptake and calcium activation comparable with those observed in human primary astrocytes. Stimulation of stem cell-derived astrocytes with interleukin-1β or tumor necrosis factor α elicits a strong and rapid pro-inflammatory response. RNA-sequencing transcriptome profiling confirmed that similar gene expression changes occurred in iPSC-derived and primary astrocytes upon stimulation with interleukin-1β. This protocol represents an important tool for modeling in-a-dish neurological diseases with an inflammatory component, allowing for the investigation of the role of diseased astrocytes in neuronal degeneration. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

The role of astrocytes in amyloid β-protein toxicity and clearance.

PubMed

Thal, Dietmar Rudolf

2012-07-01

The deposition of the amyloid β-protein (Aβ) in the brain is a pathological hallmark of Alzheimer's disease (AD). Here, Aβ deposits occur as Aβ plaques in the brain parenchyma and in the walls of cerebral and leptomeningeal blood vessels. Astrocytes are considered to be involved in the clearance of Aβ from the brain parenchyma into the perivascular space, across the blood-brain barrier, or by enzymatic degradation. As such it has been assumed that clearance of Aβ by astrocytes is beneficial. In a recent study published in Experimental Neurology Mulder et al. (2012; 233: 373-379) report changes in neprilysin and scavenger receptor class B member 1 gene expression in astrocytes exposed to fibrillar Aβ depending on the availability of amyloid-associated proteins, especially apolipoprotein E (apoE). Astrocytes from AD patients did not show this response in gene expression. Reactive astrocytes and Aβ containing astrocytes are common findings in the AD brain. A loss of excitatory amino acid transporter 2 expression in perivascular astrocytes of APOE ε4-positive AD cases and an alteration of neuronal apoE metabolism in the event of perivascular drainage of apoE-Aβ complexes has also been described. As such, reactive and compensatory changes in AD astrocytes compete with supporting functions of astrocytes finally leading to an impairment of metabolic support and transmitter recycling in the brain. In summary, exposure of astrocytes to increased amounts of Aβ over a long period in time very likely impairs the above mentioned supporting functions of astrocytes in AD patients because these cells have to clear large amounts of Aβ and, thereby, neglect their other functions. Copyright © 2012 Elsevier Inc. All rights reserved.

Calcium in the Mechanism of Ammonia-Induced Astrocyte Swelling

PubMed Central

Jayakumar, A.R.; Rao, K.V. Rama; Tong, X.Y; Norenberg, M.D.

2016-01-01

Brain edema, due largely to astrocyte swelling, is an important clinical problem in patients with acute liver failure. While mechanisms underlying astrocyte swelling in this condition are not fully understood, ammonia and associated oxidative/nitrosative stress (ONS) appear to be involved. Mechanisms responsible for the increase in reactive oxygen/nitrogen species (RONS) and their role in ammonia-induced astrocyte swelling, however, are poorly understood. Recent studies have demonstrated a transient increase in intracellular Ca2+ in cultured astrocytes exposed to ammonia. As Ca2+ is a known inducer of RONS, we investigated potential mechanisms by which Ca2+ may be responsible for the production of RONS and cell swelling in cultured astrocytes after treatment with ammonia. Exposure of cultured astrocytes to ammonia (5 mM) increased the formation of free radicals, including nitric oxide, and such increase was significantly diminished by treatment with the Ca2+ chelator BAPTA-AM. We then examined the activity of Ca2+-dependent enzymes that are known to generate RONS and found that ammonia significantly increased the activities of NADPH oxidase (NOX), constitutive nitric oxide synthase (cNOS) and phospholipase A2 (PLA2) and such increases in activity were significantly diminished by BAPTA. Pretreatment of cultures with 7-nitroindazole, apocyanin and quinacrine, respective inhibitors of cNOS, NOX and PLA2, all significantly diminished RONS production. Additionally, treatment of cultures with BAPTA or with inhibitors of cNOS, NOX and PLA2 reduced ammonia-induced astrocyte swelling. These studies suggest that the ammonia-induced rise in intracellular Ca2+ activates free radical producing enzymes that ultimately contribute to the mechanism of astrocyte swelling. PMID:19393035

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

PubMed

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

2010-01-01

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

Ghrelin Regulates Glucose and Glutamate Transporters in Hypothalamic Astrocytes

PubMed Central

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

2016-01-01

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

Astrocyte-specific DJ-1 overexpression protects against rotenone-induced neurotoxicity in a rat model of Parkinson's disease.

PubMed

De Miranda, Briana R; Rocha, Emily M; Bai, Qing; El Ayadi, Amina; Hinkle, David; Burton, Edward A; Timothy Greenamyre, J

2018-07-01

DJ-1 is a redox-sensitive protein with several putative functions important in mitochondrial physiology, protein transcription, proteasome regulation, and chaperone activity. High levels of DJ-1 immunoreactivity are reported in astrocytes surrounding pathology associated with idiopathic Parkinson's disease, possibly reflecting the glial response to oxidative damage. Previous studies showed that astrocytic over-expression of DJ-1 in vitro prevented oxidative stress and mitochondrial dysfunction in primary neurons. Based on these observations, we developed a pseudotyped lentiviral gene transfer vector with specific tropism for CNS astrocytes in vivo to overexpress human DJ-1 protein in astroglial cells. Following vector delivery to the substantia nigra and striatum of adult Lewis rats, the DJ-1 transgene was expressed robustly and specifically within astrocytes. There was no observable transgene expression in neurons or other glial cell types. Three weeks after vector infusion, animals were exposed to rotenone to induce Parkinson's disease-like pathology, including loss of dopaminergic neurons, accumulation of endogenous α-synuclein, and neuroinflammation. Animals over-expressing hDJ-1 in astrocytes were protected from rotenone-induced neurodegeneration, and displayed a marked reduction in neuronal oxidative stress and microglial activation. In addition, α-synuclein accumulation and phosphorylation were decreased within substantia nigra dopaminergic neurons in DJ-1-transduced animals, and expression of LAMP-2A, a marker of chaperone mediated autophagy, was increased. Together, these data indicate that astrocyte-specific overexpression of hDJ-1 protects neighboring neurons against multiple pathologic features of Parkinson's disease and provides the first direct evidence in vivo of a cell non-autonomous neuroprotective function of astroglial DJ-1. Copyright © 2018 Elsevier Inc. All rights reserved.

Na-K-Cl Cotransporter-1 in the Mechanism of Ammonia-induced Astrocyte Swelling*

PubMed Central

Jayakumar, Arumugam R.; Liu, Mingli; Moriyama, Mitsuaki; Ramakrishnan, Ramugounder; Forbush, Bliss; Reddy, Pichili V. B.; Norenberg, Michael D.

2008-01-01

Brain edema and the consequent increase in intracranial pressure and brain herniation are major complications of acute liver failure (fulminant hepatic failure) and a major cause of death in this condition. Ammonia has been strongly implicated as an important factor, and astrocyte swelling appears to be primarily responsible for the edema. Ammonia is known to cause cell swelling in cultured astrocytes, although the means by which this occurs has not been fully elucidated. A disturbance in one or more of these systems may result in loss of ion homeostasis and cell swelling. In particular, activation of the Na-K-Cl cotransporter (NKCC1) has been shown to be involved in cell swelling in several neurological disorders. We therefore examined the effect of ammonia on NKCC activity and its potential role in the swelling of astrocytes. Cultured astrocytes were exposed to ammonia (NH4Cl; 5 mm), and NKCC activity was measured. Ammonia increased NKCC activity at 24 h. Inhibition of this activity by bumetanide diminished ammonia-induced astrocyte swelling. Ammonia also increased total as well as phosphorylated NKCC1. Treatment with cyclohexamide, a potent inhibitor of protein synthesis, diminished NKCC1 protein expression and NKCC activity. Since ammonia is known to induce oxidative/nitrosative stress, and antioxidants and nitric-oxide synthase inhibition diminish astrocyte swelling, we also examined whether ammonia caused oxidation and/or nitration of NKCC1. Cultures exposed to ammonia increased the state of oxidation and nitration of NKCC1, whereas the antioxidants N-nitro-l-arginine methyl ester and uric acid all significantly diminished NKCC activity. These agents also reduced phosphorylated NKCC1 expression. These results suggest that activation of NKCC1 is an important factor in the mediation of astrocyte swelling by ammonia and that such activation appears to be mediated by NKCC1 abundance as well as by its oxidation/nitration and phosphorylation. PMID:18849345

Effects of MRP8, LPS, and lenalidomide on the expressions of TNF-α , brain-enriched, and inflammation-related microRNAs in the primary astrocyte culture.

PubMed

Omran, Ahmed; Ashhab, Muhammad Usman; Gan, Na; Kong, Huimin; Peng, Jing; Yin, Fei

2013-01-01

Astrocytes are now recognized as a heterogeneous class of cells with many important and diverse functions in healthy and diseased central nervous system (CNS). MicroRNAs (miRNAs) are small, noncoding RNAs which may have key roles in astrocytes activation in response to various stimuli. We performed quantitative real-time PCR (qPCR) to detect changes in the expressions of brain-enriched miRNAs (124, 134, 9, 132, and 138), inflammation-related miRNAs (146a, 21, 181a, 221, and 222), and tumor necrosis factor alpha (TNF- α ) in the rat primary astrocyte cultures after stimulation with myeloid-related protein 8 (MRP8) and lipopolysaccharides (LPS). Further, we inhibited the expression of TNF- α in the astrocytes by using TNF- α inhibitor (lenalidomide) and tested for the first time the effect of this inhibition on the expressions of the same tested miRNAs. Stimulation of the astrocytes with MRP8 or LPS leads to significant upregulation of miRNAs (124, 134, 9, 132, 146a, 21, 181a, 221, and 222), while miRNA-138 was downregulated. TNF- α inhibition with lenalidomide leads to opposite expressions of the tested miRNAs. These miRNAs may play an important role in activation of the astrocytes and may be a novel target for cell-specific therapeutic interventions in multiple CNS diseases.

Dimethyl sulfoxide damages mitochondrial integrity and membrane potential in cultured astrocytes.

PubMed

Yuan, Chan; Gao, Junying; Guo, Jichao; Bai, Lei; Marshall, Charles; Cai, Zhiyou; Wang, Linmei; Xiao, Ming

2014-01-01

Dimethyl sulfoxide (DMSO) is a polar organic solvent that is used to dissolve neuroprotective or neurotoxic agents in neuroscience research. However, DMSO itself also has pharmacological and pathological effects on the nervous system. Astrocytes play a central role in maintaining brain homeostasis, but the effect and mechanism of DMSO on astrocytes has not been studied. The present study showed that exposure of astrocyte cultures to 1% DMSO for 24 h did not significantly affect cell survival, but decreased cell viability and glial glutamate transporter expression, and caused mitochondrial swelling, membrane potential impairment and reactive oxygen species production, and subsequent cytochrome c release and caspase-3 activation. DMSO at concentrations of 5% significantly inhibited cell variability and promoted apoptosis of astrocytes, accompanied with more severe mitochondrial damage. These results suggest that mitochondrial impairment is a primary event in DMSO-induced astrocyte toxicity. The potential cytotoxic effects on astrocytes need to be carefully considered during investigating neuroprotective or neurotoxic effects of hydrophobic agents dissolved by DMSO.

Dimethyl Sulfoxide Damages Mitochondrial Integrity and Membrane Potential in Cultured Astrocytes

PubMed Central

Yuan, Chan; Gao, Junying; Guo, Jichao; Bai, Lei; Marshall, Charles; Cai, Zhiyou; Wang, Linmei; Xiao, Ming

2014-01-01

Dimethyl sulfoxide (DMSO) is a polar organic solvent that is used to dissolve neuroprotective or neurotoxic agents in neuroscience research. However, DMSO itself also has pharmacological and pathological effects on the nervous system. Astrocytes play a central role in maintaining brain homeostasis, but the effect and mechanism of DMSO on astrocytes has not been studied. The present study showed that exposure of astrocyte cultures to 1% DMSO for 24 h did not significantly affect cell survival, but decreased cell viability and glial glutamate transporter expression, and caused mitochondrial swelling, membrane potential impairment and reactive oxygen species production, and subsequent cytochrome c release and caspase-3 activation. DMSO at concentrations of 5% significantly inhibited cell variability and promoted apoptosis of astrocytes, accompanied with more severe mitochondrial damage. These results suggest that mitochondrial impairment is a primary event in DMSO-induced astrocyte toxicity. The potential cytotoxic effects on astrocytes need to be carefully considered during investigating neuroprotective or neurotoxic effects of hydrophobic agents dissolved by DMSO. PMID:25238609

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

PubMed

Howe, Charles L; Kaptzan, Tatiana; Magaña, Setty M; Ayers-Ringler, Jennifer R; LaFrance-Corey, Reghann G; Lucchinetti, Claudia F

2014-05-01

Neuromyelitis optica (NMO) is a primary astrocyte disease associated with central nervous system inflammation, demyelination, and tissue injury. Brain lesions are frequently observed in regions enriched in expression of the aquaporin-4 (AQP4) water channel, an antigenic target of the NMO IgG serologic marker. Based on observations of disease reversibility and careful characterization of NMO lesion development, we propose that the NMO IgG may induce a dynamic immunological response in astrocytes. Using primary rat astrocyte-enriched cultures and treatment with NMO patient-derived serum or purified IgG, we observed a robust pattern of gene expression changes consistent with the induction of a reactive and inflammatory phenotype in astrocytes. The reactive astrocyte factor lipocalin-2 and a broad spectrum of chemokines, cytokines, and stress response factors were induced by either NMO patient serum or purified IgG. Treatment with IgG from healthy controls had no effect. The effect is disease-specific, as serum from patients with relapsing-remitting multiple sclerosis, Sjögren's, or systemic lupus erythematosus did not induce a response in the cultures. We hypothesize that binding of the NMO IgG to AQP4 induces a cellular response that results in transcriptional and translational events within the astrocyte that are consistent with a reactive and inflammatory phenotype. Strategies aimed at reducing the inflammatory response of astrocytes may short circuit an amplification loop associated with NMO lesion development. Copyright © 2014 Wiley Periodicals, Inc.

Astrocytes mediated the nootropic and neurotrophic effects of Sarsasapogenin-AA13 via upregulating brain-derived neurotrophic factor.

PubMed

Dong, Dong; Mao, Yu; Huang, Cui; Jiao, Qian; Pan, Hui; Ma, Lei; Wang, Rui

2017-01-01

Rhizoma Anemarrhena , a widely used traditional Chinese medicine, has previously been shown to have neuroprotective effect. Sarsasapogenin-AA13 (AA13) is a novel synthetic derivative of Sarsasapogenin, which is extracted from Rhizoma Anemarrhena . The aim of this study is to investigate the nootropic and neurotrophic effects of AA13 and underlying mechanisms. In vitro , cell viability of rat primary astrocytes treated with AA13 and neurons cultured with conditioned medium of AA13-treated rat primary astrocytes was tested by MTT assays. In vivo , a pharmacological model of cognitive impairment induced by scopolamine was employed and spatial memory of the mice was assessed by Morris water maze. This study found that AA13 increased cell viability of primary astrocytes and AA13-treated astrocyte-conditioned medium enhanced the survival rate of primary neurons. Interestingly, AA13 markedly enhanced the level of BDNF in astrocytes. Furthermore, AA13 (6 mg/kg) improved the cognitive deficits in animal models (p<0.05) and BDNF and PSD95 levels were increased in brain. Therefore, we hypothesize that AA13 exerts nootropic and neurotrophic activities through astrocytes mediated upregulation of BDNF secretion. The results suggest that AA13 could be a potential compound for cognitive impairment after further research.

Transient receptor potential ankyrin 1 channels are involved in spontaneous peptide hormone release from astrocytes.

PubMed

Takizawa, Mai; Harada, Kazuki; Nakamura, Kazuaki; Tsuboi, Takashi

2018-07-02

Astrocytes, a large population of glial cells, detect neurotransmitters and respond by increasing intracellular Ca 2+ concentration ([Ca 2+ ] i ) and releasing chemical molecules called gliotransmitters. Recently discovered Ca 2+ influx through transient receptor potential ankyrin 1 (TRPA1) channels is reported to cause spontaneous [Ca 2+ ] i increase in astrocytes. While several physiological functions of TRPA1-mediated spontaneous Ca 2+ signal have been revealed, relation with gliotransmitter release, especially peptide hormone exocytosis is largely unknown. We therefore explored the [Ca 2+ ] i and exocytosis dynamics in rat astrocyte cell line C6 cells and primary astrocytes. TRPA1-mediated spontaneous [Ca 2+ ] i transients were observed in both C6 cells and primary astrocytes. Total internal reflection fluorescence microscopy revealed that Venus-tagged brain-derived neurotrophic factor and neuropeptide Y were released spontaneously from astrocytes. Activation of TRPA1 channels enhanced the frequency of peptide hormone exocytosis, and inhibition of TRPA1 channels decreased the number of peptide hormone exocytosis. These results suggest that TRPA1-mediated spontaneous [Ca 2+ ] i increase modulates the spontaneous release of peptide hormones from astrocytes. Copyright © 2018 Elsevier Inc. All rights reserved.

Tibolone Preserves Mitochondrial Functionality and Cell Morphology in Astrocytic Cells Treated with Palmitic Acid.

PubMed

González-Giraldo, Yeimy; Garcia-Segura, Luis Miguel; Echeverria, Valentina; Barreto, George E

2018-05-01

Obesity has been associated with increased chronic neuroinflammation and augmented risk of neurodegeneration. This is worsened during the normal aging process when the levels of endogenous gonadal hormones are reduced. In this study, we have assessed the protective actions of tibolone, a synthetic steroid with estrogenic actions, on T98G human astrocytic cells exposed to palmitic acid, a saturated fatty acid used to mimic obesity in vitro. Tibolone improved cell survival, and preserved mitochondrial membrane potential in palmitic acid-treated astrocytic cells. Although we did not find significant actions of tibolone on free radical production, it modulated astrocytic morphology after treatment with palmitic acid. These data suggest that tibolone protects astrocytic cells by preserving both mitochondrial functionality and morphological complexity.

Astrocytes Promote Oligodendrogenesis after White Matter Damage via Brain-Derived Neurotrophic Factor.

PubMed

Miyamoto, Nobukazu; Maki, Takakuni; Shindo, Akihiro; Liang, Anna C; Maeda, Mitsuyo; Egawa, Naohiro; Itoh, Kanako; Lo, Evan K; Lok, Josephine; Ihara, Masafumi; Arai, Ken

2015-10-14

Oligodendrocyte precursor cells (OPCs) in the adult brain contribute to white matter homeostasis. After white matter damage, OPCs compensate for oligodendrocyte loss by differentiating into mature oligodendrocytes. However, the underlying mechanisms remain to be fully defined. Here, we test the hypothesis that, during endogenous recovery from white matter ischemic injury, astrocytes support the maturation of OPCs by secreting brain-derived neurotrophic factor (BDNF). For in vitro experiments, cultured primary OPCs and astrocytes were prepared from postnatal day 2 rat cortex. When OPCs were subjected to chemical hypoxic stress by exposing them to sublethal CoCl2 for 7 d, in vitro OPC differentiation into oligodendrocytes was significantly suppressed. Conditioned medium from astrocytes (astro-medium) restored the process of OPC maturation even under the stressed conditions. When astro-medium was filtered with TrkB-Fc to remove BDNF, the BDNF-deficient astro-medium no longer supported OPC maturation. For in vivo experiments, we analyzed a transgenic mouse line (GFAP(cre)/BDNF(wt/fl)) in which BDNF expression is downregulated specifically in GFAP(+) astrocytes. Both wild-type (GFAP(wt)/BDNF(wt/fl) mice) and transgenic mice were subjected to prolonged cerebral hypoperfusion by bilateral common carotid artery stenosis. As expected, compared with wild-type mice, the transgenic mice exhibited a lower number of newly generated oligodendrocytes and larger white matter damage. Together, these findings demonstrate that, during endogenous recovery from white matter damage, astrocytes may promote oligodendrogenesis by secreting BDNF. The repair of white matter after brain injury and neurodegeneration remains a tremendous hurdle for a wide spectrum of CNS disorders. One potentially important opportunity may reside in the response of residual oligodendrocyte precursor cells (OPCs). OPCs may serve as a back-up for generating mature oligodendrocytes in damaged white matter. However

Astrocytes and energy metabolism.

PubMed

Prebil, Mateja; Jensen, Jørgen; Zorec, Robert; Kreft, Marko

2011-05-01

Astrocytes are glial cells, which play a significant role in a number of processes, including the brain energy metabolism. Their anatomical position between blood vessels and neurons make them an interface for effective glucose uptake from blood. After entering astrocytes, glucose can be involved in different metabolic pathways, e.g. in glycogen production. Glycogen in the brain is localized mainly in astrocytes and is an important energy source in hypoxic conditions and normal brain functioning. The portion of glucose metabolized into glycogen molecules in astrocytes is as high as 40%. It is thought that the release of gliotransmitters (such as glutamate, neuroactive peptides and ATP) into the extracellular space by regulated exocytosis supports a significant part of communication between astrocytes and neurons. On the other hand, neurotransmitter action on astrocytes has a significant role in brain energy metabolism. Therefore, understanding the astrocytes energy metabolism may help understanding neuron-astrocyte interactions.

Role of high-mobility group box 1 in methamphetamine-induced activation and migration of astrocytes.

PubMed

Zhang, Yuan; Zhu, Tiebing; Zhang, Xiaotian; Chao, Jie; Hu, Gang; Yao, Honghong

2015-09-04

Mounting evidence has indicated that high-mobility group box 1 (HMGB1) is involved in cell activation and migration. Our previous study demonstrated that methamphetamine mediates activation of astrocytes via sigma-1 receptor (σ-1R). However, the elements downstream of σ-1R in this process remain poorly understood. Thus, we examined the molecular mechanisms involved in astrocyte activation and migration induced by methamphetamine. The expression of HMGB1, σ-1R, and glial fibrillary acidic protein (GFAP) was examined by western blot and immunofluorescent staining. The phosphorylation of cell signaling pathways was detected by western blot, and cell migration was examined using a wound-healing assay in rat C6 astroglia-like cells transfected with lentivirus containing red fluorescent protein (LV-RFP) as well as in primary human astrocytes. The role of HMGB1 in astrocyte activation and migration was validated using a siRNA approach. Exposure of C6 cells to methamphetamine increased the expression of HMGB1 via the activation of σ-1R, Src, ERK mitogen-activated protein kinase, and downstream NF-κB p65 pathways. Moreover, methamphetamine treatment resulted in increased cell activation and migration in C6 cells and primary human astrocytes. Knockdown of HMGB1 in astrocytes transfected with HMGB1 siRNA attenuated the increased cell activation and migration induced by methamphetamine, thereby implicating the role of HMGB1 in the activation and migration of C6 cells and primary human astrocytes. This study demonstrated that methamphetamine-mediated activation and migration of astrocytes involved HMGB1 up-regulation through an autocrine mechanism. Targeting HMGB1 could provide insights into the development of a potential therapeutic approach for alleviation of cell activation and migration of astrocytes induced by methamphetamine.

Sulfocerebrosides upregulate liposome uptake in human astrocytes without inducing a proinflammatory response.

PubMed

Suesca, Elizabeth; Alejo, Jose Luis; Bolaños, Natalia I; Ocampo, Jackson; Leidy, Chad; González, John M

2013-07-01

Astrocytes are involved in the pathogenesis of demyelinating diseases, where they actively regulate the secretion of proinflammatory factors, and trigger the recruitment of immune cells in the central nervous system (CNS). Antigen presentation of myelin-derived proteins has been shown to trigger astrocyte response, suggesting that astrocytes can directly sense demyelination. However, the direct response of astrocytes to lipid-debris generated during demyelination has not been investigated. The lipid composition of the myelin sheath is distinct, presenting significant amounts of cerebrosides, sulfocerebrosides (SCB), and ceramides. Studies have shown that microglia are activated in the presence of myelin-derived lipids, pointing to the possibility of lipid-induced astrocyte activation. In this study, a human astrocyte cell line was exposed to liposomes enriched in each myelin lipid component. Although liposome uptake was observed for all compositions, astrocytes had augmented uptake for liposomes containing sulfocerebroside (SCB). This enhanced uptake did not modify their expression of human leukocyte antigen (HLA) molecules or secretion of chemokines. This was in contrast to changes observed in astrocyte cells stimulated with IFNγ. Contrary to human monocytes, astrocytes did not internalize beads in the size-range of liposomes, indicating that liposome uptake is lipid specific. Epifluorescence microscopy corroborated that liposome uptake takes place through endocytosis. Soluble SCB were found to partially block uptake of liposomes containing this same lipid. Endocytosis was not decreased when cells were treated with cytochalasin D, but it was decreased by cold temperature incubation. The specific uptake of SCB in the absence of a proinflammatory response indicates that astrocytes may participate in the trafficking and regulation of sulfocerebroside metabolism and homeostasis in the CNS. Copyright © 2013 International Society for Advancement of Cytometry.

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

PubMed

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. © 2013 International Society for Neurochemistry.

Inhibition of highly productive HIV-1 infection in T cells, primary human macrophages, microglia, and astrocytes by Sargassum fusiforme

PubMed Central

Paskaleva, Elena E; Lin, Xudong; Li, Wen; Cotter, Robin; Klein, Michael T; Roberge, Emily; Yu, Er K; Clark, Bruce; Veille, Jean-Claude; Liu, Yanze; Lee, David Y-W; Canki, Mario

2006-01-01

Background The high rate of HIV-1 mutation and increasing resistance to currently available antiretroviral (ART) therapies highlight the need for new antiviral agents. Products derived from natural sources have been shown to inhibit HIV-1 replication during various stages of the virus life cycle, and therefore represent a potential source of novel therapeutic agents. To expand our arsenal of therapeutics against HIV-1 infection, we investigated aqueous extract from Sargassum fusiforme (S. fusiforme) for ability to inhibit HIV-1 infection in the periphery, in T cells and human macrophages, and for ability to inhibit in the central nervous system (CNS), in microglia and astrocytes. Results S. fusiforme extract blocked HIV-1 infection and replication by over 90% in T cells, human macrophages and microglia, and it also inhibited pseudotyped HIV-1 (VSV/NL4-3) infection in human astrocytes by over 70%. Inhibition was mediated against both CXCR4 (X4) and CCR5 (R5)-tropic HIV-1, was dose dependant and long lasting, did not inhibit cell growth or viability, was not toxic to cells, and was comparable to inhibition by the nucleoside analogue 2', 3'-didoxycytidine (ddC). S. fusiforme treatment blocked direct cell-to-cell infection spread. To investigate at which point of the virus life cycle this inhibition occurs, we infected T cells and CD4-negative primary human astrocytes with HIV-1 pseudotyped with envelope glycoprotein of vesicular stomatitis virus (VSV), which bypasses the HIV receptor requirements. Infection by pseudotyped HIV-1 (VSV/NL4-3) was also inhibited in a dose dependant manner, although up to 57% less, as compared to inhibition of native NL4-3, indicating post-entry interferences. Conclusion This is the first report demonstrating S. fusiforme to be a potent inhibitor of highly productive HIV-1 infection and replication in T cells, in primary human macrophages, microglia, and astrocytes. Results with VSV/NL4-3 infection, suggest inhibition of both entry and

Involvement of sigma-1 receptor in astrocyte activation induced by methamphetamine via up-regulation of its own expression.

PubMed

Zhang, Yuan; Lv, Xuan; Bai, Ying; Zhu, Xinjian; Wu, Xiaodong; Chao, Jie; Duan, Ming; Buch, Shilpa; Chen, Ling; Yao, Honghong

2015-02-17

Although it has been documented that methamphetamine induces astrocyte activation, the mechanism(s) underlying this effect remain poorly understood. We thus sought to examine the molecular mechanisms involved in methamphetamine-mediated activation of astrocytes with a focus on the role of sigma-1 receptor (σ-1R) in this process. The expression of σ-1R and glial fibrillary acidic protein (GFAP) was examined by reverse transcription PCR (RT-PCR), real-time PCR, Western blot, and immunofluorescent staining; phosphorylation of cell signaling pathways was detected by Western blot analysis. Immunoprecipitation was used to determine the interaction between σ-1R and p-Src. Chromatin immunoprecipitation (ChIP) assay was employed to discern the binding of cAMP-response element-binding protein (CREB) with the promoter of σ-1R. The role of σ-1R in astrocyte activation was further validated in σ-1R knockout (KO) mice by Western blot combined with immunofluorescent staining. Exposure of primary rat astrocytes to methamphetamine increased the expression of σ-1R via the activation of Src, ERK mitogen-activated protein kinase, and downstream CREB pathways. Subsequently, CREB translocated into nucleus and interacted with the promoter of σ-1R resulting in increased expression of σ-1R with a concomitant increase in expression of GFAP. This effect was inhibited in cells treated with the σ-1R antagonist-BD1047, thereby implicating the role of σ-1R in the activation of astrocytes. In vivo relevance of these findings was further corroborated in σ-1R KO mice that were administered methamphetamine. In the methamphetamine administered mice, there was a failure of the drug to induce activation of astrocytes, an effect that was evident in wild-type (WT) mice exposed to methamphetamine. The study presented herein demonstrates that methamphetamine-mediated activation of astrocytes involved up-regulation of σ-1R through a positive-feedback mechanism. Understanding the regulation of �

Inclusion bodies in cerebral cortical astrocytes: a new change of astrocytes.

PubMed

Minagawa, M; Shioda, K; Shimizu, Y; Isshiki, T

1992-01-01

A unique pathological finding of astrocytes was observed in the brain of a 20-year-old man who had severe physical and mental retardation. The brain was malformed showing micropolygyria in several cortical areas. A large number of hypertrophic astrocytes with eosinophilic granular substances in their cytoplasm were found throughout the cerebral cortex. Several staining procedures and electron microscopical examinations were carried out on these intracytoplasmic inclusion. It was found that the appearance and staining character of these inclusions were different from other astrocytic changes, especially the Rosenthal fiber, described so far. The authors consider that these inclusion bodies in cerebral cortical astrocytes represent new pathological changes of astrocytes that appear to be associated with malformation of the brain.

Marked potentiation of cell swelling by cytokines in ammonia-sensitized cultured astrocytes

PubMed Central

2010-01-01

Background Brain edema leading to high intracranial pressure is a lethal complication of acute liver failure (ALF), which is believed to be cytotoxic due to swelling of astrocytes. In addition to the traditional view that elevated levels of blood and brain ammonia are involved in the mechanism of brain edema in ALF, emerging evidence suggests that inflammatory cytokines also contribute to this process. We earlier reported that treatment of astrocyte cultures with a pathophysiological concentration of ammonia (5 mM NH4Cl) resulted in the activation of nuclear factor-kappaB (NF-κB) and that inhibition of such activation diminished astrocyte swelling, suggesting a key role of NF-κB in the mechanism of ammonia-induced astrocyte swelling. Since cytokines are also well-known to activate NF-κB, this study examined for additive/synergistic effects of ammonia and cytokines in the activation of NF-κB and their role in astrocyte swelling. Methods Primary cultures of astrocytes were treated with ammonia and cytokines (TNF-α, IL-1, IL-6, IFN-γ, each at 10 ng/ml), individually or in combination, and cell volume was determined by the [3H]-O-methylglucose equilibration method. The effect of ammonia and cytokines on the activation of NF-κB was determined by immunoblots. Results Cell swelling was increased by ammonia (43%) and by cytokines (37%) at 24 h. Simultaneous co-treatment with cytokines and ammonia showed no additional swelling. By contrast, cultures pretreated with ammonia for 24 h and then exposed to cytokines for an additional 24 h, showed a marked increase in astrocyte swelling (129%). Treatment of cultures with ammonia or cytokines alone also activated NF-κB (80-130%), while co-treatment had no additive effect. However, in cultures pre-treated with ammonia for 24 h, cytokines induced a marked activation of NF-κB (428%). BAY 11-7082, an inhibitor of NF-κB, completely blocked the astrocyte swelling in cultures pre-treated with ammonia and followed by the

Anthocyanin-rich açaí (Euterpe oleracea Mart.) extract attenuates manganese-induced oxidative stress in rat primary astrocyte cultures.

PubMed

da Silva Santos, Vivian; Bisen-Hersh, Emily; Yu, Yingchun; Cabral, Ingridy Simone Ribeiro; Nardini, Viviani; Culbreth, Megan; Teixeira da Rocha, João Batista; Barbosa, Fernando; Aschner, Michael

2014-01-01

Manganese (Mn) is an essential element for human health. However, at high concentrations Mn may be neurotoxic. Mn accumulates in astrocytes, affecting their redox status. In view of the high antioxidant and anti-inflammatory properties of the exotic Brazilian fruit açaí (Euterpe oleracea Mart.), its methanolic extract was obtained by solid-phase extraction (SPE). This açaí extract showed considerable anthocyanins content and direct antioxidant capacity. The açaí extract scavenged 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH•) with an EC₅₀ of 19.1 ppm, showing higher antioxidant activity compared to butylated hydroxytoluene (BHT), but lower than ascorbic acid and quercetin. This obtained açaí extract also attenuated Mn-induced oxidative stress in primary cultured astrocytes. Specifically, the açaí extract at an optimal and nutritionally relevant concentration of 0.1 μg/ml prevented Mn-induced oxidative stress by (1) restoring GSH/GSSG ratio and net glutamate uptake, (2) protecting astrocytic membranes from lipid peroxidation, and (3) decreasing Mn-induced expression of erythroid 2-related factor (Nrf2) protein. A larger quantity of açaí extract exacerbated the effects of Mn on these parameters except with respect to lipid peroxidation assessed by means of F₂-isoprostanes. These studies indicate that at nutritionally relevant concentration, anthocyanins obtained from açaí protect astrocytes against Mn neurotoxicity, but at high concentrations, the "pro-oxidant" effects of its constituents likely prevail. Future studies may be profitably directed at potential protective effects of açaí anthocyanins in nutraceutical formulations.

The common insecticides cyfluthrin and chlorpyrifos alter the expression of a subset of genes with diverse functions in primary human astrocytes.

PubMed

Mense, Sarah M; Sengupta, Amitabha; Lan, Changgui; Zhou, Mei; Bentsman, Galina; Volsky, David J; Whyatt, Robin M; Perera, Frederica P; Zhang, Li

2006-09-01

Given the widespread use of insecticides in the environment, it is important to perform studies evaluating their potential effects on humans. Organophosphate insecticides, such as chlorpyrifos, are being phased out; however, the use of pyrethroids in household pest control is increasing. While chlorpyrifos is relatively well studied, much less is known about the potential neurotoxicity of cyfluthrin and other pyrethroids. To gain insights into the neurotoxicity of cyfluthrin, we compared and evaluated the toxicity profiles of chlorpyrifos and cyfluthrin in primary human fetal astrocytes. We found that at the same concentrations, cyfluthrin exerts as great as, or greater toxic effects on the growth, survival, and proper functioning of human astrocytes. By using microarray gene expression profiling, we systematically identified and compared the potential molecular targets of chlorpyrifos and cyfluthrin, at a genome-wide scale. We found that chlorpyrifos and cyfluthrin affect a similar number of transcripts. These targets include molecular chaperones, signal transducers, transcriptional regulators, transporters, and those involved in behavior and development. Further computational and biochemical analyses show that cyfluthrin and chlorpyrifos upregulate certain targets of the interferon-gamma and insulin-signaling pathways and that they increase the protein levels of activated extracellular signal-regulated kinase 1/2, a key component of insulin signaling; interleukin 6, a key inflammatory mediator; and glial fibrillary acidic protein, a marker of inflammatory astrocyte activation. These results suggest that inflammatory activation of astrocytes might be an important mechanism underlying neurotoxicity of both chlorpyrifos and cyfluthrin.

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

PubMed Central

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

2015-01-01

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

From in silico astrocyte cell models to neuron-astrocyte network models: A review.

PubMed

Oschmann, Franziska; Berry, Hugues; Obermayer, Klaus; Lenk, Kerstin

2018-01-01

The idea that astrocytes may be active partners in synaptic information processing has recently emerged from abundant experimental reports. Because of their spatial proximity to neurons and their bidirectional communication with them, astrocytes are now considered as an important third element of the synapse. Astrocytes integrate and process synaptic information and by doing so generate cytosolic calcium signals that are believed to reflect neuronal transmitter release. Moreover, they regulate neuronal information transmission by releasing gliotransmitters into the synaptic cleft affecting both pre- and postsynaptic receptors. Concurrent with the first experimental reports of the astrocytic impact on neural network dynamics, computational models describing astrocytic functions have been developed. In this review, we give an overview over the published computational models of astrocytic functions, from single-cell dynamics to the tripartite synapse level and network models of astrocytes and neurons. Copyright © 2017 Elsevier Inc. All rights reserved.

Astrocytes acquire resistance to iron-dependent oxidative stress upon proinflammatory activation

PubMed Central

2013-01-01

Background Astrocytes respond to local insults within the brain and the spinal cord with important changes in their phenotype. This process, overall known as “activation”, is observed upon proinflammatory stimulation and leads astrocytes to acquire either a detrimental phenotype, thereby contributing to the neurodegenerative process, or a protective phenotype, thus supporting neuronal survival. Within the mechanisms responsible for inflammatory neurodegeneration, oxidative stress plays a major role and has recently been recognized to be heavily influenced by changes in cytosolic iron levels. In this work, we investigated how activation affects the competence of astrocytes to handle iron overload and the ensuing oxidative stress. Methods Cultures of pure cortical astrocytes were preincubated with proinflammatory cytokines (interleukin-1β and tumor necrosis factor α) or conditioned medium from lipopolysaccharide-activated microglia to promote activation and then exposed to a protocol of iron overload. Results We demonstrate that activated astrocytes display an efficient protection against iron-mediated oxidative stress and cell death. Based on this evidence, we performed a comprehensive biochemical and molecular analysis, including a transcriptomic approach, to identify the molecular basis of this resistance. Conclusions We propose the protective phenotype acquired after activation not to involve the most common astrocytic antioxidant pathway, based on the Nrf2 transcription factor, but to result from a complex change in the expression and activity of several genes involved in the control of cellular redox state. PMID:24160637

Astrocytes increase barrier properties and ZO-1 expression in retinal vascular endothelial cells.

PubMed

Gardner, T W; Lieth, E; Khin, S A; Barber, A J; Bonsall, D J; Lesher, T; Rice, K; Brennan, W A

1997-10-01

Diabetic retinopathy and other diseases associated with retinal edema are characterized by increased microvascular leakage. Astrocytes have been proposed to maintain endothelial function in the brain, suggesting that glial impairment may underlie the development of retinal edema. The purpose of this study was to test the effects of astrocytes on barrier properties in retinal microvascular endothelial cells. Bovine retinal microvascular endothelial cells were exposed to conditioned media from rat brain astrocytes. Transendothelial electrical resistance (TER) was determined on 24-mm Transwell (Cambridge, MA) polycarbonate filters with the End-Ohm device (World Precision Instruments, Sarasota, FL). ZO-1 protein content was quantified by microtiter enzyme-linked immunosorbent assay. Astrocyte-conditioned medium (ACM) significantly increased TER (P < 0.0001) and ZO-1 content (P < 0.01). Both serum-containing and serum-free N1B defined ACM increased ZO-1 expression, but heating abolished the effect. Serum-free ACM decreased cell proliferation by 16%. Astrocytes release soluble, heat-labile factors that increase barrier properties and tight junction protein content. These results suggest that astrocytes enhance blood-retinal barrier properties, at least in part by increasing tight junction protein expression. Our findings suggest that glial malfunction plays an important role in the pathogenesis of vasogenic retinal edema.

Astrocyte Mediated Protection of Fetal Cerebral Cortical Neurons from Rotenone and Paraquat

PubMed Central

Rathinam, Mary Latha; Watts, Lora Talley; Narasimhan, Madhusudhanan; Riar, Amanjot Kaur; Mahimainathan, Lenin; Henderson, George.I.

2012-01-01

Primary cultures of fetal rat cortical neurons and astrocytes were used to test the hypothesis that astrocyte-mediated control of neuronal glutathione (GSH) is a potent factor in neuroprotection against rotenone and paraquat. In neurons, rotenone (0.025 to 1μM) for 4 and 24 h decreased viability as did paraquat (2 to 100μM). Rotenone (30nM) decreased neuronal viability and GSH by 24% and 30%, while ROS were increased by 56%. Paraquat (30μM) decreased neuronal viability and GSH by 36% and 70%, while ROS were increased by 23%. When neurons were co-cultured with astrocytes, their GSH increased 1.5 fold and 5 fold at 12 and 24 h. Co-culturing with astrocytes blocked neuronal death and damage by rotenone and paraquat. Astrocyte-mediated neuroprotection was dependent on the activity of components of the γ-glutamyl cycle. These studies illustrate the importance of astrocyte-mediated glutathione homeostasis for protection of neurons from rotenone and paraquat and the role of the γ-glutamyl cycle in this neuroprotection PMID:22301167

Decreased Astrocytic Thrombospondin-1 Secretion After Chronic Ammonia Treatment Reduces the Level of Synaptic Proteins: In Vitro and In Vivo Studies

PubMed Central

Jayakumar, A. R.; Tong, X. Y.; Curtis, K. M.; Ruiz-Cordero, R.; Shamaladevi, N.; Abuzamel, M.; Johnstone, J.; Gaidosh, G.; Rama Rao, K.V.; Norenberg, M. D.

2014-01-01

Chronic hepatic encephalopathy (CHE) is a major complication in patients with severe liver disease. Elevated blood and brain ammonia levels have been implicated in its pathogenesis, and astrocytes are the principal neural cells involved in this disorder. Since defective synthesis and release of astrocytic factors have been shown to impair synaptic integrity in other neurological conditions, we examined whether thrombospondin-1 (TSP-1), an astrocytic factor involved in the maintenance of synaptic integrity, is also altered in CHE. Cultured astrocytes were exposed to ammonia (NH4Cl, 0.5–2.5 mM) for 1–10 days, and TSP-1 content was measured in cell extracts and culture media. Astrocytes exposed to ammonia exhibited a reduction in intra- and extracellular TSP-1 levels. Exposure of cultured neurons to conditioned media (CM) from ammonia-treated astrocytes showed a decrease in synaptophysin, PSD95 and synaptotagmin levels. CM from TSP-1 overexpressing astrocytes that were treated with ammonia, when added to cultured neurons, reversed the decline in synaptic proteins. Recombinant TSP-1 similarly reversed the decrease in synaptic proteins. Metformin, an agent known to increase TSP-1 synthesis in other cell types also reversed the ammonia-induced TSP-1 reduction. Likewise, we found a significant decline in TSP-1 level in cortical astrocytes, as well as a reduction in synaptophysin content in vivo in a rat model of CHE. These findings suggest that TSP-1 may represent an important therapeutic target for CHE. PMID:25040426

Hypoxia diminishes the protective function of white-matter astrocytes in the developing brain.

PubMed

Agematsu, Kota; Korotcova, Ludmila; Morton, Paul D; Gallo, Vittorio; Jonas, Richard A; Ishibashi, Nobuyuki

2016-01-01

White-matter injury after surgery is common in neonates with cerebral immaturity secondary to in utero hypoxia. Astrocytes play a central role in brain protection; however, the reaction of astrocytes to hypothermic circulatory arrest (HCA) remains unknown. We investigated the role of astrocytes in white-matter injury after HCA and determined the effects of preoperative hypoxia on this role, using a novel mouse model. Mice were exposed to hypoxia from days 3 to 11, which is equivalent to the third trimester in humans (prehypoxia, n = 49). Brain slices were transferred to a chamber perfused by cerebrospinal fluid. Oxygen-glucose deprivation (OGD) was performed to simulate ischemia-reperfusion/reoxygenation resulting from circulatory arrest under hypothermia. Astrocyte reactions were compared with preoperative normoxia (prenormoxia; n = 45). We observed astrocyte activation after 25°C ischemia-reperfusion/reoxygenation in prenormoxia (P < .01). Astrocyte number after OGD correlated with caspase-3(+) cells (rho = .77, P = .001), confirming that astrogliosis is an important response after HCA. At 3 hours after OGD, astrocytes in prenormoxia had already proliferated and become activated (P < .05). Conversely, astrocytes that developed under hypoxia did not display these responses. At 20 hours after ischemia-reperfusion/reoxygenation, astrogliosis was not observed in prehypoxia, demonstrating that hypoxia altered the response of astrocytes to insult. In contrast to prenormoxia, caspase-3(+) cells in prehypoxia increased after ischemia reperfusion/reoxygenation, compared with control (P < .01). Caspase-3(+) cells were more common with prehypoxia than with prenormoxia (P < .001), suggesting that lack of astrogliosis permits increased white-matter injury. Preoperative hypoxia alters the neuroprotective function of astrocytes. Restoring this function before surgery may be a therapeutic option to reduce postoperative white-matter injury in the immature brain. Copyright �

Detection of the Cyanotoxins L-BMAA Uptake and Accumulation in Primary Neurons and Astrocytes.

PubMed

Tan, Vanessa X; Mazzocco, Claire; Varney, Bianca; Bodet, Dominique; Guillemin, Tristan A; Bessede, Alban; Guillemin, Gilles J

2018-01-01

We show for the first time that a newly developed polyclonal antibody (pAb) can specifically target the cyanotoxin β-methylamino-L-alanine (BMAA) and can be used to enable direct visualization of BMAA entry and accumulation in primary brain cells. We used this pAb to investigate the effect of acute and chronic accumulation, and toxicity of both BMAA and its natural isomer 2,4-diaminobutyric acid (DAB), separately or in combination, on primary cultures of rat neurons. We further present evidence that co-treatment with BMAA and DAB increased neuronal death, as measured by MAP2 fluorescence level, and appeared to reduce BMAA accumulation. DAB is likely to be acting synergistically with BMAA resulting in higher level of cellular toxicity. We also found that glial cells such as microglia and astrocytes are also able to directly uptake BMAA indicating that additional brain cell types are affected by BMAA-induced toxicity. Therefore, BMAA clearly acts at multiple cellular levels to possibly increase the risk of developing neurodegenerative diseases, including neuro- and gliotoxicity and synergetic exacerbation with other cyanotoxins.

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

PubMed

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

2015-02-01

Quinolinic acid (QUIN) is an endogenous metabolite of the kynurenine pathway involved in several neurological disorders. Among the several mechanisms involved in QUIN-mediated toxicity, disruption of the cytoskeleton has been demonstrated in striatally injected rats and in striatal slices. The present work searched for the actions of QUIN in primary striatal neurons. Neurons exposed to 10 µM QUIN presented hyperphosphorylated neurofilament (NF) subunits (NFL, NFM, and NFH). Hyperphosphorylation was abrogated in the presence of protein kinase A and protein kinase C inhibitors H89 (20 μM) and staurosporine (10 nM), respectively, as well as by specific antagonists to N-methyl-D-aspartate (50 µM DL-AP5) and metabotropic glutamate receptor 1 (100 µM MPEP). Also, intra- and extracellular Ca(2+) chelators (10 µM BAPTA-AM and 1 mM EGTA, respectively) and Ca(2+) influx through L-type voltage-dependent Ca(2+) channel (10 µM verapamil) are implicated in QUIN-mediated effects. Cells immunostained for the neuronal markers βIII-tubulin and microtubule-associated protein 2 showed altered neurite/neuron ratios and neurite outgrowth. NF hyperphosphorylation and morphological alterations were totally prevented by conditioned medium from QUIN-treated astrocytes. Cocultured astrocytes and neurons interacted with one another reciprocally, protecting them against QUIN injury. Cocultured cells preserved their cytoskeletal organization and cell morphology together with unaltered activity of the phosphorylating system associated with the cytoskeleton. This article describes cytoskeletal disruption as one of the most relevant actions of QUIN toxicity in striatal neurons in culture with soluble factors secreted by astrocytes, with neuron-astrocyte interaction playing a role in neuroprotection. © 2014 Wiley Periodicals, Inc.

Toll-Like Receptor 4 Mediates Methamphetamine-Induced Neuroinflammation through Caspase-11 Signaling Pathway in Astrocytes

PubMed Central

Du, Si-Hao; Qiao, Dong-Fang; Chen, Chuan-Xiang; Chen, Si; Liu, Chao; Lin, Zhoumeng; Wang, Huijun; Xie, Wei-Bing

2017-01-01

Methamphetamine (METH) is an amphetamine-typed stimulant drug that is increasingly being abused worldwide. Previous studies have shown that METH toxicity is systemic, especially targeting dopaminergic neurons in the central nervous system (CNS). However, the role of neuroinflammation in METH neurotoxicity remains unclear. We hypothesized that Toll-like receptor 4 (TLR4) and Caspase-11 are involved in METH-induced astrocyte-related neuroinflammation. We tested our hypothesis by examining the changes of TLR4 and Caspase-11 protein expression in primary cultured C57BL/6 mouse astrocytes and in the midbrain and striatum of mice exposed to METH with western blot and double immunofluorescence labeling. We also determined the effects of blocking Caspase-11 expression with wedelolactone (a specific inhibitor of Caspase-11) or siRNA on METH-induced neuroinflammation in astrocytes. Furthermore, we determined the effects of blocking TLR4 expression with TAK-242 (a specific inhibitor of TLR4) or siRNA on METH-induced neuroinflammation in astrocytes. METH exposure increased Caspase-11 and TLR4 expression both in vitro and in vivo, with the effects in vitro being dose-dependent. Inhibition of Caspase-11 expression with either wedelolactone or siRNAs reduced the expression of inflammasome NLRP3 and pro-inflammatory cytokines. In addition, blocking TLR4 expression inhibited METH-induced activation of NF-κB and Caspase-11 in vitro and in vivo, suggesting that TLR4-Caspase-11 pathway is involved in METH-induced neuroinflammation. These results indicate that Caspase-11 and TLR4 play an important role in METH-induced neuroinflammation and may be potential gene targets for therapeutics in METH-caused neurotoxicity. PMID:29311802

Astrocytes Can Adopt Endothelial Cell Fates in a p53-Dependent Manner.

PubMed

Brumm, Andrew J; Nunez, Stefanie; Doroudchi, Mehdi M; Kawaguchi, Riki; Duan, Jinhzu; Pellegrini, Matteo; Lam, Larry; Carmichael, S Thomas; Deb, Arjun; Hinman, Jason D

2017-08-01

Astrocytes respond to a variety of CNS injuries by cellular enlargement, process outgrowth, and upregulation of extracellular matrix proteins that function to prevent expansion of the injured region. This astrocytic response, though critical to the acute injury response, results in the formation of a glial scar that inhibits neural repair. Scar-forming cells (fibroblasts) in the heart can undergo mesenchymal-endothelial transition into endothelial cell fates following cardiac injury in a process dependent on p53 that can be modulated to augment cardiac repair. Here, we sought to determine whether astrocytes, as the primary scar-forming cell of the CNS, are able to undergo a similar cellular phenotypic transition and adopt endothelial cell fates. Serum deprivation of differentiated astrocytes resulted in a change in cellular morphology and upregulation of endothelial cell marker genes. In a tube formation assay, serum-deprived astrocytes showed a substantial increase in vessel-like morphology that was comparable to human umbilical vein endothelial cells and dependent on p53. RNA sequencing of serum-deprived astrocytes demonstrated an expression profile that mimicked an endothelial rather than astrocyte transcriptome and identified p53 and angiogenic pathways as specifically upregulated. Inhibition of p53 with genetic or pharmacologic strategies inhibited astrocyte-endothelial transition. Astrocyte-endothelial cell transition could also be modulated by miR-194, a microRNA downstream of p53 that affects expression of genes regulating angiogenesis. Together, these studies demonstrate that differentiated astrocytes retain a stimulus-dependent mechanism for cellular transition into an endothelial phenotype that may modulate formation of the glial scar and promote injury-induced angiogenesis.

Neuropharmacological effects of Phoneutria nigriventer venom on astrocytes.

PubMed

Rapôso, Catarina; Björklund, Ulrika; Kalapothakis, Evanguedes; Biber, Björn; Alice da Cruz-Höfling, Maria; Hansson, Elisabeth

2016-06-01

Bites from genus Phoneutria (Ctenidae, Araneomorpha) are the second most frequent source of spider accidents in Southeast Brazil. Severe envenoming from Phoneutria nigriventer produces vision disturbance, tremor and convulsion, suggesting that the CNS is involved; however, the mechanisms by which P. nigriventer venom (PNV) affects the CNS remain poorly understood. The present study aimed to investigate whether PNV directly impairs astrocytes. Cultured astrocytes were exposed to PNV, and intracellular Ca(2+) release and signaling were measured (Fura-2/AM), Na(+)/K(+)-ATPase and Toll-like receptor 4 (TLR4) involvement were investigated, actin filaments were stained (Alexa™ 488-conjugated phalloidin probe), the G-actin/F-actin ratio was determined, and the expression level of connexin 43 (Cx43) was assessed. Incubation in Ca(2+)-free buffer did not change the Ca(2+) responses. However, pre-incubation in thapsigargin/caffeine completely abolished these responses, suggesting that PNV-evoked Ca(2+) transients were from intracellular Ca(2+) stores. Pretreatment with a Na(+)/K(+)-ATPase antagonist (ouabain) or a TLR4 antagonist (LPS-RS) decreased or increased the Ca(2+)-evoked transients, respectively. Astrocytes showed altered actin filament structure after PNV exposure. PNV treatment increased the expression levels of Na(+)/K(+)-ATPase and Cx43 but decreased those of TLR4. The present results suggest that PNV directly affects astrocytes. Na(+)/K(+)-ATPase may thus represent a more specific drug target for controlling the neurotoxicity of PNV. Copyright © 2016 Elsevier Ltd. All rights reserved.

Multiple Oxygen Tension Environments Reveal Diverse Patterns of Transcriptional Regulation in Primary Astrocytes

PubMed Central

Zhou, Yu; Wang, Liyun; Park, Sung-Soo; Martin, Bronwen; Wang, Rui; Becker, Kevin G.; Wood, William H.; Zhang, Yongqing; Peers, Chris; Maudsley, Stuart

2011-01-01

The central nervous system normally functions at O2 levels which would be regarded as hypoxic by most other tissues. However, most in vitro studies of neurons and astrocytes are conducted under hyperoxic conditions without consideration of O2-dependent cellular adaptation. We analyzed the reactivity of astrocytes to 1, 4 and 9% O2 tensions compared to the cell culture standard of 20% O2, to investigate their ability to sense and translate this O2 information to transcriptional activity. Variance of ambient O2 tension for rat astrocytes resulted in profound changes in ribosomal activity, cytoskeletal and energy-regulatory mechanisms and cytokine-related signaling. Clustering of transcriptional regulation patterns revealed four distinct response pattern groups that directionally pivoted around the 4% O2 tension, or demonstrated coherent ascending/decreasing gene expression patterns in response to diverse oxygen tensions. Immune response and cell cycle/cancer-related signaling pathway transcriptomic subsets were significantly activated with increasing hypoxia, whilst hemostatic and cardiovascular signaling mechanisms were attenuated with increasing hypoxia. Our data indicate that variant O2 tensions induce specific and physiologically-focused transcript regulation patterns that may underpin important physiological mechanisms that connect higher neurological activity to astrocytic function and ambient oxygen environments. These strongly defined patterns demonstrate a strong bias for physiological transcript programs to pivot around the 4% O2 tension, while uni-modal programs that do not, appear more related to pathological actions. The functional interaction of these transcriptional ‘programs’ may serve to regulate the dynamic vascular responsivity of the central nervous system during periods of stress or heightened activity. PMID:21738745

Neuro-Compatible Metabolic Glycan Labeling of Primary Hippocampal Neurons in Noncontact, Sandwich-Type Neuron-Astrocyte Coculture.

PubMed

Choi, Ji Yu; Park, Matthew; Cho, Hyeoncheol; Kim, Mi-Hee; Kang, Kyungtae; Choi, Insung S

2017-12-20

Glycans are intimately involved in several facets of neuronal development and neuropathology. However, the metabolic labeling of surface glycans in primary neurons is a difficult task because of the neurotoxicity of unnatural monosaccharides that are used as a metabolic precursor, hindering the progress of metabolic engineering in neuron-related fields. Therefore, in this paper, we report a neurosupportive, neuron-astrocyte coculture system that neutralizes the neurotoxic effects of unnatural monosaccharides, allowing for the long-term observation and characterization of glycans in primary neurons in vitro. Polysialic acids in neurons are selectively imaged, via the metabolic labeling of sialoglycans with peracetylated N-azidoacetyl-d-mannosamine (Ac 4 ManNAz), for up to 21 DIV. Two-color labeling shows that neuronal activities, such as neurite outgrowth and recycling of membrane components, are highly dynamic and change over time during development. In addition, the insertion sites of membrane components are suggested to not be random, but be predominantly localized in developing neurites. This work provides a new research platform and also suggests advanced 3D systems for metabolic-labeling studies of glycans in primary neurons.

Endothelial cell-derived nitric oxide enhances aerobic glycolysis in astrocytes via HIF-1α-mediated target gene activation.

PubMed

Brix, Britta; Mesters, Jeroen R; Pellerin, Luc; Jöhren, Olaf

2012-07-11

Astrocytes exhibit a prominent glycolytic activity, but whether such a metabolic profile is influenced by intercellular communication is unknown. Treatment of primary cultures of mouse cortical astrocytes with the nitric oxide (NO) donor DetaNONOate induced a time-dependent enhancement in the expression of genes encoding various glycolytic enzymes as well as transporters for glucose and lactate. Such an effect was shown to be dependent on the hypoxia-inducible factor HIF-1α, which is stabilized and translocated to the nucleus to exert its transcriptional regulation. NO action was dependent on both the PI3K/Akt/mTOR and MEK signaling pathways and required the activation of COX, but was independent of the soluble guanylate cyclase pathway. Furthermore, as a consequence of NO treatment, an enhanced lactate production and release by astrocytes was evidenced, which was prevented by downregulating HIF-1α. Several brain cell types represent possible sources of NO. It was found that endothelial cells, which express the endothelial NO synthase (eNOS) isoform, constitutively produced the largest amount of NO in culture. When astrocytes were cocultured with primary cultures of brain vascular endothelial cells, stabilization of HIF-1α and an enhancement in glucose transporter-1, hexokinase-2, and monocarboxylate transporter-4 expression as well as increased lactate production was found in astrocytes. This effect was inhibited by the NOS inhibitor l-NAME and was not seen when astrocytes were cocultured with primary cultures of cortical neurons. Our findings suggest that endothelial cell-derived NO participates to the maintenance of a high glycolytic activity in astrocytes mediated by astrocytic HIF-1α activation.

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

PubMed Central

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

2016-01-01

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

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

PubMed

Ling, Yun-Zhi; Li, Xiao-Hong; Yu, Li; Zhang, Ye; Liang, Qi-Sheng; Yang, Xiao-di; Wang, Hong-Tao

2016-09-01

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

Insulin-like growth factor-I gene delivery to astrocytes reduces their inflammatory response to lipopolysaccharide

PubMed Central

2011-01-01

Background Insulin-like growth factor-I (IGF-I) exerts neuroprotective actions in the central nervous system that are mediated at least in part by control of activation of astrocytes. In this study we have assessed the efficacy of exogenous IGF-I and IGF-I gene therapy in reducing the inflammatory response of astrocytes from cerebral cortex. Methods An adenoviral vector harboring the rat IGF-I gene and a control adenoviral vector harboring a hybrid gene encoding the herpes simplex virus type 1 thymidine kinase fused to Aequorea victoria enhanced green fluorescent protein were used in this study. Primary astrocytes from mice cerebral cortex were incubated for 24 h or 72 h with vehicle, IGF-I, the IGF-I adenoviral vector, or control vector; and exposed to bacterial lipopolysaccharide to induce an inflammatory response. IGF-I levels were measured by radioimmunoassay. Levels of interleukin 6, tumor necrosis factor-α, interleukin-1β and toll-like receptor 4 mRNA were assessed by quantitative real-time polymerase chain reaction. Levels of IGF-I receptor and IGF binding proteins 2 and 3 were assessed by western blotting. The subcellular distribution of nuclear factor κB (p65) was assessed by immunocytochemistry. Statistical significance was assessed by one way analysis of variance followed by the Bonferroni pot hoc test. Results IGF-I gene therapy increased IGF-I levels without affecting IGF-I receptors or IGF binding proteins. Exogenous IGF-I, and IGF-I gene therapy, decreased expression of toll-like receptor 4 and counteracted the lipopolysaccharide-induced inflammatory response of astrocytes. In addition, IGF-I gene therapy decreased lipopolysaccharide-induced translocation of nuclear factor κB (p65) to the cell nucleus. Conclusion These findings demonstrate efficacy of exogenous IGF-I and of IGF-I gene therapy in reducing the inflammatory response of astrocytes. IGF-I gene therapy may represent a new approach to reduce inflammatory reactions in glial cells. PMID

Zika virus propagation and release in human fetal astrocytes can be suppressed by neutral sphingomyelinase-2 inhibitor GW4869.

PubMed

Huang, Yunlong; Li, Yuju; Zhang, Hainan; Zhao, Runze; Jing, Ran; Xu, Yinghua; He, Miao; Peer, Justin; Kim, Yeong C; Luo, Jiangtao; Tong, Zenghan; Zheng, Jialin

2018-01-01

Zika virus (ZIKV) is a neurotrophic flavivirus that is capable of infecting humans, leading to brain abnormalities during fetal development. The ZIKV infectivity in neural target cells remains poorly understood. Here, we found that ZIKV specifically infected glial fibrillary acidic protein- and S100B-positive primary human astrocytes derived from fetal brains. In contrast, neuron-specific Class III β-tubulin (TuJ1)-positive neurons in the astrocyte cultures and SOX2-positive neural progenitor cells derived from the fetal brains were less susceptible to ZIKV infection compared with astrocytes. The infected astrocytes released competent viral particles and manifested programmed cell death with a progressive cytopathic effect. Interestingly, ZIKV infection in human fetal astrocytes induced a significant increase of extracellular vesicles (EVs). Treatment with GW4869, a specific inhibitor of neutral sphingomyelinase-2, decreased EV levels, suppressed ZIKV propagation, and reduced the release of infectious virions in astrocytes. Therefore, ZIKV infects primary human fetal astrocytes and the infection can be suppressed by neutral sphingomyelinase-2 inhibitor GW4869. Further investigation into sphingomyelin metabolism and EVs may provide insights to the therapeutic treatment of ZIKV infection.

Dysbalance of Astrocyte Calcium under Hyperammonemic Conditions

PubMed Central

Haack, Nicole; Dublin, Pavel; Rose, Christine R.

2014-01-01

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

NFκB in the Mechanism of Ammonia-Induced Astrocyte Swelling in Culture

PubMed Central

Sinke, A.P.; Jayakumar, A.R.; Panickar, K.S.; Moriyama, M.; Reddy, P.V.B.; Norenberg, M.D.

2008-01-01

Astrocyte swelling and brain edema are major neuropathological findings in the acute form of hepatic encephalopathy (fulminant hepatic failure, FHF), and substantial evidence supports the view that elevated brain ammonia level is an important etiological factor in this condition. Although the mechanism by which ammonia brings about astrocyte swelling remains to be determined, oxidative/nitrosative stress and mitogen-activated protein kinases (MAPKs) have been considered as important elements in this process. One factor known to be activated by both oxidative stress and MAPKs is nuclear factor κB (NFκB), a transcription factor that activates many genes, including inducible nitric oxide synthase (iNOS). Since the product of iNOS, nitric oxide (NO), is known to cause astrocyte swelling, we examined the potential involvement of NFκB in ammonia-induced astrocyte swelling. Western blot analysis of cultured astrocytes showed a significant increase in NFκB nuclear translocation (a measure of NFκB activation) from 12 h to 2 days after treatment with NH4Cl (5 mM). Cultures treated with antioxidants, including superoxide dismutase, catalase and vitamin E, as well as the MAPKs inhibitors SB239063 (an inhibitor of p38-MAPK), and SP600125 (an inhibitor of c-Jun N-terminal kinase, JNK) significantly diminished NFκB activation by ammonia, supporting a role of oxidative stress and MAPKs in NFκB activation. The activation of NFκB was associated with increased iNOS protein expression and NO generation, and these changes were blocked by BAY 11-7082, an inhibitor of NFκB. Additionally, ammonia-induced astrocyte swelling was inhibited by the NFκB inhibitors BAY 11-7082 and SN-50, thereby implicating NFκB in the mechanism of astrocyte swelling. Our studies indicate that cultured astrocytes exposed to ammonia display NFκB activation, which is likely a consequence of oxidative stress and activation of MAPKs. NFκB activation appears to contribute to the mechanism of ammonia

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

PubMed

Yoder, Elizabeth J

2002-04-15

Astrocytes extend specialized endfoot processes to perisynaptic and perivascular regions, and thus are positioned to mediate the bidirectional flow of metabolic, ionic, and other transmissive substances between neurons and the blood stream. While mutual structural and functional interactions between neurons and astrocytes have been documented, less is known about the interactions between astrocytes and cerebrovascular cells. For example, although the ability of astrocytes to induce structural and functional changes in endothelial cells is established, the reciprocity of brain endothelial cells to induce changes in astrocytes is undetermined. This issue is addressed in the present study. Changes in primary cultures of neonatal mouse cortical astrocytes were investigated following their coculture with mouse brain capillary endothelial (bEnd3) cells. The presence of bEnd3 cells altered the morphology of astrocytes by transforming them from confluent monolayers into networks of elongated multicellular columns. These columns did not occur when either bEnd3 cells or astrocytes were cocultured with other cell types, suggesting that astrocytes undergo specific morphological consequences when placed in close proximity to brain endothelial cells. In addition to these structural changes, the pharmacological profile of astrocytes was modified by coculture with bEnd3 cells. Astrocytes in the cocultures showed an increased Ca2+ responsiveness to bradykinin and glutamate, but no change in responsiveness to ATP, as compared to controls. Coculturing the astrocytes with a neuronal cell line resulted in increased responsiveness of the glial responses to glutamate but not to bradykinin. These studies indicate that brain endothelial cells induce changes in astrocyte morphology and pharmacology. Copyright 2002 Wiley-Liss, Inc.

Astrocyte-Mediated Neuronal Synchronization Properties Revealed by False Gliotransmitter Release.

PubMed

Pirttimaki, Tiina M; Sims, Robert E; Saunders, Gregory; Antonio, Serena A; Codadu, Neela Krushna; Parri, H Rheinallt

2017-10-11

Astrocytes spontaneously release glutamate (Glut) as a gliotransmitter (GT), resulting in the generation of extrasynaptic NMDAR-mediated slow inward currents (SICs) in neighboring neurons, which can increase local neuronal excitability. However, there is a deficit in our knowledge of the factors that control spontaneous astrocyte GT release and the extent of its influence. We found that, in rat brain slices, increasing the supply of the physiological transmitter Glut increased the frequency and signaling charge of SICs over an extended period. This phenomenon was replicated by exogenous preexposure to the amino acid D-aspartate (D-Asp). Using D-Asp as a "false" GT, we determined the extent of local neuron excitation by GT release in ventrobasal thalamus, CA1 hippocampus, and somatosensory cortex. By analyzing synchronized neuronal NMDAR-mediated excitation, we found that the properties of the excitation were conserved in different brain areas. In the three areas, astrocyte-derived GT release synchronized groups of neurons at distances of >;200 μm. Individual neurons participated in more than one synchronized population, indicating that individual neurons can be excited by more than one astrocyte and that individual astrocytes may determine a neuron's synchronized network. The results confirm that astrocytes can act as excitatory nodes that can influence neurons over a significant range in a number of brain regions. Our findings further suggest that chronic elevation of ambient Glut levels can lead to increased GT Glut release, which may be relevant in some pathological states. SIGNIFICANCE STATEMENT Astrocytes spontaneously release glutamate (Glut) and other gliotransmitters (GTs) that can modify neuronal activity. Exposing brain slices to Glut and D-aspartate (D-Asp) before recording resulted in an increase in frequency of GT-mediated astrocyte-neuron signaling. Using D-Asp, it was possible to investigate the effects of specific GT release at neuronal NMDARs

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

PubMed Central

Wang, Yueming; Li, Guanjun; Wang, Lihua; Li, Huafang

2015-01-01

MK-801, also known as dizocilpine, is a noncompetitive N-methyl-D-aspartic acid (NMDA) receptor antagonist that induces schizophrenia-like symptoms. While astrocytes have been implicated in the pathophysiology of psychiatric disorders, including schizophrenia, astrocytic responses to MK-801 and their significance to schizotypic symptoms are unclear. Changes in the expression levels of glial fibrillary acid protein (GFAP), a marker of astrocyte activation in response to a variety of pathogenic stimuli, were examined in the hippocampus of rats treated with the repeated MK-801 injection (0.5 mg/10ml/kg body weight for 6 days) and in primary cultured hippocampal astrocytes incubated with MK-801 (5 or 20 μM for 24 h). Moreover, the expression levels of BDNF and its receptors TrkB and p75 were examined in MK-801-treated astrocyte cultures. MK-801 treatment enhanced GFAP expression in the rat hippocampus and also increased the levels of GFAP protein and mRNA in hippocampal astrocytes in vitro. Treatment of cultured hippocampal astrocytes with MK-801 enhanced protein and mRNA levels of BDNF, TrkB, and p75. Collectively, our results suggest that hippocampal astrocytes may contribute to the pathophysiology of schizophrenia symptoms associated with NMDA receptor hypofunction by reactive transformation and altered BDNF signaling. PMID:26700309

Ferulic acid and its water-soluble derivatives inhibit nitric oxide production and inducible nitric oxide synthase expression in rat primary astrocytes.

PubMed

Kikugawa, Masaki; Ida, Tomoaki; Ihara, Hideshi; Sakamoto, Tatsuji

2017-08-01

We recently reported that two water-soluble derivatives of ferulic acid (1-feruloyl glycerol, 1-feruloyl diglycerol) previously developed by our group exhibited protective effects against amyloid-β-induced neurodegeneration in vitro and in vivo. In the current study, we aimed to further understand this process by examining the derivatives' ability to suppress abnormal activation of astrocytes, the key event of neurodegeneration. We investigated the effects of ferulic acid (FA) derivatives on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in rat primary astrocytes. The results showed that these compounds inhibited NO production and iNOS expression in a concentration-dependent manner and that the mechanism underlying these effects was the suppression of the nuclear factor-κB pathway. This evidence suggests that FA and its derivatives may be effective neuroprotective agents and could be useful in the treatment of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.

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

PubMed

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

2014-08-01

Astrocytes play a key role in removing the synaptically released glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. However, high concentration of glutamate leads to toxicity in astrocytes, and the underlying mechanisms are unclear. The purpose of this study was to investigate whether energy metabolism disorder, especially impairment of mitochondrial respiration, is involved in the glutamate-induced gliotoxicity. Exposure to 10-mM glutamate for 48 h stimulated glycolysis and respiration in astrocytes. However, the increased oxygen consumption was used for proton leak and non-mitochondrial respiration, but not for oxidative phosphorylation and ATP generation. When the exposure time extended to 72 h, glycolysis was still activated for ATP generation, but the mitochondrial ATP-linked respiration of astrocytes was reduced. The glutamate-induced astrocyte damage can be mimicked by the non-metabolized substrate d-aspartate but reversed by the non-selective glutamate transporter inhibitor TBOA. In addition, the glutamate toxicity can be partially reversed by vitamin E. These findings demonstrate that changes of bioenergetic profile occur in cultured cortical astrocytes exposed to high concentration of glutamate and highlight the role of mitochondria respiration in glutamate-induced gliotoxicity in cortical astrocytes. Copyright © 2014 John Wiley & Sons, Ltd.

Astrocytic control of synaptic function.

PubMed

Papouin, Thomas; Dunphy, Jaclyn; Tolman, Michaela; Foley, Jeannine C; Haydon, Philip G

2017-03-05

Astrocytes intimately interact with synapses, both morphologically and, as evidenced in the past 20 years, at the functional level. Ultrathin astrocytic processes contact and sometimes enwrap the synaptic elements, sense synaptic transmission and shape or alter the synaptic signal by releasing signalling molecules. Yet, the consequences of such interactions in terms of information processing in the brain remain very elusive. This is largely due to two major constraints: (i) the exquisitely complex, dynamic and ultrathin nature of distal astrocytic processes that renders their investigation highly challenging and (ii) our lack of understanding of how information is encoded by local and global fluctuations of intracellular calcium concentrations in astrocytes. Here, we will review the existing anatomical and functional evidence of local interactions between astrocytes and synapses, and how it underlies a role for astrocytes in the computation of synaptic information.This article is part of the themed issue 'Integrating Hebbian and homeostatic plasticity'. © 2017 The Author(s).

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wan, Zhongxiao; Mah, Dorrian; Simtchouk, Svetlana

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 observedmore » 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.« less

Nitric Oxide in Astrocyte-Neuron Signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Nianzhen

Astrocytes, a subtype of glial cell, have recently been shown to exhibit Ca 2+ elevations in response to neurotransmitters. A Ca 2+ elevation can propagate to adjacent astrocytes as a Ca 2+ wave, which allows an astrocyte to communicate with its neighbors. Additionally, glutamate can be released from astrocytes via a Ca 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 2+ signaling by imaging NO in purified murine cortical astrocytemore » cultures. Physiological concentrations of a natural messenger, ATP, caused a Ca 2+-dependent NO production. To test the roles of NO in astrocytic Ca 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 2+, possibly through store-operated Ca 2+ channels. The NO-induced Ca 2+ signaling is cGMP-independent since 8-Br-cGMP, an agonistic analog of cGMP, did not induce a detectable Ca 2+ change. The consequence of this NO-induced Ca 2+ influx was assessed by simultaneously monitoring of cytosolic and internal store Ca 2+ using fluorescent Ca 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 2+ release, suggesting that NO modulates internal store refilling. Furthermore, locally photo-release of NO to a single astrocyte led to a Ca 2+ elevation in the stimulated astrocyte and a subsequent Ca 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). Although NO is not required for the SIC,PTIO reduced SIC amplitude, suggesting

Nonproductive human immunodeficiency virus type 1 infection of human fetal astrocytes: independence from CD4 and major chemokine receptors.

PubMed

Sabri, F; Tresoldi, E; Di Stefano, M; Polo, S; Monaco, M C; Verani, A; Fiore, J R; Lusso, P; Major, E; Chiodi, F; Scarlatti, G

1999-11-25

Human immunodeficiency virus type 1 (HIV-1) infection of the brain is associated with neurological manifestations both in adults and in children. The primary target for HIV-1 infection in the brain is the microglia, but astrocytes can also be infected. We tested 26 primary HIV-1 isolates for their capacity to infect human fetal astrocytes in culture. Eight of these isolates, independent of their biological phenotype and chemokine receptor usage, were able to infect astrocytes. Although no sustained viral replication could be demonstrated, the virus was recovered by coculture with receptive cells such as macrophages or on stimulation with interleukin-1beta. To gain knowledge into the molecular events that regulate attachment and penetration of HIV-1 in astrocytes, we investigated the expression of several chemokine receptors. Fluorocytometry and calcium-mobilization assay did not provide evidence of expression of any of the major HIV-1 coreceptors, including CXCR4, CCR5, CCR3, and CCR2b, as well as the CD4 molecule on the cell surface of human fetal astrocytes. However, mRNA transcripts for CXCR4, CCR5, Bonzo/STRL33/TYMSTR, and APJ were detected by RT-PCR. Furthermore, infection of astrocytes by HIV-1 isolates with different chemokine receptor usage was not inhibited by the chemokines SDF-1beta, RANTES, MIP-1beta, or MCP-1 or by antibodies directed against the third variable region or the CD4 binding site of gp120. These data show that astrocytes can be infected by primary HIV-1 isolates via a mechanism independent of CD4 or major chemokine receptors. Furthermore, astrocytes are potential carriers of latent HIV-1 and on activation may be implicated in spreading the infection to other neighbouring cells, such as microglia or macrophages. Copyright 1999 Academic Press.

Phosphorylation status of pyruvate dehydrogenase distinguishes metabolic phenotypes of cultured rat brain astrocytes and neurons.

PubMed

Halim, Nader D; Mcfate, Thomas; Mohyeldin, Ahmed; Okagaki, Peter; Korotchkina, Lioubov G; Patel, Mulchand S; Jeoung, Nam Ho; Harris, Robert A; Schell, Michael J; Verma, Ajay

2010-08-01

Glucose metabolism in nervous tissue has been proposed to occur in a compartmentalized manner with astrocytes contributing largely to glycolysis and neurons being the primary site of glucose oxidation. However, mammalian astrocytes and neurons both contain mitochondria, and it remains unclear why in culture neurons oxidize glucose, lactate, and pyruvate to a much larger extent than astrocytes. The objective of this study was to determine whether pyruvate metabolism is differentially regulated in cultured neurons versus astrocytes. Expression of all components of the pyruvate dehydrogenase complex (PDC), the rate-limiting step for pyruvate entry into the Krebs cycle, was determined in cultured astrocytes and neurons. In addition, regulation of PDC enzymatic activity in the two cell types via protein phosphorylation was examined. We show that all components of the PDC are expressed in both cell types in culture, but that PDC activity is kept strongly inhibited in astrocytes through phosphorylation of the pyruvate dehydrogenase alpha subunit (PDH alpha). In contrast, neuronal PDC operates close to maximal levels with much lower levels of phosphorylated PDH alpha. Dephosphorylation of astrocytic PDH alpha restores PDC activity and lowers lactate production. Our findings suggest that the glucose metabolism of astrocytes and neurons may be far more flexible than previously believed. (c) 2010 Wiley-Liss, Inc.

Glycogen synthase kinase-3 regulates inflammatory tolerance in astrocytes

PubMed Central

Beurel, Eléonore; Jope, Richard S.

2010-01-01

Inflammatory tolerance is the down-regulation of inflammation upon repeated stimuli, which is well-established to occur in peripheral immune cells. However, less is known about inflammatory tolerance in the brain although it may provide an important protective mechanism from detrimental consequences of prolonged inflammation, which appears to occur in many psychiatric and neurodegenerative conditions. Array analysis of 308 inflammatory molecules produced by mouse primary astrocytes after two sequential stimulations with lipopolysaccharide (LPS) distinguished three classes, tolerant, sensitized and unaltered groups. For many of these inflammatory molecules, inhibition of glycogen synthase kinase-3 (GSK3) increased tolerance and reduced sensitization. Focusing on LPS-tolerance in interleukin-6 (IL-6) production, we found that microglia exhibited a strong tolerance response that matched that of macrophages, whereas astrocytes exhibited only partial tolerance. The astrocyte semi-tolerance was found to be regulated by GSK3. GSK3 inhibitors or knocking down GSK3 levels promoted LPS-tolerance and astrocytes expressing constitutively active GSK3 did not develop LPS-tolerance. These findings identify the critical role of GSK3 in counteracting IL-6 inflammatory tolerance in cells of the CNS, supporting the therapeutic potential of GSK3 inhibitors to reduce neuroinflammation by promoting tolerance. PMID:20553816

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

PubMed

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

2014-05-01

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

Freshly dissociated mature hippocampal astrocytes exhibit passive membrane conductance and low membrane resistance similarly to syncytial coupled astrocytes

PubMed Central

Du, Yixing; Ma, Baofeng; Kiyoshi, Conrad M.; Alford, Catherine C.; Wang, Wei

2015-01-01

Mature astrocytes exhibit a linear current-to-voltage K+ membrane conductance (passive conductance) and an extremely low membrane resistance (Rm) in situ. The combination of these electrophysiological characteristics establishes a highly negative and stable membrane potential that is essential for basic functions, such as K+ spatial buffering and neurotransmitter uptake. However, astrocytes are coupled extensively in situ. It remains to be determined whether the observed passive behavior and low Rm are attributable to the intrinsic properties of membrane ion channels or to gap junction coupling in functionally mature astrocytes. In the present study, freshly dissociated hippocampal tissues were used as a new model to examine this basic question in young adult animals. The morphologically intact single astrocytes could be reliably dissociated from animals postnatal day 21 and older. At this animal age, dissociated single astrocytes exhibit passive conductance and resting membrane potential similar to those exhibited by astrocytes in situ. To precisely measure the Rm from single astrocytes, dual-patch single-astrocyte recording was performed. We show that dissociated single astrocytes exhibit a low Rm similarly to syncytial coupled astrocytes. Functionally, the symmetric expression of high-K+ conductance enabled rapid change in the intracellular K+ concentrations in response to changing K+ drive force. Altogether, we demonstrate that freshly dissociated tissue preparation is a highly useful model for study of the functional expression and regulation of ion channels, receptors, and transporters in astrocytes and that passive behavior and low Rm are the intrinsic properties of mature astrocytes. PMID:25810481

α₂-Adrenoceptors activate noradrenaline-mediated glycogen turnover in chick astrocytes.

PubMed

Hutchinson, Dana S; Catus, Stephanie L; Merlin, Jon; Summers, Roger J; Gibbs, Marie E

2011-06-01

In the brain, glycogen is primarily stored in astrocytes where it is regulated by several hormones/neurotransmitters, including noradrenaline that controls glycogen breakdown (in the short term) and synthesis. Here, we have examined the adrenoceptor (AR) subtype that mediates the glycogenic effect of noradrenaline in chick primary astrocytes by the measurement of glycogen turnover (total (14) C incorporation of glucose into glycogen) following noradrenergic activation. Noradrenaline and insulin increased glycogen turnover in a concentration-dependent manner. The effect of noradrenaline was mimicked by stimulation of α(2) -ARs (and to a lesser degree by β(3) -ARs), but not by stimulation of α(1) -, β(1) -, or β(2) -ARs, and occurred only in astrocytes and not neurons. In chick astrocytes, studies using RT-PCR and radioligand binding showed that α(2A) - and α(2C) -AR mRNA and protein were present. α(2) -AR- or insulin-mediated glycogen turnover was inhibited by phosphatidylinositol-3 kinase inhibitors, and both insulin and clonidine caused phosphorylation of Akt and glycogen synthase kinase-3 in chick astrocytes. α(2) -AR but not insulin-mediated glycogen turnover was inhibited by pertussis toxin pre-treatment indicating involvement of Gi/o proteins. These results show that the increase in glycogen turnover caused by noradrenaline is because of activation of α(2) -ARs that increase glycogen turnover in astrocytes utilizing a Gi/o-PI3K pathway. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

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

PubMed

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

2016-03-01

A significant role of brain-derived neurotrophic factor (BDNF) has been previously implicated in the therapeutic effect of antidepressants. To ascertain the contribution of specific cell types in the brain that produce BDNF following antidepressant treatment, the effects of the tricyclic antidepressant amitriptyline on rat primary neuronal, astrocytic and microglial cortical cultures were examined. Amitriptyline increased the expression of BDNF mRNA in astrocytic and microglial cultures but not neuronal cultures. Antidepressants with distinct mechanisms of action, such as clomipramine, duloxetine and fluvoxamine, also increased BDNF mRNA expression in astrocytic and microglial cultures. There are multiple BDNF mRNA variants (exon I, IIA, IV and VI) expressed in astrocytes and microglia and the variant induced by antidepressants has yet to be elaborated. Treatment with antidepressants increased the expression of exon I, IV and VI in astrocyte and microglia. Clomipramine alone significantly upregulated expression of exon IIA. The amitriptyline-induced expression of both total and individual BDNF mRNA variants (exon I, IV and VI) were blocked by MEK inhibitor U0126, indicating MEK/ERK signaling is required in the expression of BDNF. These findings indicate that non-neural cells are a significant target of antidepressants and further support the contention that glial production of BDNF is crucial role in the therapeutic effect of antidepressants. The current data suggest that targeting of glial function could lead to the development of antidepressants with a truly novel mechanism of action. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-09-01

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

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

PubMed Central

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

2013-01-01

Amyotrophic lateral sclerosis (ALS) is a fatal paralytic disorder caused by dysfunction and degeneration of motor neurons. Multiple disease-causing mutations, including in the genes for SOD1 and TDP-43, have been identified in ALS. Astrocytes expressing mutant SOD1 are strongly implicated in the pathogenesis of ALS: we have shown that media conditioned by astrocytes carrying mutant SOD1G93A contains toxic factor(s) that kill motoneurons by activating voltage-sensitive sodium (Nav) channels. In contrast, a recent study suggests that astrocytes expressing mutated TDP43 contribute to ALS pathology, but do so via cell-autonomous processes and lack non-cell-autonomous toxicity. Here we investigate whether astrocytes that express diverse ALS-causing mutations release toxic factor(s) that induce motoneuron death, and if so, whether they do so via a common pathogenic pathway. We exposed primary cultures of wild-type spinal cord cells to conditioned medium derived from astrocytes (ACM) that express SOD1 (ACM-SOD1G93A and ACM-SOD1G86R) or TDP43 (ACM-TDP43A315T) mutants; we show that such exposure rapidly (within 30–60 min) increases dichlorofluorescein (DCF) fluorescence (indicative of nitroxidative stress) and leads to extensive motoneuron-specific death within a few days. Co-application of the diverse ACMs with anti-oxidants Trolox or esculetin (but not with resveratrol) strongly improves motoneuron survival. We also find that co-incubation of the cultures in the ACMs with Nav channel blockers (including mexiletine, spermidine, or riluzole) prevents both intracellular nitroxidative stress and motoneuron death. Together, our data document that two completely unrelated ALS models lead to the death of motoneuron via non-cell-autonomous processes, and show that astrocytes expressing mutations in SOD1 and TDP43 trigger such cell death through a common pathogenic pathway that involves nitroxidative stress, induced at least in part by Nav channel activity. PMID:24570655

Astrocyte-Mediated Neuronal Synchronization Properties Revealed by False Gliotransmitter Release

PubMed Central

Pirttimaki, Tiina M.; Sims, Robert E.; Saunders, Gregory; Antonio, Serena A.; Codadu, Neela Krushna

2017-01-01

Astrocytes spontaneously release glutamate (Glut) as a gliotransmitter (GT), resulting in the generation of extrasynaptic NMDAR-mediated slow inward currents (SICs) in neighboring neurons, which can increase local neuronal excitability. However, there is a deficit in our knowledge of the factors that control spontaneous astrocyte GT release and the extent of its influence. We found that, in rat brain slices, increasing the supply of the physiological transmitter Glut increased the frequency and signaling charge of SICs over an extended period. This phenomenon was replicated by exogenous preexposure to the amino acid D-aspartate (D-Asp). Using D-Asp as a “false” GT, we determined the extent of local neuron excitation by GT release in ventrobasal thalamus, CA1 hippocampus, and somatosensory cortex. By analyzing synchronized neuronal NMDAR-mediated excitation, we found that the properties of the excitation were conserved in different brain areas. In the three areas, astrocyte-derived GT release synchronized groups of neurons at distances of >;200 μm. Individual neurons participated in more than one synchronized population, indicating that individual neurons can be excited by more than one astrocyte and that individual astrocytes may determine a neuron's synchronized network. The results confirm that astrocytes can act as excitatory nodes that can influence neurons over a significant range in a number of brain regions. Our findings further suggest that chronic elevation of ambient Glut levels can lead to increased GT Glut release, which may be relevant in some pathological states. SIGNIFICANCE STATEMENT Astrocytes spontaneously release glutamate (Glut) and other gliotransmitters (GTs) that can modify neuronal activity. Exposing brain slices to Glut and D-aspartate (D-Asp) before recording resulted in an increase in frequency of GT-mediated astrocyte–neuron signaling. Using D-Asp, it was possible to investigate the effects of specific GT release at neuronal

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

PubMed

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.

Rat astrocytes during anoxia: Secretome profile of cytokines and chemokines.

PubMed

Samy, Zeinab Adel; Al-Abdullah, Lulwa; Turcani, Marian; Craik, James; Redzic, Zoran

2018-06-04

The precise mechanisms of the inflammatory responses after cerebral ischemia in vivo are difficult to elucidate because of the complex nature of multiple series of interactions between cells and molecules. This study explored temporal patterns of secretion of 30 cytokines and chemokines from Sprague Dawley rat astrocytes in primary culture in order to elucidate signaling pathways that are triggered by astrocytes during anoxia. Primary cultures of rat brain astrocytes were incubated for periods of 2-24 hr in the absence of oxygen (anoxia) or under normal partial pressure of oxygen (controls). Simultaneous detection of 29 cytokines and chemokines in the samples was performed using a rat cytokine array panel, while the temporal pattern of angiopoietin-1 (Ang-1) secretion was determined separately using ELISA. Wilcoxon-Mann-Whitney test was used to compare normoxic and anoxic samples and the Hodge-Lehman estimator with exact 95% confidence intervals was computed to assess the size of differences in cytokine secretion. The obtained data were imported into the Core Analysis tool of Ingenuity Pathways Analysis software in order to relate changes in secretion of cytokines and chemokines from astrocytes during anoxia to potential molecular signal networks. With the exception of Ang-1, concentrations of all cytokines/chemokines in samples collected after anoxia exposure were either the same, or higher, than in control groups. No clear pattern of changes could be established for groups of cytokines with similar effects (i.e., pro- or anti-inflammatory cytokines). The pattern of changes in cytokine secretion during anoxia was associated with the HIF-1α-mediated response, as well as cytokines IL-1β and cathepsin S pathways, which are related to initiation of inflammation and antigen presentation, respectively, and to ciliary neurotrophic factor. These in vitro findings suggest that astrocytes may play a role in triggering inflammation during anoxia/ischemia of the brain. �

Hypothermia protects against oxygen-glucose deprivation-induced neuronal injury by down-regulating the reverse transport of glutamate by astrocytes as mediated by neurons.

PubMed

Wang, D; Zhao, Y; Zhang, Y; Zhang, T; Shang, X; Wang, J; Liu, Y; Kong, Q; Sun, B; Mu, L; Liu, X; Wang, G; Li, H

2013-05-01

Glutamate is the major mediator of excitotoxic neuronal death following cerebral ischemia. Under severe ischemic conditions, glutamate transporters can functionally reverse to release glutamate, thereby inducing further neuronal injury. Hypothermia has been shown to protect neurons from brain ischemia. However, the mechanism(s) involved remain unclear. Therefore, the aim of this study was to investigate the mechanism(s) mediating glutamate release during brain ischemia-reperfusion injury under hypothermic conditions. Neuron/astrocyte co-cultures were exposed to oxygen-glucose deprivation (OGD) at various temperatures for 2h, and cell viability was assayed 12h after reoxygenation. PI and MAP-2 staining demonstrated that hypothermia significantly decreased neuronal injury. Furthermore, [(3)H]-glutamate uptake assays showed that hypothermia protected rat primary cortical cultures against OGD reoxygenation-induced injury. Protein levels of the astrocytic glutamate transporter, GLT-1, which is primarily responsible for the clearance of extracellular glutamate, were also found to be reduced in a temperature-dependent manner. In contrast, expression of GLT-1 in astrocyte-enriched cultures was found to significantly increase following the addition of neuron-conditioned medium maintained at 37 °C, and to a lesser extent with neuron-conditioned medium at 33 °C. In conclusion, the neuroprotective effects of hypothermia against brain ischemia-reperfusion injury involve down-regulation of astrocytic GLT-1, which mediates the reverse transport of glutamate. Moreover, this process may be regulated by molecules secreted by stressed neurons. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Regulation of NF-{kappa}B activity in astrocytes: effects of flavonoids at dietary-relevant concentrations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spilsbury, Alison; Vauzour, David; Spencer, Jeremy P.E.

Highlights: Black-Right-Pointing-Pointer We tested the hypothesis that low concentrations of flavonoids inhibit NF-{kappa}B in astrocytes. Black-Right-Pointing-Pointer Primary cultured astrocytes possess a functional {kappa}B-system, measured using luciferase assays. Black-Right-Pointing-Pointer Seven flavonoids (100 nM-1 {mu}M) failed to reduce NF-{kappa}B activity in astrocytes. Black-Right-Pointing-Pointer Four flavonoids (100 nM-1 {mu}M) failed to reduce TNFa-stimulated NF-{kappa}B activity in astrocytes. Black-Right-Pointing-Pointer (-)-Epicatechin did not regulate nuclear translocation of the NF-{kappa}B subunit, p65. -- Abstract: Neuroinflammation plays an important role in the progression of neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Sustained activation of nuclear transcription factor {kappa}B (NF-{kappa}B) is thought to play an importantmore » role in the pathogenesis of neurodegenerative disorders. Flavonoids have been shown to possess antioxidant and anti-inflammatory properties and we investigated whether flavonoids, at submicromolar concentrations relevant to their bioavailability from the diet, were able to modulate NF-{kappa}B signalling in astrocytes. Using luciferase reporter assays, we found that tumour necrosis factor (TNF{alpha}, 150 ng/ml) increased NF-{kappa}B-mediated transcription in primary cultures of mouse cortical astrocytes, which was abolished on co-transfection of a dominant-negative I{kappa}B{alpha} construct. In addition, TNF{alpha} increased nuclear localisation of p65 as shown by immunocytochemistry. To investigate potential flavonoid modulation of NF-{kappa}B activity, astrocytes were treated with flavonoids from different classes; flavan-3-ols ((-)-epicatechin and (+)-catechin), flavones (luteolin and chrysin), a flavonol (kaempferol) or the flavanones (naringenin and hesperetin) at dietary-relevant concentrations (0.1-1 {mu}M) for 18 h. None of the flavonoids modulated constitutive or

Spatial organization of astrocytes in ferret visual cortex

PubMed Central

López‐Hidalgo, Mónica; Hoover, Walter B.

2016-01-01

ABSTRACT Astrocytes form an intricate partnership with neural circuits to influence numerous cellular and synaptic processes. One prominent organizational feature of astrocytes is the “tiling” of the brain with non‐overlapping territories. There are some documented species and brain region–specific astrocyte specializations, but the extent of astrocyte diversity and circuit specificity are still unknown. We quantitatively defined the rules that govern the spatial arrangement of astrocyte somata and territory overlap in ferret visual cortex using a combination of in vivo two‐photon imaging, morphological reconstruction, immunostaining, and model simulations. We found that ferret astrocytes share, on average, half of their territory with other astrocytes. However, a specific class of astrocytes, abundant in thalamo‐recipient cortical layers (“kissing” astrocytes), overlap markedly less. Together, these results demonstrate novel features of astrocyte organization indicating that different classes of astrocytes are arranged in a circuit‐specific manner and that tiling does not apply universally across brain regions and species. J. Comp. Neurol. 524:3561–3576, 2016. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc. PMID:27072916

Astrocytic GABA transporter activity modulates excitatory neurotransmission

PubMed Central

Boddum, Kim; Jensen, Thomas P.; Magloire, Vincent; Kristiansen, Uffe; Rusakov, Dmitri A.; Pavlov, Ivan; Walker, Matthew C.

2016-01-01

Astrocytes are ideally placed to detect and respond to network activity. They express ionotropic and metabotropic receptors, and can release gliotransmitters. Astrocytes also express transporters that regulate the extracellular concentration of neurotransmitters. Here we report a previously unrecognized role for the astrocytic GABA transporter, GAT-3. GAT-3 activity results in a rise in astrocytic Na+ concentrations and a consequent increase in astrocytic Ca2+ through Na+/Ca2+ exchange. This leads to the release of ATP/adenosine by astrocytes, which then diffusely inhibits neuronal glutamate release via activation of presynaptic adenosine receptors. Through this mechanism, increases in astrocytic GAT-3 activity due to GABA released from interneurons contribute to 'diffuse' heterosynaptic depression. This provides a mechanism for homeostatic regulation of excitatory transmission in the hippocampus. PMID:27886179

High-Throughput Phenotypic Screening of Human Astrocytes to Identify Compounds That Protect Against Oxidative Stress.

PubMed

Thorne, Natasha; Malik, Nasir; Shah, Sonia; Zhao, Jean; Class, Bradley; Aguisanda, Francis; Southall, Noel; Xia, Menghang; McKew, John C; Rao, Mahendra; Zheng, Wei

2016-05-01

optimized astrocyte differentiation protocol, it was possible to screen approximately 4,100 compounds in titration to identify 22 that are cytoprotective of astrocytes. This study is the largest-scale high-throughput screen conducted using human astrocytes, with a total of 17,536 data points collected in the primary screen. The results demonstrate the relevancy and utility of using astrocytes differentiated from human stem cells as a disease model for drug discovery and development. ©AlphaMed Press.

α-Synuclein transfer between neurons and astrocytes indicates that astrocytes play a role in degradation rather than in spreading.

PubMed

Loria, Frida; Vargas, Jessica Y; Bousset, Luc; Syan, Sylvie; Salles, Audrey; Melki, Ronald; Zurzolo, Chiara

2017-11-01

Recent evidence suggests that disease progression in Parkinson's disease (PD) could occur by the spreading of α-synuclein (α-syn) aggregates between neurons. Here we studied the role of astrocytes in the intercellular transfer and fate of α-syn fibrils, using in vitro and ex vivo models. α-Syn fibrils can be transferred to neighboring cells; however, the transfer efficiency changes depending on the cell types. We found that α-syn is efficiently transferred from astrocytes to astrocytes and from neurons to astrocytes, but less efficiently from astrocytes to neurons. Interestingly, α-syn puncta are mainly found inside the lysosomal compartments of the recipient cells. However, differently from neurons, astrocytes are able to efficiently degrade fibrillar α-syn, suggesting an active role for these cells in clearing α-syn deposits. Astrocytes co-cultured with organotypic brain slices are able to take up α-syn fibrils from the slices. Altogether our data support a role for astrocytes in trapping and clearing α-syn pathological deposits in PD.

STAT3 labels a subpopulation of reactive astrocytes required for brain metastasis.

PubMed

Priego, Neibla; Zhu, Lucía; Monteiro, Cátia; Mulders, Manon; Wasilewski, David; Bindeman, Wendy; Doglio, Laura; Martínez, Liliana; Martínez-Saez, Elena; Cajal, Santiago Ramón Y; Megías, Diego; Hernández-Encinas, Elena; Blanco-Aparicio, Carmen; Martínez, Lola; Zarzuela, Eduardo; Muñoz, Javier; Fustero-Torres, Coral; Pineiro, Elena; Hernández-Laín, Aurelio; Bertero, Luca; Poli, Valeria; Sánchez-Martínez, Melchor; Menendez, Javier A; Soffietti, Riccardo; Bosch-Barrera, Joaquim; Valiente, Manuel

2018-06-11

The brain microenvironment imposes a particularly intense selective pressure on metastasis-initiating cells, but successful metastases bypass this control through mechanisms that are poorly understood. Reactive astrocytes are key components of this microenvironment that confine brain metastasis without infiltrating the lesion. Here, we describe that brain metastatic cells induce and maintain the co-option of a pro-metastatic program driven by signal transducer and activator of transcription 3 (STAT3) in a subpopulation of reactive astrocytes surrounding metastatic lesions. These reactive astrocytes benefit metastatic cells by their modulatory effect on the innate and acquired immune system. In patients, active STAT3 in reactive astrocytes correlates with reduced survival from diagnosis of intracranial metastases. Blocking STAT3 signaling in reactive astrocytes reduces experimental brain metastasis from different primary tumor sources, even at advanced stages of colonization. We also show that a safe and orally bioavailable treatment that inhibits STAT3 exhibits significant antitumor effects in patients with advanced systemic disease that included brain metastasis. Responses to this therapy were notable in the central nervous system, where several complete responses were achieved. Given that brain metastasis causes substantial morbidity and mortality, our results identify a novel treatment for increasing survival in patients with secondary brain tumors.

Derivation of Functional Human Astrocytes from Cerebral Organoids

PubMed Central

Dezonne, Rômulo Sperduto; Sartore, Rafaela Costa; Nascimento, Juliana Minardi; Saia-Cereda, Verônica M.; Romão, Luciana Ferreira; Alves-Leon, Soniza Vieira; de Souza, Jorge Marcondes; Martins-de-Souza, Daniel; Rehen, Stevens Kastrup; Gomes, Flávia Carvalho Alcantara

2017-01-01

Astrocytes play a critical role in the development and homeostasis of the central nervous system (CNS). Astrocyte dysfunction results in several neurological and degenerative diseases. However, a major challenge to our understanding of astrocyte physiology and pathology is the restriction of studies to animal models, human post-mortem brain tissues, or samples obtained from invasive surgical procedures. Here, we report a protocol to generate human functional astrocytes from cerebral organoids derived from human pluripotent stem cells. The cellular isolation of cerebral organoids yielded cells that were morphologically and functionally like astrocytes. Immunolabelling and proteomic assays revealed that human organoid-derived astrocytes express the main astrocytic molecular markers, including glutamate transporters, specific enzymes and cytoskeletal proteins. We found that organoid-derived astrocytes strongly supported neuronal survival and neurite outgrowth and responded to ATP through transient calcium wave elevations, which are hallmarks of astrocyte physiology. Additionally, these astrocytes presented similar functional pathways to those isolated from adult human cortex by surgical procedures. This is the first study to provide proteomic and functional analyses of astrocytes isolated from human cerebral organoids. The isolation of these astrocytes holds great potential for the investigation of developmental and evolutionary features of the human brain and provides a useful approach to drug screening and neurodegenerative disease modelling. PMID:28345587

Spatial organization of astrocytes in ferret visual cortex.

PubMed

López-Hidalgo, Mónica; Hoover, Walter B; Schummers, James

2016-12-01

Astrocytes form an intricate partnership with neural circuits to influence numerous cellular and synaptic processes. One prominent organizational feature of astrocytes is the "tiling" of the brain with non-overlapping territories. There are some documented species and brain region-specific astrocyte specializations, but the extent of astrocyte diversity and circuit specificity are still unknown. We quantitatively defined the rules that govern the spatial arrangement of astrocyte somata and territory overlap in ferret visual cortex using a combination of in vivo two-photon imaging, morphological reconstruction, immunostaining, and model simulations. We found that ferret astrocytes share, on average, half of their territory with other astrocytes. However, a specific class of astrocytes, abundant in thalamo-recipient cortical layers ("kissing" astrocytes), overlap markedly less. Together, these results demonstrate novel features of astrocyte organization indicating that different classes of astrocytes are arranged in a circuit-specific manner and that tiling does not apply universally across brain regions and species. J. Comp. Neurol. 524:3561-3576, 2016. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

DJ-1 KNOCK-DOWN IMPAIRS ASTROCYTE MITOCHONDRIAL FUNCTION

PubMed Central

LARSEN, N. J.; AMBROSI, G.; MULLETT, S. J.; BERMAN, S. B.; HINKLE, D. A.

2012-01-01

Mitochondrial dysfunction has long been implicated in the pathogenesis of Parkinson’s disease (PD). PD brain tissues show evidence for mitochondrial respiratory chain Complex I deficiency. Pharmacological inhibitors of Complex I, such as rotenone, cause experimental parkinsonism. The cytoprotective protein DJ-1, whose deletion is sufficient to cause genetic PD, is also known to have mitochondria-stabilizing properties. We have previously shown that DJ-1 is over-expressed in PD astrocytes, and that DJ-1 deficiency impairs the capacity of astrocytes to protect co-cultured neurons against rotenone. Since DJ-1 modulated, astrocyte-mediated neuroprotection against rotenone may depend upon proper astrocytic mitochondrial functioning, we hypothesized that DJ-1 deficiency would impair astrocyte mitochondrial motility, fission/fusion dynamics, membrane potential maintenance, and respiration, both at baseline and as an enhancement of rotenone-induced mitochondrial dysfunction. In astrocyte-enriched cultures, we observed that DJ-1 knock-down reduced mitochondrial motility primarily in the cellular processes of both untreated and rotenone treated cells. In these same cultures, DJ-1 knock-down did not appreciably affect mitochondrial fission, fusion, or respiration, but did enhance rotenone-induced reductions in the mitochondrial membrane potential. In neuron–astrocyte co-cultures, astrocytic DJ-1 knock-down reduced astrocyte process mitochondrial motility in untreated cells, but this effect was not maintained in the presence of rotenone. In the same co-cultures, astrocytic DJ-1 knock-down significantly reduced mitochondrial fusion in the astrocyte cell bodies, but not the processes, under the same conditions of rotenone treatment in which DJ-1 deficiency is known to impair astrocyte-mediated neuroprotection. Our studies therefore demonstrated the following new findings: (i) DJ-1 deficiency can impair astrocyte mitochondrial physiology at multiple levels, (ii) astrocyte

Astrocyte glycogen and brain energy metabolism.

PubMed

Brown, Angus M; Ransom, Bruce R

2007-09-01

The brain contains glycogen but at low concentration compared with liver and muscle. In the adult brain, glycogen is found predominantly in astrocytes. Astrocyte glycogen content is modulated by a number of factors including some neurotransmitters and ambient glucose concentration. Compelling evidence indicates that astrocyte glycogen breaks down during hypoglycemia to lactate that is transferred to adjacent neurons or axons where it is used aerobically as fuel. In the case of CNS white matter, this source of energy can extend axon function for 20 min or longer. Likewise, during periods of intense neural activity when energy demand exceeds glucose supply, astrocyte glycogen is degraded to lactate, a portion of which is transferred to axons for fuel. Astrocyte glycogen, therefore, offers some protection against hypoglycemic neural injury and ensures that neurons and axons can maintain their function during very intense periods of activation. These emerging principles about the roles of astrocyte glycogen contradict the long held belief that this metabolic pool has little or no functional significance.

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

PubMed Central

Szlachetka, Adam M.; Haorah, James

2011-01-01

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

Computational Models for Calcium-Mediated Astrocyte Functions.

PubMed

Manninen, Tiina; Havela, Riikka; Linne, Marja-Leena

2018-01-01

The computational neuroscience field has heavily concentrated on the modeling of neuronal functions, largely ignoring other brain cells, including one type of glial cell, the astrocytes. Despite the short history of modeling astrocytic functions, we were delighted about the hundreds of models developed so far to study the role of astrocytes, most often in calcium dynamics, synchronization, information transfer, and plasticity in vitro , but also in vascular events, hyperexcitability, and homeostasis. Our goal here is to present the state-of-the-art in computational modeling of astrocytes in order to facilitate better understanding of the functions and dynamics of astrocytes in the brain. Due to the large number of models, we concentrated on a hundred models that include biophysical descriptions for calcium signaling and dynamics in astrocytes. We categorized the models into four groups: single astrocyte models, astrocyte network models, neuron-astrocyte synapse models, and neuron-astrocyte network models to ease their use in future modeling projects. We characterized the models based on which earlier models were used for building the models and which type of biological entities were described in the astrocyte models. Features of the models were compared and contrasted so that similarities and differences were more readily apparent. We discovered that most of the models were basically generated from a small set of previously published models with small variations. However, neither citations to all the previous models with similar core structure nor explanations of what was built on top of the previous models were provided, which made it possible, in some cases, to have the same models published several times without an explicit intention to make new predictions about the roles of astrocytes in brain functions. Furthermore, only a few of the models are available online which makes it difficult to reproduce the simulation results and further develop the models. Thus

Computational Models for Calcium-Mediated Astrocyte Functions

PubMed Central

Manninen, Tiina; Havela, Riikka; Linne, Marja-Leena

2018-01-01

The computational neuroscience field has heavily concentrated on the modeling of neuronal functions, largely ignoring other brain cells, including one type of glial cell, the astrocytes. Despite the short history of modeling astrocytic functions, we were delighted about the hundreds of models developed so far to study the role of astrocytes, most often in calcium dynamics, synchronization, information transfer, and plasticity in vitro, but also in vascular events, hyperexcitability, and homeostasis. Our goal here is to present the state-of-the-art in computational modeling of astrocytes in order to facilitate better understanding of the functions and dynamics of astrocytes in the brain. Due to the large number of models, we concentrated on a hundred models that include biophysical descriptions for calcium signaling and dynamics in astrocytes. We categorized the models into four groups: single astrocyte models, astrocyte network models, neuron-astrocyte synapse models, and neuron-astrocyte network models to ease their use in future modeling projects. We characterized the models based on which earlier models were used for building the models and which type of biological entities were described in the astrocyte models. Features of the models were compared and contrasted so that similarities and differences were more readily apparent. We discovered that most of the models were basically generated from a small set of previously published models with small variations. However, neither citations to all the previous models with similar core structure nor explanations of what was built on top of the previous models were provided, which made it possible, in some cases, to have the same models published several times without an explicit intention to make new predictions about the roles of astrocytes in brain functions. Furthermore, only a few of the models are available online which makes it difficult to reproduce the simulation results and further develop the models. Thus

Zinc promotes the death of hypoxic astrocytes by upregulating hypoxia-induced hypoxiainducible factor-1alpha expression via Poly(ADP-ribose) polymerase -1

PubMed Central

Pan, Rong; Chen, Chen; Liu, Wenlan; Liu, Ke Jian

2013-01-01

Aim Pathological release of excess zinc ions has been implicated in ischemic brain cell death. However, the underlying mechanisms remain to be elucidated. In stroke, ischemia-induced zinc release and hypoxia-inducible factor-1 (HIF-1) accumulation concurrently occur in the ischemic tissue. The present study testes the hypothesis that the presence of high intracellular zinc concentration is a major cause of modifications to PARP-1 and HIF-1α during hypoxia, which significantly contributes to cell death during ischemia. Methods Primary cortical astrocytes and C8-D1A cells were exposed to different concentrations of zinc chloride. Cell death rate and protein expression of HIF-1 and Poly(ADP-ribose) polymerase (PARP)-1 were examined after 3-hour hypoxic treatment. Results Although 3-hr hypoxia or 100 μM of zinc alone did not induce noticeable cytotoxicity, their combination led to a dramatic increase in astrocytic cell death in a zinc concentration dependent manner. Exposure of astrocytes to hypoxia for 3-hr remarkably increased the levels of intracellular zinc and HIF-1α protein, which was further augmented by added exogenous zinc. Notably HIF-1α knockdown blocked zinc-induced astrocyte death. Moreover, knockdown of PARP-1, another important protein in the response of hypoxia, attenuated the overexpression of HIF-1α and reduced the cell death rate. Conclusions Our studies show that zinc promotes hypoxic cell death through overexpression of the hypoxia response factor HIF-1α via the cell fate determine factor PARP-1 modification, which provides a novel mechanism for zinc-mediated ischemic brain injury. PMID:23582235

Expression of decay-accelerating factor (CD55), membrane cofactor protein (CD46) and CD59 in the human astroglioma cell line, D54-MG, and primary rat astrocytes.

PubMed

Yang, C; Jones, J L; Barnum, S R

1993-09-01

In this report, we have shown the expression of the complement regulatory proteins decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and CD59 on human D54-MG astroglioma cells by several methods, including immunofluorescence, flow cytometry and Western blotting and Northern blot analysis. These studies demonstrate that all three proteins are structurally and antigenically similar to their counterparts expressed on HepG2 and SW480 cells (hepatocyte and epithelial cell lines, respectively). D54-MG cells express mRNA for all three proteins of the appropriate size(s). The phosphatidylinositol-specific enzyme, PIPLC, cleaved DAF from the surface of D54-MG cells, demonstrating that DAF is linked by a glycophospholipid anchor as has been shown for other cell types. Flow cytometry demonstrates that primary rat astrocytes also constitutively express all three regulatory proteins. These data are the first to demonstrate the expression of CD59 on astrocytes, and the presence of all three regulatory proteins on astrocytes suggests that regulation of complement activation in the central nervous system is important in neural host defense mechanisms.

Monocarboxylate transporter-dependent mechanism confers resistance to oxygen- and glucose-deprivation injury in astrocyte-neuron co-cultures.

PubMed

Gao, Chen; Zhou, Liya; Zhu, Wenxia; Wang, Hongyun; Wang, Ruijuan; He, Yunfei; Li, Zhiyun

2015-05-06

Hypoxic and low-glucose stressors contribute to neuronal death in many brain diseases. Astrocytes are anatomically well-positioned to shield neurons from hypoxic injury. During hypoxia/ischemia, lactate released from astrocytes is taken up by neurons and stored for energy. This process is mediated by monocarboxylate transporters (MCTs) in the central nervous system. In the present study, we investigated the ability of astrocytes to protect neurons from oxygen- and glucose-deprivation (OGD) injury via an MCT-dependent mechanism in vitro. Primary cultures of neurons, astrocytes, and astrocytes-neurons derived from rat hippocampus were subjected to OGD, MCT inhibition with small interfering (si)RNA. Cell survival and expression of MCT4, MCT2, glial fibrillary acidic protein, and neuronal nuclear antigen were evaluated. OGD significantly increased cell death in neuronal cultures and up-regulated MCT4 expression in astrocyte cultures, but no increased cell death was observed in neuron-astrocyte co-cultures or astrocyte cultures. However, neuronal cell death in co-cultures was increased by exposure to MCT4- or MCT2-specific siRNA, and this effect was attenuated by the addition of lactate into the extracellular medium of neuronal cultures prior to OGD. These findings demonstrate that resistance to OGD injury in astrocyte-neuron co-cultures occurs via an MCT-dependent mechanism. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Tlx acts as a proangiogenic switch by regulating extracellular assembly of fibronectin matrices in retinal astrocytes.

PubMed

Uemura, Akiyoshi; Kusuhara, Sentaro; Wiegand, Stanley J; Yu, Ruth T; Nishikawa, Shin-ichi

2006-02-01

In response to hypoxia, hypoxia-inducible factors act as the primary proangiogenic triggers by regulating transcription levels of target genes, including VEGF. However, little is known about the specific factors that control other components of the angiogenic process, particularly formation of matrix scaffolds that promote adhesion and migration of endothelial cells. We show that in the postnatal mouse retina, the orphan nuclear receptor tailless (Tlx) is strongly expressed in the proangiogenic astrocytes, which secrete VEGF and fibronectin. Tlx expression by retinal astrocytes is controlled by oxygen concentration and rapidly downregulated upon contact with blood vessels. In mice null for Tlx, retinal astrocytes maintain VEGF expression; however, the extracellular assembly of fibronectin matrices by astrocytes is severely impaired, leading to defective scaffold formation and a complete failure of normal retinal vascular development. This work identifies Tlx as an essential component of the molecular network involved in the hypoxia-inducible proangiogenic switch in retinal astrocytes.

Tlx acts as a proangiogenic switch by regulating extracellular assembly of fibronectin matrices in retinal astrocytes

PubMed Central

Uemura, Akiyoshi; Kusuhara, Sentaro; Wiegand, Stanley J.; Yu, Ruth T.; Nishikawa, Shin-Ichi

2006-01-01

In response to hypoxia, hypoxia-inducible factors act as the primary proangiogenic triggers by regulating transcription levels of target genes, including VEGF. However, little is known about the specific factors that control other components of the angiogenic process, particularly formation of matrix scaffolds that promote adhesion and migration of endothelial cells. We show that in the postnatal mouse retina, the orphan nuclear receptor tailless (Tlx) is strongly expressed in the proangiogenic astrocytes, which secrete VEGF and fibronectin. Tlx expression by retinal astrocytes is controlled by oxygen concentration and rapidly downregulated upon contact with blood vessels. In mice null for Tlx, retinal astrocytes maintain VEGF expression; however, the extracellular assembly of fibronectin matrices by astrocytes is severely impaired, leading to defective scaffold formation and a complete failure of normal retinal vascular development. This work identifies Tlx as an essential component of the molecular network involved in the hypoxia-inducible proangiogenic switch in retinal astrocytes. PMID:16424942

Rat Nucleus Accumbens Core Astrocytes Modulate Reward and the Motivation to Self-Administer Ethanol after Abstinence

PubMed Central

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

2014-01-01

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

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

PubMed

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

2014-11-01

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

Differential Pro-Inflammatory Responses of Astrocytes and Microglia Involve STAT3 Activation in Response to 1800 MHz Radiofrequency Fields

PubMed Central

Lu, Yonghui; He, Mindi; Zhang, Yang; Xu, Shangcheng; Zhang, Lei; He, Yue; Chen, Chunhai; Liu, Chuan; Pi, Huifeng; Yu, Zhengping; Zhou, Zhou

2014-01-01

Microglia and astrocytes play important role in maintaining the homeostasis of central nervous system (CNS). Several CNS impacts have been postulated to be associated with radiofrequency (RF) electromagnetic fields exposure. Given the important role of inflammation in neural physiopathologic processes, we investigated the pro-inflammatory responses of microglia and astrocytes and the involved mechanism in response to RF fields. Microglial N9 and astroglial C8-D1A cells were exposed to 1800 MHz RF for different time with or without pretreatment with STAT3 inhibitor. Microglia and astrocytes were activated by RF exposure indicated by up-regulated CD11b and glial fibrillary acidic protein (GFAP). However, RF exposure induced differential pro-inflammatory responses in astrocytes and microglia, characterized by different expression and release profiles of IL-1β, TNF-α, IL-6, PGE2, nitric oxide (NO), inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2). Moreover, the RF exposure activated STAT3 in microglia but not in astrocytes. Furthermore, the STAT3 inhibitor Stattic ameliorated the RF-induced release of pro-inflammatory cytokines in microglia but not in astrocytes. Our results demonstrated that RF exposure differentially induced pro-inflammatory responses in microglia and astrocytes, which involved differential activation of STAT3 in microglia and astrocytes. Our data provide novel insights into the potential mechanisms of the reported CNS impacts associated with mobile phone use and present STAT3 as a promising target to protect humans against increasing RF exposure. PMID:25275372

Differential pro-inflammatory responses of astrocytes and microglia involve STAT3 activation in response to 1800 MHz radiofrequency fields.

PubMed

Lu, Yonghui; He, Mindi; Zhang, Yang; Xu, Shangcheng; Zhang, Lei; He, Yue; Chen, Chunhai; Liu, Chuan; Pi, Huifeng; Yu, Zhengping; Zhou, Zhou

2014-01-01

Microglia and astrocytes play important role in maintaining the homeostasis of central nervous system (CNS). Several CNS impacts have been postulated to be associated with radiofrequency (RF) electromagnetic fields exposure. Given the important role of inflammation in neural physiopathologic processes, we investigated the pro-inflammatory responses of microglia and astrocytes and the involved mechanism in response to RF fields. Microglial N9 and astroglial C8-D1A cells were exposed to 1800 MHz RF for different time with or without pretreatment with STAT3 inhibitor. Microglia and astrocytes were activated by RF exposure indicated by up-regulated CD11b and glial fibrillary acidic protein (GFAP). However, RF exposure induced differential pro-inflammatory responses in astrocytes and microglia, characterized by different expression and release profiles of IL-1β, TNF-α, IL-6, PGE2, nitric oxide (NO), inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2). Moreover, the RF exposure activated STAT3 in microglia but not in astrocytes. Furthermore, the STAT3 inhibitor Stattic ameliorated the RF-induced release of pro-inflammatory cytokines in microglia but not in astrocytes. Our results demonstrated that RF exposure differentially induced pro-inflammatory responses in microglia and astrocytes, which involved differential activation of STAT3 in microglia and astrocytes. Our data provide novel insights into the potential mechanisms of the reported CNS impacts associated with mobile phone use and present STAT3 as a promising target to protect humans against increasing RF exposure.

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

PubMed

Vardjan, Nina; Kreft, Marko; Zorec, Robert

2014-04-01

The morphology of astrocytes, likely regulated by cAMP, determines the structural association between astrocytes and the synapse, consequently modulating synaptic function. β-Adrenergic receptors (β-AR), which increase cytosolic cAMP concentration ([cAMP]i ), may affect cell morphology. However, the real-time dynamics of β-AR-mediated cAMP signaling in single live astrocytes and its effect on cell morphology have not been studied. We used the fluorescence resonance energy transfer (FRET)-based cAMP biosensor Epac1-camps to study time-dependent changes in [cAMP]i ; morphological changes in primary rat astrocytes were monitored by real-time confocal microscopy. Stimulation of β-AR by adrenaline, noradrenaline, and isoprenaline, a specific agonist of β-AR, rapidly increased [cAMP]i (∼15 s). The FRET signal response, mediated via β-AR, was faster than in the presence of forskolin (twofold) and dibutyryl-cAMP (>35-fold), which directly activate adenylyl cyclase and Epac1-camps, respectively, likely due to slow entry of these agents into the cytosol. Oscillations in [cAMP]i have not been recorded, indicating that cAMP-dependent processes operate in a slow time domain. Most Epac1-camps expressing astrocytes revealed a morphological change upon β-AR activation and attained a stellate morphology within 1 h. The morphological changes exhibited a bell-shaped dependency on [cAMP]i . The 5-10% decrease in cell cross-sectional area and the 30-50% increase in cell perimeter are likely due to withdrawal of the cytoplasm to the perinuclear region and the appearance of protrusions on the surface of astrocytes. Because astrocyte processes ensheath neurons, β-AR/cAMP-mediated morphological changes can modify the geometry of the extracellular space, affecting synaptic, neuronal, and astrocyte functions in health and disease. Copyright © 2014 Wiley Periodicals, Inc.

Epilepsy and astrocyte energy metabolism.

PubMed

Boison, Detlev; Steinhäuser, Christian

2018-06-01

Epilepsy is a complex neurological syndrome characterized by neuronal hyperexcitability and sudden, synchronized electrical discharges that can manifest as seizures. It is now increasingly recognized that impaired astrocyte function and energy homeostasis play key roles in the pathogenesis of epilepsy. Excessive neuronal discharges can only happen, if adequate energy sources are made available to neurons. Conversely, energy depletion during seizures is an endogenous mechanism of seizure termination. Astrocytes control neuronal energy homeostasis through neurometabolic coupling. In this review, we will discuss how astrocyte dysfunction in epilepsy leads to distortion of key metabolic and biochemical mechanisms. Dysfunctional glutamate metabolism in astrocytes can directly contribute to neuronal hyperexcitability. Closure of astrocyte intercellular gap junction coupling as observed early during epileptogenesis limits activity-dependent trafficking of energy metabolites, but also impairs clearance of the extracellular space from accumulation of K + and glutamate. Dysfunctional astrocytes also increase the metabolism of adenosine, a metabolic product of ATP degradation that broadly inhibits energy-consuming processes as an evolutionary adaptation to conserve energy. Due to the critical role of astroglial energy homeostasis in the control of neuronal excitability, metabolic therapeutic approaches that prevent the utilization of glucose might represent a potent antiepileptic strategy. In particular, high fat low carbohydrate "ketogenic diets" as well as inhibitors of glycolysis and lactate metabolism are of growing interest for the therapy of epilepsy. © 2017 Wiley Periodicals, Inc.

Glucose-Coated Gold Nanoparticles Transfer across Human Brain Endothelium and Enter Astrocytes In Vitro

PubMed Central

Gromnicova, Radka; Davies, Heather A.; Sreekanthreddy, Peddagangannagari; Romero, Ignacio A.; Lund, Torben; Roitt, Ivan M.; Phillips, James B.; Male, David K.

2013-01-01

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. PMID:24339894

Adrenoceptors in Brain: Cellular Gene Expression and Effects on Astrocytic Metabolism and [Ca2+]i

PubMed Central

Hertz, Leif; Lovatt, Ditte; Goldman, Steven A.; Nedergaard, Maiken

2010-01-01

Recent in vivo studies have established astrocytes as a major target for locus coeruleus activation (Bekar et al., Cereb. Cortex 18, 2789–2795), renewing interest in cell culture studies on noradrenergic effects on astrocytes in primary cultures and calling for additional information about the expression of adrenoceptor subtypes on different types of brain cells. In the present communication, mRNA expression of α1-, α2- and β-adrenergic receptors and their subtypes was determined in freshly-isolated, cell marker-defined populations of astrocytes, NG2-positive cells, microglia, endothelial cells, and Thy1-positive neurons (mainly glutamatergic projection neurons) in murine cerebral cortex. Immediately after dissection of frontal, parietal and occipital cortex of 10–12-week-old transgenic mice, which combined each cell-type marker with a specific fluorescent signal, the tissue was digested, triturated and centrifuged, yielding a solution of dissociated cells of all types, which were separated by fluorescence-activated cell sorting (FACS). mRNA expression in each cell fraction was determined by microarray analysis. α1A-Receptors were unequivocally expressed in astrocytes and NG2-positive cells, but absent in other cell types, and α1B-receptors were not expressed in any cell population. Among α2-receptors only α2A-receptors were expressed, unequivocally in astrocytes and NG-positive cells, tentatively in microglia and questionably in Thy1-positive neurons and endothelial cells. β1-Receptors were unequivocally expressed in astrocytes, tentatively in microglia, and questionably in neurons and endothelial cells, whereas β2-adrenergic receptors showed tentative expression in neurons and astrocytes and unequivocal expression in other cell types. This distribution was supported by immunochemical data and its relevance established by previous studies in well-differentiated primary cultures of mouse astrocytes, showing that stimulation of α2-adrenoceptors

Astrocytes.

ERIC Educational Resources Information Center

Kimelberg, Harold K.; Norenberg, Michael D.

1989-01-01

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

Synapse-specific astrocyte gating of amygdala-related behavior.

PubMed

Martin-Fernandez, Mario; Jamison, Stephanie; Robin, Laurie M; Zhao, Zhe; Martin, Eduardo D; Aguilar, Juan; Benneyworth, Michael A; Marsicano, Giovanni; Araque, Alfonso

2017-11-01

The amygdala plays key roles in fear and anxiety. Studies of the amygdala have largely focused on neuronal function and connectivity. Astrocytes functionally interact with neurons, but their role in the amygdala remains largely unknown. We show that astrocytes in the medial subdivision of the central amygdala (CeM) determine the synaptic and behavioral outputs of amygdala circuits. To investigate the role of astrocytes in amygdala-related behavior and identify the underlying synaptic mechanisms, we used exogenous or endogenous signaling to selectively activate CeM astrocytes. Astrocytes depressed excitatory synapses from basolateral amygdala via A 1 adenosine receptor activation and enhanced inhibitory synapses from the lateral subdivision of the central amygdala via A 2A receptor activation. Furthermore, astrocytic activation decreased the firing rate of CeM neurons and reduced fear expression in a fear-conditioning paradigm. Therefore, we conclude that astrocyte activity determines fear responses by selectively regulating specific synapses, which indicates that animal behavior results from the coordinated activity of neurons and astrocytes.

Astrocytes in the tempest of multiple sclerosis.

PubMed

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

2011-12-01

Astrocytes are the most abundant cell population within the CNS of mammals. Their glial role is perfectly performed in the healthy CNS as they support functions of neurons. The omnipresence of astrocytes throughout the white and grey matter and their intimate relation with blood vessels of the CNS, as well as numerous immunity-related actions that these cells are capable of, imply that astrocytes should have a prominent role in neuroinflammatory disorders, such as multiple sclerosis (MS). The role of astrocytes in MS is rather ambiguous, as they have the capacity to both stimulate and restrain neuroinflammation and tissue destruction. In this paper we present some of the proved and the proposed functions of astrocytes in neuroinflammation and discuss the effect of MS therapeutics on astrocytes. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Why are astrocytes important?

PubMed

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.

Erythropoietin modulation of astrocyte water permeability as a component of neuroprotection

PubMed Central

Gunnarson, Eli; Song, Yutong; Kowalewski, Jacob M.; Brismar, Hjalmar; Brines, Michael; Cerami, Anthony; Andersson, Ulf; Zelenina, Marina; Aperia, Anita

2009-01-01

Disturbed brain water homeostasis with swelling of astroglial cells is a common complication in stroke, trauma, and meningitis and is considered to be a major cause of permanent brain damage. Astroglial cells possess the water channel aquaporin 4 (AQP4). Recent studies from our laboratory have shown that glutamate, acting on group I metabotropic glutamate receptors (mGluRs), increases the permeability of astrocyte AQP4, which, in situations of hypoxia-ischemia, will increase astrocyte water uptake. Here we report that erythropoietin (EPO), which in recent years has emerged as a potent neuro-protective agent, antagonizes the effect of a group I mGluR agonist on astrocyte water permeability. Activation of group I mGluRs triggers fast and highly regular intracellular calcium oscillations and we show that EPO interferes with this signaling event by altering the frequency of the oscillations. These effects of EPO are immediate, in contrast to the neuroprotective effects of EPO that are known to depend upon gene activation. Our findings indicate that EPO may directly reduce the risk of astrocyte swelling in stroke and other brain insults. In support of this conclusion we found that EPO reduced the neurological symptoms in a mouse model of primary brain edema known to depend upon AQP4 water transport. PMID:19164545

Elusive roles for reactive astrocytes in neurodegenerative diseases

PubMed Central

Ben Haim, Lucile; Carrillo-de Sauvage, Maria-Angeles; Ceyzériat, Kelly; Escartin, Carole

2015-01-01

Astrocytes play crucial roles in the brain and are involved in the neuroinflammatory response. They become reactive in response to virtually all pathological situations in the brain such as axotomy, ischemia, infection, and neurodegenerative diseases (ND). Astrocyte reactivity was originally characterized by morphological changes (hypertrophy, remodeling of processes) and the overexpression of the intermediate filament glial fibrillary acidic protein (GFAP). However, it is unclear how the normal supportive functions of astrocytes are altered by their reactive state. In ND, in which neuronal dysfunction and astrocyte reactivity take place over several years or decades, the issue is even more complex and highly debated, with several conflicting reports published recently. In this review, we discuss studies addressing the contribution of reactive astrocytes to ND. We describe the molecular triggers leading to astrocyte reactivity during ND, examine how some key astrocyte functions may be enhanced or altered during the disease process, and discuss how astrocyte reactivity may globally affect ND progression. Finally we will consider the anticipated developments in this important field. With this review, we aim to show that the detailed study of reactive astrocytes may open new perspectives for ND. PMID:26283915

Metallothionein-3 modulates the amyloid β endocytosis of astrocytes through its effects on actin polymerization.

PubMed

Lee, Sook-Jeong; Seo, Bo-Ra; Koh, Jae-Young

2015-12-04

Astrocytes may play important roles in the pathogenesis of Alzheimer's disease (AD) by clearing extracellular amyloid beta (Aβ) through endocytosis and degradation. We recently showed that metallothionein 3 (Mt3), a zinc-binding metallothionein that is enriched in the central nervous system, contributes to actin polymerization in astrocytes. Because actin is likely involved in the endocytosis of Aβ, we investigated the possible role of Mt3 in Aβ endocytosis by cortical astrocytes in this study. To assess the route of Aβ uptake, we exposed cultured astrocytes to fluorescently labeled Aβ1-40 or Aβ1-42 together with chloropromazine (CP) or methyl-beta-cyclodextrin (MβCD), inhibitors of clathrin- and caveolin-dependent endocytosis, respectively. CP treatment almost completely blocked Aβ1-40 and Aβ1-42 endocytosis, whereas exposure to MβCD had no significant effect. Actin disruption with cytochalasin D (CytD) or latrunculin B also completely blocked Aβ1-40 and Aβ1-42 endocytosis. Because the absence of Mt3 also results in actin disruption, we examined Aβ1-40 and Aβ1-42 uptake and expression in Mt3 (-/-) astrocytes. Compared with wild-type (WT) cells, Mt3 (-/-) cells exhibited markedly reduced Aβ1-40 and Aβ1-42 endocytosis and expression of Aβ1-42 monomers and oligomers. A similar reduction was observed in CytD-treated WT cells. Finally, actin disruption and Mt3 knockout each increased the overall levels of clathrin and the associated protein phosphatidylinositol-binding clathrin assembly protein (PICALM) in astrocytes. Our results suggest that the absence of Mt3 reduces Aβ uptake in astrocytes through an abnormality in actin polymerization. In light of evidence that Mt3 is downregulated in AD, our findings indicate that this mechanism may contribute to the extracellular accumulation of Aβ in this disease.

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

PubMed

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

2016-10-10

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

Hydrostatic pressure-dependent changes in cyclic AMP signaling in optic nerve head astrocytes from Caucasian and African American donors

PubMed Central

Chen, Lin; Hernandez, M. Rosario

2009-01-01

Purpose Investigate the effect of hydrostatic pressure (HP) on 3′, 5′-cyclic adenosine monophosphate (cAMP) levels and downstream signaling in cultures of normal optic nerve head (ONH) astrocytes from Caucasian American (CA) and African American (AA) donors. Methods Intracellular cAMP levels were assayed after exposing ONH astrocytes to HP for varying times. Quantitative RT–PCR was used to determine the expression levels of selected cAMP pathway genes in human ONH astrocytes after HP treatment. Western blots were used to measure changes in the phosphorylation state of cAMP response element binding protein (CREB) in astrocytes subjected to HP, ATP, and phosphodiesterase or kinase inhibitors. Results The basal intracellular cAMP level is similar among AA and CA astrocytes. After exposure to HP for 15 min and 30 min in the presence of a phosphodiesterase inhibitor a further increase of intracellular cAMP was observed in AA astrocytes, but not in CA astrocytes. Consistent with activation of the cAMP-dependent protein kinase pathway, CREB phosphorylation (Ser-133) was increased to a greater extent in AA than in CA astrocytes after 3 h of HP. Exposure to elevated HP for 3–6 h differentially altered the expression levels of selected cAMP pathway genes (ADCY3, ADCY9, PTHLH, PDE7B) in AA compared to CA astrocytes. Treatment with ATP increased more CREB phosphorylation in CA than in AA astrocytes, suggesting differential Ca2+ signaling in these populations. Conclusions Activation of the cAMP-dependent signaling pathway by pressure may be an important contributor to increased susceptibility to elevated intraocular pressure and glaucoma in AA, a population at higher risk for the disease. PMID:19710943

αB-crystallin negative astrocytic inclusions.

PubMed

Barnett, Brad P; Bressler, Joseph; Chen, Terina; Hutchins, Grover M; Crain, Barbara J; Kaufmann, Walter E

2011-04-01

We report on an unusual pathological finding of astrocytes, observed in the brain of a 16-year-old African-American male with severe intellectual disability and spastic quadriplegia. The brain showed bilateral pericentral, perisylvian polymicrogyria and pachygyria, in conjunction with a large number of hypertrophic astrocytes with eosinophilic granular cytoplasmic inclusions. The astrocytic abnormality was more severe in the dysgenetic area but present throughout the cerebral cortex. Astrocytic inclusions stained with acid fuchsin, azocarmine and Holzer's stain, and were immunoreactive for GFAP, S-100, and ubiquitin, but not for αB-crystallin, filamin, vimentin, nestin, tau or α-synuclein. Based on the case and a review of the literature, the authors postulate that these astrocytic inclusions in the cerebral cortex reflect abnormalities in radial glial developmental processes, such as migration, differentiation, or glial-neuronal interaction function during neuronal migration. Copyright © 2010 The Japanese Society of Child Neurology. All rights reserved.

Patterning of functional human astrocytes onto parylene-C/SiO2 substrates for the study of Ca2+ dynamics in astrocytic networks

NASA Astrophysics Data System (ADS)

Raos, B. J.; Simpson, M. C.; Doyle, C. S.; Murray, A. F.; Graham, E. S.; Unsworth, C. P.

2018-06-01

Objective. Recent literature suggests that astrocytes form organized functional networks and communicate through transient changes in cytosolic Ca2+. Traditional techniques to investigate network activity, such as pharmacological blocking or genetic knockout, are difficult to restrict to individual cells. The objective of this work is to develop cell-patterning techniques to physically manipulate astrocytic interactions to enable the study of Ca2+ in astrocytic networks. Approach. We investigate how an in vitro cell-patterning platform that utilizes geometric patterns of parylene-C on SiO2 can be used to physically isolate single astrocytes and small astrocytic networks. Main results. We report that single astrocytes are effectively isolated on 75  ×  75 µm square parylene nodes, whereas multi-cellular astrocytic networks are isolated on larger nodes, with the mean number of astrocytes per cluster increasing as a function of node size. Additionally, we report that astrocytes in small multi-cellular clusters exhibit spatio-temporal clustering of Ca2+ transients. Finally, we report that the frequency and regularity of Ca2+ transients was positively correlated with astrocyte connectivity. Significance. The significance of this work is to demonstrate how patterning hNT astrocytes replicates spatio-temporal clustering of Ca2+ signalling that is observed in vivo but not in dissociated in vitro cultures. We therefore highlight the importance of the structure of astrocytic networks in determining ensemble Ca2+ behaviour.

Methylmercury-induced alterations in astrocyte functions are attenuated by ebselen.

PubMed

Yin, Zhaobao; Lee, Eunsook; Ni, Mingwei; Jiang, Haiyan; Milatovic, Dejan; Rongzhu, Lu; Farina, Marcelo; Rocha, Joao B T; Aschner, Michael

2011-06-01

Methylmercury (MeHg) preferentially accumulates in glia of the central nervous system (CNS), but its toxic mechanisms have yet to be fully recognized. In the present study, we tested the hypothesis that MeHg induces neurotoxicity via oxidative stress mechanisms, and that these effects are attenuated by the antioxidant, ebselen. Rat neonatal primary cortical astrocytes were pretreated with or without 10 μM ebselen for 2h followed by MeHg (0, 1, 5, and 10 μM) treatments. MeHg-induced changes in astrocytic [(3)H]-glutamine uptake were assessed along with changes in mitochondrial membrane potential (ΔΨ(m)), using the potentiometric dye tetramethylrhodamine ethyl ester (TMRE). Western blot analysis was used to detect MeHg-induced ERK (extracellular-signal related kinase) phosphorylation and caspase-3 activation. MeHg treatment significantly decreased (p<0.05) astrocytic [(3)H]-glutamine uptake at all time points and concentrations. Ebselen fully reversed MeHg's (1 μM) effect on [(3)H]-glutamine uptake at 1 min. At higher MeHg concentrations, ebselen partially reversed the MeHg-induced astrocytic inhibition of [(3)H]-glutamine uptake [at 1 min (5 and 10 μM) (p<0.05); 5 min (1, 5 and 10 μM) (p<0.05)]. MeHg treatment (1h) significantly (p<0.05) dissipated the ΔΨ(m) in astrocytes as evidenced by a decrease in mitochondrial TMRE fluorescence. Ebselen fully reversed the effect of 1 μM MeHg treatment for 1h on astrocytic ΔΨ(m) and partially reversed the effect of 5 and 10 μM MeHg treatments for 1h on ΔΨ(m). In addition, ebselen inhibited MeHg-induced phosphorylation of ERK (p<0.05) and blocked MeHg-induced activation of caspase-3 (p<0.05-0.01). These results are consistent with the hypothesis that MeHg exerts its toxic effects via oxidative stress and that the phosphorylation of ERK and the dissipation of the astrocytic mitochondrial membrane potential are involved in MeHg toxicity. In addition, the protective effects elicited by ebselen reinforce the idea that

Artificial astrocytes improve neural network performance.

PubMed

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

2011-04-19

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

Artificial Astrocytes Improve Neural Network Performance

PubMed Central

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

2011-01-01

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

From the Cover: AstrocytesAre Protective Against Chlorpyrifos Developmental Neurotoxicity in Human Pluripotent Stem Cell-Derived Astrocyte-Neuron Cocultures.

PubMed

Wu, Xian; Yang, Xiangkun; Majumder, Anirban; Swetenburg, Raymond; Goodfellow, Forrest T; Bartlett, Michael G; Stice, Steven L

2017-06-01

Human neural progenitor cells are capable of independent, directed differentiation into astrocytes, oligodendrocytes and neurons and thus offer a potential cell source for developmental neurotoxicity (DNT) systems. Human neural progenitor-derived astrocyte-neuron cocultured at defined ratios mimic cellular heterogeneity and interaction in the central nervous system. Cytochrome P450 enzymes are expressed at a relatively high level in astrocytes and may play a critical role in the biotransformation of endogenous or exogenous compounds, including chlorpyrifos, an organophosphate insecticide that affects the central nervous system. P450 enzymes metabolize chlorpyrifos to chlorpyrifos-oxon, which is then metabolized primarily to 3, 5, 6-trichloropyridinol in addition to diethylphosphate and diethylthiophosphate. These end metabolites are less neurotoxic than chlorpyrifos and chlorpyrifos-oxon. Our objective was to identify the interactive role of astrocytes and neurons in chlorpyrifos-induced human DNT. In neuron-only cultures, chlorpyrifos inhibited neurite length, neurite number and branch points per neuron in a dose-dependent manner during a 48 h exposure, starting at 10 μM. However, in astrocyte-neuron cocultures, astrocytes protected neurons from the effects of chlorpyrifos at higher concentrations, up to and including 30 μM chlorpyrifos and endogenous astrocyte P450 enzymes effectively metabolized chlorpyrifos. The P450 inhibitor SKF525A partly negated the protective effect of astrocytes, allowing reduction in branch points with chlorpyrifos (10 μM). Thus, the scalable and defined astrocyte-neuron cocultures model that we established here has potentially identified a role for P450 enzymes in astrocytic neuroprotection against chlorpyrifos and provides a novel model for addressing DNT in a more accurate multicellular environment. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For

The multi-dimensional roles of astrocytes in ALS.

PubMed

Yamanaka, Koji; Komine, Okiru

2018-01-01

Despite significant progress in understanding the molecular and genetic aspects of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by the progressive loss of motor neurons, the precise and comprehensive pathomechanisms remain largely unknown. In addition to motor neuron involvement, recent studies using cellular and animal models of ALS indicate that there is a complex interplay between motor neurons and neighboring non-neuronal cells, such as astrocytes, in non-cell autonomous neurodegeneration. Astrocytes are key homeostatic cells that play numerous supportive roles in maintaining the brain environment. In neurodegenerative diseases such as ALS, astrocytes change their shape and molecular expression patterns and are referred to as reactive or activated astrocytes. Reactive astrocytes in ALS lose their beneficial functions and gain detrimental roles. In addition, interactions between motor neurons and astrocytes are impaired in ALS. In this review, we summarize growing evidence that astrocytes are critically involved in the survival and demise of motor neurons through several key molecules and cascades in astrocytes in both sporadic and inherited ALS. These observations strongly suggest that astrocytes have multi-dimensional roles in disease and are a viable therapeutic target for ALS. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Astrocyte Resilience to Oxidative Stress Induced by Insulin-like Growth Factor I (IGF-I) Involves Preserved AKT (Protein Kinase B) Activity*

PubMed Central

Dávila, David; Fernández, Silvia; Torres-Alemán, Ignacio

2016-01-01

Disruption of insulin-like growth factor I (IGF-I) signaling is a key step in the development of cancer or neurodegeneration. For example, interference of the prosurvival IGF-I/AKT/FOXO3 pathway by redox activation of the stress kinases p38 and JNK is instrumental in neuronal death by oxidative stress. However, in astrocytes, IGF-I retains its protective action against oxidative stress. The molecular mechanisms underlying this cell-specific protection remain obscure but may be relevant to unveil new ways to combat IGF-I/insulin resistance. Here, we describe that, in astrocytes exposed to oxidative stress by hydrogen peroxide (H2O2), p38 activation did not inhibit AKT (protein kinase B) activation by IGF-I, which is in contrast to our previous observations in neurons. Rather, stimulation of AKT by IGF-I was significantly higher and more sustained in astrocytes than in neurons either under normal or oxidative conditions. This may be explained by phosphorylation of the phosphatase PTEN at the plasma membrane in response to IGF-I, inducing its cytosolic translocation and preserving in this way AKT activity. Stimulation of AKT by IGF-I, mimicked also by a constitutively active AKT mutant, reduced oxidative stress levels and cell death in H2O2-exposed astrocytes, boosting their neuroprotective action in co-cultured neurons. These results indicate that armoring of AKT activation by IGF-I is crucial to preserve its cytoprotective effect in astrocytes and may form part of the brain defense mechanism against oxidative stress injury. PMID:26631726

Astrocyte atrophy and immune dysfunction in self-harming macaques.

PubMed

Lee, Kim M; Chiu, Kevin B; Sansing, Hope A; Inglis, Fiona M; Baker, Kate C; MacLean, Andrew G

2013-01-01

Self-injurious behavior (SIB) is a complex condition that exhibits a spectrum of abnormal neuropsychological and locomotor behaviors. Mechanisms for neuropathogenesis could include irregular immune activation, host soluble factors, and astrocyte dysfunction. We examined the role of astrocytes as modulators of immune function in macaques with SIB. We measured changes in astrocyte morphology and function. Paraffin sections of frontal cortices from rhesus macaques identified with SIB were stained for glial fibrillary acidic protein (GFAP) and Toll-like receptor 2 (TLR2). Morphologic features of astrocytes were determined using computer-assisted camera lucida. There was atrophy of white matter astrocyte cell bodies, decreased arbor length in both white and gray matter astrocytes, and decreased bifurcations and tips on astrocytes in animals with SIB. This was combined with a five-fold increase in the proportion of astrocytes immunopositive for TLR2. These results provide direct evidence that SIB induces immune activation of astrocytes concomitant with quantifiably different morphology.

The BCL-2 family protein Bid is critical for pro-inflammatory signaling in astrocytes.

PubMed

König, Hans-Georg; Coughlan, Karen S; Kinsella, Sinéad; Breen, Bridget A; Prehn, Jochen H M

2014-10-01

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motoneurons in the spinal cord, brainstem and motor cortex. Mutations in the superoxide dismutase 1 (SOD1) gene represent a frequent genetic determinant and recapitulate a disease phenotype similar to ALS when expressed in mice. Previous studies using SOD1(G93A) transgenic mice have suggested a paracrine mechanism of neuronal loss, in which cytokines and other toxic factors released from astroglia or microglia trigger motoneuron degeneration. Several pro-inflammatory cytokines activate death receptors and may downstream from this activate the Bcl-2 family protein, Bid. We here sought to investigate the role of Bid in astrocyte activation and non-cell autonomous motoneuron degeneration. We found that spinal cord Bid protein levels increased significantly during disease progression in SOD1(G93A) mice. Subsequent experiments in vitro indicated that Bid was expressed at relatively low levels in motoneurons, but was enriched in astrocytes and microglia. Bid was strongly induced in astrocytes in response to pro-inflammatory cytokines or exposure to lipopolysaccharide. Experiments in bid-deficient astrocytes or astrocytes treated with a small molecule Bid inhibitor demonstrated that Bid was required for the efficient activation of transcription factor nuclear factor-κB in response to these pro-inflammatory stimuli. Finally, we found that conditioned medium from wild-type astrocytes, but not from bid-deficient astrocytes, was toxic when applied to primary motoneuron cultures. Collectively, our data demonstrate a new role for the Bcl-2 family protein Bid as a mediator of astrocyte activation during neuroinflammation, and suggest that Bid activation may contribute to non-cell autonomous motoneuron degeneration in ALS. Copyright © 2014 Elsevier Inc. All rights reserved.

Human astrocytes/astrocyte conditioned medium and shear stress enhance the barrier properties of human brain microvascular endothelial cells

PubMed Central

Siddharthan, Venkatraman; V. Kim, Yuri; Liu, Suyi; Kim, Kwang Sik

2009-01-01

The blood-brain barrier (BBB) is a structural and functional barrier that regulates the passage of molecules into and out of the brain to maintain the neural microenvironment. We have previously developed the in vitro BBB model with human brain microvascular endothelial cells (HBMEC). However, in vivo HBMEC are shown to interact with astrocytes and also exposed to shear stress through blood flow. In an attempt to develop the BBB model to mimic the in vivo condition we constructed the flow-based in vitro BBB model using HBMEC and human fetal astrocytes (HFA). We also examined the effect of astrocyte conditioned medium (ACM) in lieu of HFA to study the role of secreted factor(s) on the BBB properties. The tightness of HBMEC monolayer was assessed by the permeability of dextran and propidium iodide as well as by measuring the transendothelial electrical resistance (TEER). We showed that the HBMEC permeability was reduced and TEER was increased by non-contact, co-cultivation with HFA and ACM. The exposure of HBMEC to shear stress also exhibited decreased permeability. Moreover, HFA/ACM and shear flow exhibited additive effect of decreasing the permeability of HBMEC monolayer. In addition, we showed that the HBMEC expression of ZO-1 (tight junction protein) was increased by co-cultivation with ACM and in response to shear stress. These findings suggest that the non-contact co-cultivation with HFA helps maintain the barrier properties of HBMEC by secreting factor(s) into the medium. Our in vitro flow model system with the cells of human origin should be useful for studying the interactions between endothelial cells, glial cells, and secreted factor(s) as well as the role of shear stress in the barrier property of HBMEC. PMID:17368578

Quinolinic acid neurotoxicity: Differential roles of astrocytes and microglia via FGF-2-mediated signaling in redox-linked cytoskeletal changes.

PubMed

Pierozan, Paula; Biasibetti, Helena; Schmitz, Felipe; Ávila, Helena; Parisi, Mariana M; Barbe-Tuana, Florencia; Wyse, Angela T S; Pessoa-Pureur, Regina

2016-12-01

QUIN is a glutamate agonist playing a role in the misregulation of the cytoskeleton, which is associated with neurodegeneration in rats. In this study, we focused on microglial activation, FGF2/Erk signaling, gap junctions (GJs), inflammatory parameters and redox imbalance acting on cytoskeletal dynamics of the in QUIN-treated neural cells of rat striatum. FGF-2/Erk signaling was not altered in QUIN-treated primary astrocytes or neurons, however cytoskeleton was disrupted. In co-cultured astrocytes and neurons, QUIN-activated FGF2/Erk signaling prevented the cytoskeleton from remodeling. In mixed cultures (astrocyte, neuron, microglia), QUIN-induced FGF-2 increased level failed to activate Erk and promoted cytoskeletal destabilization. The effects of QUIN in mixed cultures involved redox imbalance upstream of Erk activation. Decreased connexin 43 (Cx43) immunocontent and functional GJs, was also coincident with disruption of the cytoskeleton in primary astrocytes and mixed cultures. We postulate that in interacting astrocytes and neurons the cytoskeleton is preserved against the insult of QUIN by activation of FGF-2/Erk signaling and proper cell-cell interaction through GJs. In mixed cultures, the FGF-2/Erk signaling is blocked by the redox imbalance associated with microglial activation and disturbed cell communication, disrupting the cytoskeleton. Thus, QUIN signal activates differential mechanisms that could stabilize or destabilize the cytoskeleton of striatal astrocytes and neurons in culture, and glial cells play a pivotal role in these responses preserving or disrupting a combination of signaling pathways and cell-cell interactions. Taken together, our findings shed light into the complex role of the active interaction of astrocytes, neurons and microglia in the neurotoxicity of QUIN. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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

2015-01-01

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

Inflammatory Mediators Alter the Astrocyte Transcriptome and Calcium Signaling Elicited by Multiple G-Protein-Coupled Receptors

PubMed Central

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

2012-01-01

Inflammation features in CNS disorders such as stroke, trauma, neurodegeneration, infection, and autoimmunity in which astrocytes play critical roles. To elucidate how inflammatory mediators alter astrocyte functions, we examined effects of transforming growth factor-β1 (TGF-β1), lipopolysaccharide (LPS), and interferon-gamma (IFNγ), alone and in combination, on purified, mouse primary cortical astrocyte cultures. We used microarrays to conduct whole-genome expression profiling, and measured calcium signaling, which is implicated in mediating dynamic astrocyte functions. Combinatorial exposure to TGF-β1, LPS, and IFNγ significantly modulated astrocyte expression of >6800 gene probes, including >380 synergistic changes not predicted by summing individual treatment effects. Bioinformatic analyses revealed significantly and markedly upregulated molecular networks and pathways associated in particular with immune signaling and regulation of cell injury, death, growth, and proliferation. Highly regulated genes included chemokines, growth factors, enzymes, channels, transporters, and intercellular and intracellular signal transducers. Notably, numerous genes for G-protein-coupled receptors (GPCRs) and G-protein effectors involved in calcium signaling were significantly regulated, mostly down (for example, Cxcr4, Adra2a, Ednra, P2y1, Gnao1, Gng7), but some up (for example, P2y14, P2y6, Ccrl2, Gnb4). We tested selected cases and found that changes in GPCR gene expression were accompanied by significant, parallel changes in astrocyte calcium signaling evoked by corresponding GPCR-specific ligands. These findings identify pronounced changes in the astrocyte transcriptome induced by TGF-β1, LPS, and IFNγ, and show that these inflammatory stimuli upregulate astrocyte molecular networks associated with immune- and injury-related functions and significantly alter astrocyte calcium signaling stimulated by multiple GPCRs. PMID:23077035

HIV and drug abuse mediate astrocyte senescence in a β-catenin-dependent manner leading to neuronal toxicity.

PubMed

Yu, Chunjiang; Narasipura, Srinivas D; Richards, Maureen H; Hu, Xiu-Ti; Yamamoto, Bryan; Al-Harthi, Lena

2017-10-01

Emerging evidence suggests that cell senescence plays an important role in aging-associated diseases including neurodegenerative diseases. HIV leads to a spectrum of neurologic diseases collectively termed HIV-associated neurocognitive disorders (HAND). Drug abuse, particularly methamphetamine (meth), is a frequently abused psychostimulant among HIV+ individuals and its abuse exacerbates HAND. The mechanism by which HIV and meth lead to brain cell dysregulation is not entirely clear. In this study, we evaluated the impact of HIV and meth on astrocyte senescence using in vitro and several animal models. Astrocytes constitute up to 50% of brain cells and play a pivotal role in marinating brain homeostasis. We show here that HIV and meth induce significant senescence of primary human fetal astrocytes, as evaluated by induction of senescence markers (β-galactosidase and p16 INK 4A ), senescence-associated morphologic changes, and cell cycle arrest. HIV- and meth-mediated astrocyte senescence was also demonstrated in three small animal models (humanized mouse model of HIV/NSG-huPBMCs, HIV-transgenic rats, and in a meth administration rat model). Senescent astrocytes in turn mediated neuronal toxicity. Further, we show that β-catenin, a pro-survival/proliferation transcriptional co-activator, is downregulated by HIV and meth in human astrocytes and this downregulation promotes astrocyte senescence while induction of β-catenin blocks HIV- and meth-mediated astrocyte senescence. These studies, for the first time, demonstrate that HIV and meth induce astrocyte senescence and implicate the β-catenin pathway as potential therapeutic target to overcome astrocyte senescence. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Hippocampal neuronal subtypes develop abnormal dendritic arbors in the presence of Fragile X astrocytes.

PubMed

Jacobs, S; Cheng, C; Doering, L C

2016-06-02

Astrocytes are now recognized as key players in the neurobiology of neurodevelopmental disorders such as Fragile X syndrome. However, the nature of Fragile X astrocyte-mediated control of dendrite development in subtypes of hippocampal neurons is not yet known. We used a co-culture procedure in which wildtype primary hippocampal neurons were cultured with astrocytes from either a wildtype or Fragile X mouse, for either 7, 14 or 21 days. The neurons were processed for immunocytochemistry with the dendritic marker MAP2, classified by morphological criteria into one of five neuronal subtypes, and subjected to Sholl analyses. Both linear and semi-log methods of Sholl analyses were applied to the neurons in order to provide an in depth analysis of the dendritic arborizations. We found that Fragile X astrocytes affect the development of dendritic arborization of all subtypes of wildtype hippocampal neurons. Furthermore, we show that hippocampal neurons with spiny stellate neuron morphology exhibit the most pervasive developmental delays, with significant dendritic arbor alterations persisting at 21 days in culture. The results further dictate the critical role astrocytes play in governing neuronal morphology including altered dendrite development in Fragile X. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

A Novel Method for Assessing Sex-Specific and Genotype-Specific Response to Injury in Astrocyte Culture

PubMed Central

Liu, Mingyue; Oyarzabal, Esteban; Yang, Rui; Murphy, Stephanie J; Hurn, Patricia D.

2008-01-01

Female astrocytes sustain less cell death from oxygen-glucose deprivation (OGD) than male astrocytes. Arimidex, an aromatase inhibitor, abolishes these sex differences. To verify sex-dependent differences in P450 aromatase function in astrocyte cell death following OGD, we developed a novel method that uses sex-specific and genotype-specific single pup primary astrocyte cultures from wild-type (WT) and aromatase-knockout (ArKO) mice. After determining sex by external and internal examination as well as PCR and genotype by PCR amplification of tail cDNA, we established cultures from 1−3 day-old male and female, WT and ArKO mice pups and grew them to confluence in estrogen-free media. Cell death was measured by lactate dehydrogenase (LDH) assay. Our study shows that, while WT female astrocytes are more resistant to OGD than WT male cells, sex differences disappear in ArKO cells. Cell death is significantly increased in ArKO compared to WT in female astrocytes but not male cells. Therefore, P450 aromatase appears to be essential in endogenous neuroprotection in females, and this finding may have clinical implications. This innovative technique may also be applied to other in vitro studies of sex-related functional differences. PMID:18436308

The role of astrocytes in multiple sclerosis pathogenesis.

PubMed

Guerrero-García, J J

2017-09-25

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system (CNS), in which astrocytes play an important role as CNS immune cells. However, the activity of astrocytes as antigen-presenting cells (APC) continues to be subject to debate. This review analyses the existing evidence on the participation of astrocytes in CNS inflammation in MS and on several mechanisms that modify astrocyte activity in the disease. Astrocytes play a crucial role in the pathogenesis of MS because they express toll-like receptors (TLR) and major histocompatibility complex (MHC) classI andII. In addition, astrocytes participate in regulating the blood-brain barrier (BBB) and in modulating T cell activity through the production of cytokines. Future studies should focus on the role of astrocytes in order to find new therapeutic targets for the treatment of MS. Copyright © 2017 Sociedad Española de Neurología. Publicado por Elsevier España, S.L.U. All rights reserved.

Disentangling the role of astrocytes in alcohol use disorder

PubMed Central

Adermark, Louise; Bowers, M. Scott

2016-01-01

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

[ARTCEREB irrigation and perfusion solution for cerebrospinal surgery: pharmacological assessment using human astrocytes exposed to test solutions].

PubMed

Nishimura, Masuhiro; Doi, Kazuhisa; Enomoto, Riyo; Lee, Eibai; Naito, Shinsaku; Yamauchi, Aiko

2009-09-01

ARTCEREB irrigation and perfusion solution (Artcereb) is a preparation intended for the irrigation and perfusion of the cerebral ventricles, and it is therefore important to evaluate the effects of Artcereb on brain cells. In vitro assessment of the effects of Artcereb in cell cultures of human fetal astrocytes was conducted in comparison with normal saline and lactated Ringer's solution. The effects of exposure to Artcereb were evaluated based on microscopic images of the mitochondria stained with rhodamine 123. The effects of exposure to Artcereb on cell function were also evaluated by quantitative analysis of mitochondrial activity based on rhodamine 123 and (3)H-thymidine incorporation. Morphological changes in nuclear structure were also evaluated. The results of the present study showed that cell function in cell cultures of human astrocytes was relatively unaffected by exposure to Artcereb as compared with normal saline or lactated Ringer's solution, suggesting that Artcereb has less effect on brain cells than normal saline or lactated Ringer's solution when used for the irrigation or perfusion of the cerebral ventricles.

Effect of chronic antipsychotic exposure on astrocyte and oligodendrocyte numbers in macaque monkeys

PubMed Central

Konopaske, Glenn T.; Dorph-Petersen, Karl-Anton; Sweet, Robert A.; Pierri, Joseph N.; Zhang, Wei; Sampson, Allan R.; Lewis, David A.

2008-01-01

Background Both in vivo and post-mortem studies suggest that oligodendrocyte and myelination alterations are present in individuals with schizophrenia. However, it is unclear whether prolonged treatment with antipsychotic medications contributes to these disturbances. We recently reported that chronic exposure of macaque monkeys to haloperidol or olanzapine was associated with a 10−18% lower glial cell number in the parietal grey matter. Consequently, in this study we sought to determine whether the lower glial cell number was due to fewer oligodendrocytes as opposed to lower numbers of astrocytes. Methods Using fluorescent immunocytochemical techniques, we optimized the visualization of each cell type throughout the entire thickness of tissue sections, while minimizing final tissue shrinkage. As a result, we were able to obtain robust stereological estimates of total oligodendrocyte and astrocyte numbers in the parietal grey matter using the optical fractionator method. Results We found a significant 20.5% lower astrocyte number with a non-significant 12.9% lower oligodendrocyte number in the antipsychotic-exposed monkeys. Similar effects were seen in both the haloperidol and olanzapine groups. Conclusion These findings suggest that studies investigating glial cell alterations in schizophrenia must take into account the effect of antipsychotic treatment. PMID:17945195

Zinc is released by cultured astrocytes as a gliotransmitter under hypoosmotic stress-loaded conditions and regulates microglial activity.

PubMed

Segawa, Shohei; Nishiura, Takeshi; Furuta, Takahiro; Ohsato, Yuki; Tani, Misaki; Nishida, Kentaro; Nagasawa, Kazuki

2014-01-17

Astrocytes contribute to the maintenance of brain homeostasis via the release of gliotransmitters such as ATP and glutamate. Here we examined whether zinc was released from astrocytes under stress-loaded conditions, and was involved in the regulation of microglial activity as a gliotransmitter. Hypoosmotic stress was loaded to astrocytes using balanced salt solution prepared to 214-314 mOsmol/L, and then intra- and extra-cellular zinc levels were assessed using Newport Green DCF diacetate (NG) and ICP-MS, respectively. Microglial activation by the astrocytic supernatant was assessed by their morphological changes and poly(ADP-ribose) (PAR) polymer accumulation. Exposure of astrocytes to hypoosmotic buffer, increased the extracellular ATP level in osmolarity-dependent manners, indicating a load of hypoosmotic stress. In hypoosmotic stress-loaded astrocytes, there were apparent increases in the intra- and extra-cellular zinc levels. Incubation of microglia in the astrocytic conditioned medium transformed them into the activated "amoeboid" form and induced PAR formation. Administration of an extracellular zinc chelator, CaEDTA, to the astrocytic conditioned medium almost completely prevented the microglial activation. Treatment of astrocytes with an intracellular zinc chelator, TPEN, suppressed the hypoosmotic stress-increased intracellular, but not the extracellular, zinc level, and the increase in the intracellular zinc level was blocked partially by a nitric oxide synthase inhibitor, but not by CaEDTA, indicating that the mechanisms underlying the increases in the intra- and extra-cellular zinc levels might be different. These findings suggest that under hypoosmotic stress-loaded conditions, zinc is released from astrocytes and then plays a primary role in microglial activation as a gliotransmitter. Copyright © 2013 Elsevier Inc. All rights reserved.

A Novel Optical Intracellular Imaging Approach for Potassium Dynamics in Astrocytes

PubMed Central

Rimmele, Theresa S.; Chatton, Jean-Yves

2014-01-01

Astrocytes fulfill a central role in regulating K+ and glutamate, both released by neurons into the extracellular space during activity. Glial glutamate uptake is a secondary active process that involves the influx of three Na+ ions and one proton and the efflux of one K+ ion. Thus, intracellular K+ concentration ([K+]i) is potentially influenced both by extracellular K+ concentration ([K+]o) fluctuations and glutamate transport in astrocytes. We evaluated the impact of these K+ ion movements on [K+]i in primary mouse astrocytes by microspectrofluorimetry. We established a new noninvasive and reliable approach to monitor and quantify [K+]i using the recently developed K+ sensitive fluorescent indicator Asante Potassium Green-1 (APG-1). An in situ calibration procedure enabled us to estimate the resting [K+]i at 133±1 mM. We first investigated the dependency of [K+]i levels on [K+]o. We found that [K+]i followed [K+]o changes nearly proportionally in the range 3–10 mM, which is consistent with previously reported microelectrode measurements of intracellular K+ concentration changes in astrocytes. We then found that glutamate superfusion caused a reversible drop of [K+]i that depended on the glutamate concentration with an apparent EC50 of 11.1±1.4 µM, corresponding to the affinity of astrocyte glutamate transporters. The amplitude of the [K+]i drop was found to be 2.3±0.1 mM for 200 µM glutamate applications. Overall, this study shows that the fluorescent K+ indicator APG-1 is a powerful new tool for addressing important questions regarding fine [K+]i regulation with excellent spatial resolution. PMID:25275375

Simultaneous neuron- and astrocyte-specific fluorescent marking

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schulze, Wiebke; Hayata-Takano, Atsuko; Kamo, Toshihiko

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 alignmentmore » 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.« less

Methylmercury-induced inhibition of regulatory volume decrease in astrocytes: characterization of osmoregulator efflux and its reversal by amiloride.

PubMed

Aschner, M; Vitarella, D; Allen, J W; Conklin, D R; Cowan, K S

1998-11-16

Swelling of neonatal rat primary astrocyte cultures by hypotonic media leads to regulatory volume decrease (RVD) and the resumption of resting cell volume. RVD is associated with activation of conductive K+ and Cl- channels, allowing for the escape of KCl, as well as the release of osmoregulators, such as taurine and myoinositol. As we have previously shown [D. Vitarella, H.K. Kimelberg, M. Aschner, Inhibition of RVD in swollen rat primary astrocyte cultures by methylmercury (MeHg) is due to increase amiloride-sensitive Na+ uptake, Brain Res. 732 (1996) 169-178.], MeHg, when added to hypotonic buffer inhibits RVD, primarily due to increased cellular permeability to Na+ via the Na+/H+ antiporter. The present study was, therefore, undertaken to assess the ability of cation-anion cotransport blockers to reverse the inhibitory effect of MeHg on RVD in swollen astrocytes, and to further characterize MeHg-induced changes in astrocytic osmoregulatory release processes. The studies demonstrate the following: (1) MeHg-induced inhibition of RVD is partially inhibited by the Na+/H+ antiporter blocker, amiloride, but not SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid), DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid), furosemide or bumetanide; (2) exposure of swollen astrocytes to MeHg is associated with specific effects on osmoregulatory release, leading to significant inhibition of taurine release and a significant increase in potassium and myoinositol release compared with release in hypotonic conditions. Copyright 1998 Elsevier Science B.V.

New Transgenic Mouse Lines for Selectively Targeting Astrocytes and Studying Calcium Signals in Astrocyte Processes In Situ and In Vivo.

PubMed

Srinivasan, Rahul; Lu, Tsai-Yi; Chai, Hua; Xu, Ji; Huang, Ben S; Golshani, Peyman; Coppola, Giovanni; Khakh, Baljit S

2016-12-21

Astrocytes exist throughout the nervous system and are proposed to affect neural circuits and behavior. However, studying astrocytes has proven difficult because of the lack of tools permitting astrocyte-selective genetic manipulations. Here, we report the generation of Aldh1l1-Cre/ERT2 transgenic mice to selectively target astrocytes in vivo. We characterized Aldh1l1-Cre/ERT2 mice using imaging, immunohistochemistry, AAV-FLEX-GFP microinjections, and crosses to RiboTag, Ai95, and new Cre-dependent membrane-tethered Lck-GCaMP6f knockin mice that we also generated. Two to three weeks after tamoxifen induction, Aldh1l1-Cre/ERT2 selectively targeted essentially all adult (P80) brain astrocytes with no detectable neuronal contamination, resulting in expression of cytosolic and Lck-GCaMP6f, and permitting subcellular astrocyte calcium imaging during startle responses in vivo. Crosses with RiboTag mice allowed sequencing of actively translated mRNAs and determination of the adult cortical astrocyte transcriptome. Thus, we provide well-characterized, easy-to-use resources with which to selectively study astrocytes in situ and in vivo in multiple experimental scenarios. Copyright © 2016 Elsevier Inc. All rights reserved.

Minocycline attenuates bone cancer pain in rats by inhibiting NF-κB in spinal astrocytes

PubMed Central

Song, Zhen-peng; Xiong, Bing-rui; Guan, Xue-hai; Cao, Fei; Manyande, Anne; Zhou, Ya-qun; Zheng, Hua; Tian, Yu-ke

2016-01-01

Aim: To investigate the mechanisms underlying the anti-nociceptive effect of minocycline on bone cancer pain (BCP) in rats. Methods: A rat model of BCP was established by inoculating Walker 256 mammary carcinoma cells into tibial medullary canal. Two weeks later, the rats were injected with minocycline (50, 100 μg, intrathecally; or 40, 80 mg/kg, ip) twice daily for 3 consecutive days. Mechanical paw withdrawal threshold (PWT) was used to assess pain behavior. After the rats were euthanized, spinal cords were harvested for immunoblotting analyses. The effects of minocycline on NF-κB activation were also examined in primary rat astrocytes stimulated with IL-1β in vitro. Results: BCP rats had marked bone destruction, and showed mechanical tactile allodynia on d 7 and d 14 after the operation. Intrathecal injection of minocycline (100 μg) or intraperitoneal injection of minocycline (80 mg/kg) reversed BCP-induced mechanical tactile allodynia. Furthermore, intraperitoneal injection of minocycline (80 mg/kg) reversed BCP-induced upregulation of GFAP (astrocyte marker) and PSD95 in spinal cord. Moreover, intraperitoneal injection of minocycline (80 mg/kg) reversed BCP-induced upregulation of NF-κB, p-IKKα and IκBα in spinal cord. In IL-1β-stimulated primary rat astrocytes, pretreatment with minocycline (75, 100 μmol/L) significantly inhibited the translocation of NF-κB to nucleus. Conclusion: Minocycline effectively alleviates BCP by inhibiting the NF-κB signaling pathway in spinal astrocytes. PMID:27157092

Minocycline attenuates bone cancer pain in rats by inhibiting NF-κB in spinal astrocytes.

PubMed

Song, Zhen-Peng; Xiong, Bing-Rui; Guan, Xue-Hai; Cao, Fei; Manyande, Anne; Zhou, Ya-Qun; Zheng, Hua; Tian, Yu-Ke

2016-06-01

To investigate the mechanisms underlying the anti-nociceptive effect of minocycline on bone cancer pain (BCP) in rats. A rat model of BCP was established by inoculating Walker 256 mammary carcinoma cells into tibial medullary canal. Two weeks later, the rats were injected with minocycline (50, 100 μg, intrathecally; or 40, 80 mg/kg, ip) twice daily for 3 consecutive days. Mechanical paw withdrawal threshold (PWT) was used to assess pain behavior. After the rats were euthanized, spinal cords were harvested for immunoblotting analyses. The effects of minocycline on NF-κB activation were also examined in primary rat astrocytes stimulated with IL-1β in vitro. BCP rats had marked bone destruction, and showed mechanical tactile allodynia on d 7 and d 14 after the operation. Intrathecal injection of minocycline (100 μg) or intraperitoneal injection of minocycline (80 mg/kg) reversed BCP-induced mechanical tactile allodynia. Furthermore, intraperitoneal injection of minocycline (80 mg/kg) reversed BCP-induced upregulation of GFAP (astrocyte marker) and PSD95 in spinal cord. Moreover, intraperitoneal injection of minocycline (80 mg/kg) reversed BCP-induced upregulation of NF-κB, p-IKKα and IκBα in spinal cord. In IL-1β-stimulated primary rat astrocytes, pretreatment with minocycline (75, 100 μmol/L) significantly inhibited the translocation of NF-κB to nucleus. Minocycline effectively alleviates BCP by inhibiting the NF-κB signaling pathway in spinal astrocytes.

Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses

PubMed Central

Shigetomi, Eiji; Bushong, Eric A.; Haustein, Martin D.; Tong, Xiaoping; Jackson-Weaver, Olan; Kracun, Sebastian; Xu, Ji; Sofroniew, Michael V.; Ellisman, Mark H.

2013-01-01

Intracellular Ca2+ transients are considered a primary signal by which astrocytes interact with neurons and blood vessels. With existing commonly used methods, Ca2+ has been studied only within astrocyte somata and thick branches, leaving the distal fine branchlets and endfeet that are most proximate to neuronal synapses and blood vessels largely unexplored. Here, using cytosolic and membrane-tethered forms of genetically encoded Ca2+ indicators (GECIs; cyto-GCaMP3 and Lck-GCaMP3), we report well-characterized approaches that overcome these limitations. We used in vivo microinjections of adeno-associated viruses to express GECIs in astrocytes and studied Ca2+ signals in acute hippocampal slices in vitro from adult mice (aged ∼P80) two weeks after infection. Our data reveal a sparkling panorama of unexpectedly numerous, frequent, equivalently scaled, and highly localized Ca2+ microdomains within entire astrocyte territories in situ within acute hippocampal slices, consistent with the distribution of perisynaptic branchlets described using electron microscopy. Signals from endfeet were revealed with particular clarity. The tools and experimental approaches we describe in detail allow for the systematic study of Ca2+ signals within entire astrocytes, including within fine perisynaptic branchlets and vessel-associated endfeet, permitting rigorous evaluation of how astrocytes contribute to brain function. PMID:23589582

Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses.

PubMed

Shigetomi, Eiji; Bushong, Eric A; Haustein, Martin D; Tong, Xiaoping; Jackson-Weaver, Olan; Kracun, Sebastian; Xu, Ji; Sofroniew, Michael V; Ellisman, Mark H; Khakh, Baljit S

2013-05-01

Intracellular Ca(2+) transients are considered a primary signal by which astrocytes interact with neurons and blood vessels. With existing commonly used methods, Ca(2+) has been studied only within astrocyte somata and thick branches, leaving the distal fine branchlets and endfeet that are most proximate to neuronal synapses and blood vessels largely unexplored. Here, using cytosolic and membrane-tethered forms of genetically encoded Ca(2+) indicators (GECIs; cyto-GCaMP3 and Lck-GCaMP3), we report well-characterized approaches that overcome these limitations. We used in vivo microinjections of adeno-associated viruses to express GECIs in astrocytes and studied Ca(2+) signals in acute hippocampal slices in vitro from adult mice (aged ∼P80) two weeks after infection. Our data reveal a sparkling panorama of unexpectedly numerous, frequent, equivalently scaled, and highly localized Ca(2+) microdomains within entire astrocyte territories in situ within acute hippocampal slices, consistent with the distribution of perisynaptic branchlets described using electron microscopy. Signals from endfeet were revealed with particular clarity. The tools and experimental approaches we describe in detail allow for the systematic study of Ca(2+) signals within entire astrocytes, including within fine perisynaptic branchlets and vessel-associated endfeet, permitting rigorous evaluation of how astrocytes contribute to brain function.

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

PubMed

Gotoh, Hitoshi; Nomura, Tadashi; Ono, Katsuhiko

2017-06-01

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

Extracellular Electrophysiological Measurements of Cooperative Signals in Astrocytes Populations

PubMed Central

Mestre, Ana L. G.; Inácio, Pedro M. C.; Elamine, Youssef; Asgarifar, Sanaz; Lourenço, Ana S.; Cristiano, Maria L. S.; Aguiar, Paulo; Medeiros, Maria C. R.; Araújo, Inês M.; Ventura, João; Gomes, Henrique L.

2017-01-01

Astrocytes are neuroglial cells that exhibit functional electrical properties sensitive to neuronal activity and capable of modulating neurotransmission. Thus, electrophysiological recordings of astroglial activity are very attractive to study the dynamics of glial signaling. This contribution reports on the use of ultra-sensitive planar electrodes combined with low noise and low frequency amplifiers that enable the detection of extracellular signals produced by primary cultures of astrocytes isolated from mouse cerebral cortex. Recorded activity is characterized by spontaneous bursts comprised of discrete signals with pronounced changes on the signal rate and amplitude. Weak and sporadic signals become synchronized and evolve with time to higher amplitude signals with a quasi-periodic behavior, revealing a cooperative signaling process. The methodology presented herewith enables the study of ionic fluctuations of population of cells, complementing the single cells observation by calcium imaging as well as by patch-clamp techniques. PMID:29109679

Extracellular Electrophysiological Measurements of Cooperative Signals in Astrocytes Populations.

PubMed

Mestre, Ana L G; Inácio, Pedro M C; Elamine, Youssef; Asgarifar, Sanaz; Lourenço, Ana S; Cristiano, Maria L S; Aguiar, Paulo; Medeiros, Maria C R; Araújo, Inês M; Ventura, João; Gomes, Henrique L

2017-01-01

Astrocytes are neuroglial cells that exhibit functional electrical properties sensitive to neuronal activity and capable of modulating neurotransmission. Thus, electrophysiological recordings of astroglial activity are very attractive to study the dynamics of glial signaling. This contribution reports on the use of ultra-sensitive planar electrodes combined with low noise and low frequency amplifiers that enable the detection of extracellular signals produced by primary cultures of astrocytes isolated from mouse cerebral cortex. Recorded activity is characterized by spontaneous bursts comprised of discrete signals with pronounced changes on the signal rate and amplitude. Weak and sporadic signals become synchronized and evolve with time to higher amplitude signals with a quasi-periodic behavior, revealing a cooperative signaling process. The methodology presented herewith enables the study of ionic fluctuations of population of cells, complementing the single cells observation by calcium imaging as well as by patch-clamp techniques.

Neuron-astrocyte signaling is preserved in the aging brain.

PubMed

Gómez-Gonzalo, Marta; Martin-Fernandez, Mario; Martínez-Murillo, Ricardo; Mederos, Sara; Hernández-Vivanco, Alicia; Jamison, Stephanie; Fernandez, Ana P; Serrano, Julia; Calero, Pilar; Futch, Hunter S; Corpas, Rubén; Sanfeliu, Coral; Perea, Gertrudis; Araque, Alfonso

2017-04-01

Astrocytes play crucial roles in brain homeostasis and are emerging as regulatory elements of neuronal and synaptic physiology by responding to neurotransmitters with Ca 2+ elevations and releasing gliotransmitters that activate neuronal receptors. Aging involves neuronal and astrocytic alterations, being considered risk factor for neurodegenerative diseases. Most evidence of the astrocyte-neuron signaling is derived from studies with young animals; however, the features of astrocyte-neuron signaling in adult and aging brain remain largely unknown. We have investigated the existence and properties of astrocyte-neuron signaling in physiologically and pathologically aging mouse hippocampal and cortical slices at different lifetime points (0.5 to 20 month-old animals). We found that astrocytes preserved their ability to express spontaneous and neurotransmitter-dependent intracellular Ca 2+ signals from juvenile to aging brains. Likewise, resting levels of gliotransmission, assessed by neuronal NMDAR activation by glutamate released from astrocytes, were largely preserved with similar properties in all tested age groups, but DHPG-induced gliotransmission was reduced in aged mice. In contrast, gliotransmission was enhanced in the APP/PS1 mouse model of Alzheimer's disease, indicating a dysregulation of astrocyte-neuron signaling in pathological conditions. Disruption of the astrocytic IP 3 R2 mediated-signaling, which is required for neurotransmitter-induced astrocyte Ca 2+ signals and gliotransmission, boosted the progression of amyloid plaque deposits and synaptic plasticity impairments in APP/PS1 mice at early stages of the disease. Therefore, astrocyte-neuron interaction is a fundamental signaling, largely conserved in the adult and aging brain of healthy animals, but it is altered in Alzheimer's disease, suggesting that dysfunctions of astrocyte Ca 2+ physiology may contribute to this neurodegenerative disease. GLIA 2017 GLIA 2017;65:569-580. © 2017 Wiley

Reactive astrocytes and therapeutic potential in focal ischemic stroke

PubMed Central

Choudhury, Gourav Roy; Ding, Shinghua

2015-01-01

Astrocytes are specialized and the most abundant cell type in the central nervous system (CNS). They play important roles in the physiology of the brain. Astrocytes are also critically involved in many CNS disorders including focal ischemic stroke, the leading cause of brain injury and death in patients. One of the prominent pathological features of a focal ischemic stroke is reactive astrogliosis and glial scar formation. Reactive astrogliosis is accompanied with changes in morphology, proliferation and gene expression in the reactive astrocytes. This study provides an overview of the most recent advances in astrocytic Ca2+ signaling, spatial and temporal dynamics of the morphology and proliferation of reactive astrocytes as well as signaling pathways involved in the reactive astrogliosis after ischemic stroke based on results from experimental studies performed in various animal models. This review also discusses the therapeutic potential of reactive astrocytes in a focal ischemic stroke. As reactive astrocytes exhibit high plasticity, we suggest that modulation of local reactive astrocytes is a promising strategy for cell-based stroke therapy. PMID:25982835

Role of metabotropic glutamate receptor 5 signaling and homer in oxygen glucose deprivation-mediated astrocyte apoptosis.

PubMed

Paquet, Maryse; Ribeiro, Fabiola M; Guadagno, Jennifer; Esseltine, Jessica L; Ferguson, Stephen S G; Cregan, Sean P

2013-02-14

Group I metabotropic glutamate receptors (mGluR) are coupled via Gαq/11 to the activation of phospholipase Cβ, which hydrolyzes membrane phospholipids to form inositol 1,4,5 trisphosphate and diacylglycerol. In addition to functioning as neurotransmitter receptors to modulate synaptic activity, pathological mGluR5 signaling has been implicated in a number of disease processes including Fragile X, amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, epilepsy, and drug addiction. The expression of mGluR5 in astrocytes has been shown to be increased in several acute and chronic neurodegenerative conditions, but little is known about the functional relevance of mGluR5 up-regulation in astrocytes following injury. In the current study, we investigated primary mouse cortical astrocyte cell death in response to oxygen glucose deprivation (OGD) and found that OGD induced both necrotic and apoptotic cell death of astrocytes. OGD resulted in an increase in astrocytic mGluR5 protein expression, inositol phosphate formation and extracellular regulated kinase (ERK1/2) phosphorylation, but only inositol phosphate formation was blocked with the mGluR5 selective antagonist MPEP. Cortical astrocytes derived from mGluR5 knockout mice exhibited resistance to OGD-stimulated apoptosis, but a lack of mGluR5 expression did not confer protection against necrotic cell death. The antagonism of the inositol 1,4,5 trisphosphate receptor also reduced apoptotic cell death in wild-type astrocytes, but did not provide any additional protection to astrocytes derived from mGluR5 null mice. Moreover, the disruption of Homer protein interactions with mGluR5 also reduced astrocyte apoptosis. Taken together these observations indicated that mGluR5 up-regulation contributed selectively to the apoptosis of astrocytes via the activation of phospholipase C and the release of calcium from intracellular stores as well as via the association with

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

PubMed Central

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

2011-01-01

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

Astrocytes Secrete Exosomes Enriched with Proapoptotic Ceramide and Prostate Apoptosis Response 4 (PAR-4)

PubMed Central

Wang, Guanghu; Dinkins, Michael; He, Qian; Zhu, Gu; Poirier, Christophe; Campbell, Andrew; Mayer-Proschel, Margot; Bieberich, Erhard

2012-01-01

Amyloid protein is well known to induce neuronal cell death, whereas only little is known about its effect on astrocytes. We found that amyloid peptides activated caspase 3 and induced apoptosis in primary cultured astrocytes, which was prevented by caspase 3 inhibition. Apoptosis was also prevented by shRNA-mediated down-regulation of PAR-4, a protein sensitizing cells to the sphingolipid ceramide. Consistent with a potentially proapoptotic effect of PAR-4 and ceramide, astrocytes surrounding amyloid plaques in brain sections of the 5xFAD mouse (and Alzheimer disease patient brain) showed caspase 3 activation and were apoptotic when co-expressing PAR-4 and ceramide. Apoptosis was not observed in astrocytes with deficient neutral sphingomyelinase 2 (nSMase2), indicating that ceramide generated by nSMase2 is critical for amyloid-induced apoptosis. Antibodies against PAR-4 and ceramide prevented amyloid-induced apoptosis in vitro and in vivo, suggesting that apoptosis was mediated by exogenous PAR-4 and ceramide, potentially associated with secreted lipid vesicles. This was confirmed by the analysis of lipid vesicles from conditioned medium showing that amyloid peptide induced the secretion of PAR-4 and C18 ceramide-enriched exosomes. Exosomes were not secreted by nSMase2-deficient astrocytes, indicating that ceramide generated by nSMase2 is critical for exosome secretion. Consistent with the ceramide composition in amyloid-induced exosomes, exogenously added C18 ceramide restored PAR-4-containing exosome secretion in nSMase2-deficient astrocytes. Moreover, isolated PAR-4/ceramide-enriched exosomes were taken up by astrocytes and induced apoptosis in the absence of amyloid peptide. Taken together, we report a novel mechanism of apoptosis induction by PAR-4/ceramide-enriched exosomes, which may critically contribute to Alzheimer disease. PMID:22532571

T cells' immunological synapses induce polarization of brain astrocytes in vivo and in vitro: a novel astrocyte response mechanism to cellular injury.

PubMed

Barcia, Carlos; Sanderson, Nicholas S R; Barrett, Robert J; Wawrowsky, Kolja; Kroeger, Kurt M; Puntel, Mariana; Liu, Chunyan; Castro, Maria G; Lowenstein, Pedro R

2008-08-20

Astrocytes usually respond to trauma, stroke, or neurodegeneration by undergoing cellular hypertrophy, yet, their response to a specific immune attack by T cells is poorly understood. Effector T cells establish specific contacts with target cells, known as immunological synapses, during clearance of virally infected cells from the brain. Immunological synapses mediate intercellular communication between T cells and target cells, both in vitro and in vivo. How target virally infected astrocytes respond to the formation of immunological synapses established by effector T cells is unknown. Herein we demonstrate that, as a consequence of T cell attack, infected astrocytes undergo dramatic morphological changes. From normally multipolar cells, they become unipolar, extending a major protrusion towards the immunological synapse formed by the effector T cells, and withdrawing most of their finer processes. Thus, target astrocytes become polarized towards the contacting T cells. The MTOC, the organizer of cell polarity, is localized to the base of the protrusion, and Golgi stacks are distributed throughout the protrusion, reaching distally towards the immunological synapse. Thus, rather than causing astrocyte hypertrophy, antiviral T cells cause a major structural reorganization of target virally infected astrocytes. Astrocyte polarization, as opposed to hypertrophy, in response to T cell attack may be due to T cells providing a very focused attack, and thus, astrocytes responding in a polarized manner. A similar polarization of Golgi stacks towards contacting T cells was also detected using an in vitro allogeneic model. Thus, different T cells are able to induce polarization of target astrocytes. Polarization of target astrocytes in response to immunological synapses may play an important role in regulating the outcome of the response of astrocytes to attacking effector T cells, whether during antiviral (e.g. infected during HIV, HTLV-1, HSV-1 or LCMV infection), anti

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

PubMed

Thevenet, Jonathan; De Marchi, Umberto; Domingo, Jaime Santo; Christinat, Nicolas; Bultot, Laurent; Lefebvre, Gregory; Sakamoto, Kei; Descombes, Patrick; Masoodi, Mojgan; Wiederkehr, Andreas

2016-05-01

Medium-chain triglycerides have been used as part of a ketogenic diet effective in reducing epileptic episodes. The health benefits of the derived medium-chain fatty acids (MCFAs) are thought to result from the stimulation of liver ketogenesis providing fuel for the brain. We tested whether MCFAs have direct effects on energy metabolism in induced pluripotent stem cell-derived human astrocytes and neurons. Using single-cell imaging, we observed an acute pronounced reduction of the mitochondrial electrical potential and a concomitant drop of the NAD(P)H signal in astrocytes, but not in neurons. Despite the observed effects on mitochondrial function, MCFAs did not lower intracellular ATP levels or activate the energy sensor AMP-activated protein kinase. ATP concentrations in astrocytes were unaltered, even when blocking the respiratory chain, suggesting compensation through accelerated glycolysis. The MCFA decanoic acid (300 μM) promoted glycolysis and augmented lactate formation by 49.6%. The shorter fatty acid octanoic acid (300 μM) did not affect glycolysis but increased the rates of astrocyte ketogenesis 2.17-fold compared with that of control cells. MCFAs may have brain health benefits through the modulation of astrocyte metabolism leading to activation of shuttle systems that provide fuel to neighboring neurons in the form of lactate and ketone bodies.-Thevenet, J., De Marchi, U., Santo Domingo, J., Christinat, N., Bultot, L., Lefebvre, G., Sakamoto, K., Descombes, P., Masoodi, M., Wiederkehr, A. Medium-chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte-neuron lactate and ketone body shuttle systems. © FASEB.

BMP Signaling in Astrocytes Downregulates EGFR to Modulate Survival and Maturation

PubMed Central

Scholze, Anja R.; Foo, Lynette C.; Mulinyawe, Sara; Barres, Ben A.

2014-01-01

Astrocytes constitute a major cell population in the brain with a myriad of essential functions, yet we know remarkably little about the signaling pathways and mechanisms that direct astrocyte maturation. To explore the signals regulating astrocyte development, we prospectively purified and cultured immature postnatal rodent astrocytes. We identified fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs) as robust trophic factors for immature astrocytes. We showed that astrocytes respond directly to BMPs via phosphorylation of the smad1/5/8 pathway. In vitro, BMP signaling promoted immature astrocytes to adopt multiple characteristics of mature astrocytes, including a more process-bearing morphology, aquaporin-4 (AQP4) and S100β immunoreactivity, limited proliferation, and strong downregulation of epidermal growth factor receptor (EGFR). In vivo, activation of the smad1/5/8 pathway in astrocytes was seen during early postnatal development, but inhibition of astrocyte proliferation was not observed. These insights can aid in the further dissection of the mechanisms and pathways controlling astrocyte biology and development. PMID:25330173

Intrinsic Astrocyte Heterogeneity Influences Tumor Growth in Glioma Mouse Models.

PubMed

Irvin, David M; McNeill, Robert S; Bash, Ryan E; Miller, C Ryan

2017-01-01

The influence of cellular origin on glioma pathogenesis remains elusive. We previously showed that mutations inactivating Rb and Pten and activating Kras transform astrocytes and induce tumorigenesis throughout the adult mouse brain. However, it remained unclear whether astrocyte subpopulations were susceptible to these mutations. We therefore used genetic lineage tracing and fate mapping in adult conditional, inducible genetically engineered mice to monitor transformation of glial fibrillary acidic protein (GFAP) and glutamate aspartate transporter (GLAST) astrocytes and immunofluorescence to monitor cellular composition of the tumor microenvironment over time. Because considerable regional heterogeneity exists among astrocytes, we also examined the influence of brain region on tumor growth. GFAP astrocyte transformation induced uniformly rapid, regionally independent tumor growth, but transformation of GLAST astrocytes induced slowly growing tumors with significant regional bias. Transformed GLAST astrocytes had reduced proliferative response in culture and in vivo and malignant progression was delayed in these tumors. Recruited glial cells, including proliferating astrocytes, oligodendrocyte progenitors and microglia, were the majority of GLAST, but not GFAP astrocyte-derived tumors and their abundance dynamically changed over time. These results suggest that intrinsic astrocyte heterogeneity, and perhaps regional brain microenvironment, significantly contributes to glioma pathogenesis. © 2016 International Society of Neuropathology.

Spinal astrocyte gap junctions contribute to oxaliplatin-induced mechanical hypersensitivity.

PubMed

Yoon, Seo-Yeon; Robinson, Caleb R; Zhang, Haijun; Dougherty, Patrick M

2013-02-01

Spinal glial cells contribute to the development of many types of inflammatory and neuropathic pain. Here the contribution of spinal astrocytes and astrocyte gap junctions to oxaliplatin-induced mechanical hypersensitivity was explored. The expression of glial fibrillary acidic protein (GFAP) in spinal dorsal horn was significantly increased at day 7 but recovered at day 14 after oxaliplatin treatment, suggesting a transient activation of spinal astrocytes by chemotherapy. Astrocyte-specific gap junction protein connexin 43 (Cx43) was significantly increased in dorsal horn at both day 7 and day 14 following chemotherapy, but neuronal (connexin 36 [Cx36]) and oligodendrocyte (connexin 32 [Cx32]) gap junction proteins did not show any change. Blockade of astrocyte gap junction with carbenoxolone (CBX) prevented oxaliplatin-induced mechanical hypersensitivity in a dose-dependent manner and the increase of spinal GFAP expression, but had no effect once the mechanical hypersensitivity induced by oxaliplatin had fully developed. These results suggest that oxaliplatin chemotherapy induces the activation of spinal astrocytes and this is accompanied by increased expression of astrocyte-astrocyte gap junction connections via Cx43. These alterations in spinal astrocytes appear to contribute to the induction but not the maintenance of oxaliplatin-induced mechanical hypersensitivity. Combined, these results suggest that targeting spinal astrocyte/astrocyte-specific gap junction could be a new therapeutic strategy to prevent oxaliplatin-induced neuropathy. Spinal astrocytes but not microglia were recently shown to be recruited in paclitaxel-related chemoneuropathy. Here, spinal astrocyte gap junctions are shown to play an important role in the induction of oxaliplatin neuropathy. Copyright © 2013 American Pain Society. Published by Elsevier Inc. All rights reserved.

Astrocytes express functional TRPV2 ion channels.

PubMed

Shibasaki, Koji; Ishizaki, Yasuki; Mandadi, Sravan

2013-11-15

Thermosensitive transient receptor potential (thermo TRP) channels are important for sensory transduction. Among them, TRPV2 has an interesting characteristic of being activated by very high temperature (>52 °C). In addition to the heat sensor function, TRPV2 also acts as a mechanosensor, an osomosensor and a lipid sensor. It has been reported that TRPV2 is expressed in heart, intestine, pancreas and sensory nerves. In the central nervous system, neuronal TRPV2 expression was reported, however, glial expression and the precise roles of TRPV2 have not been determined. To explore the functional expression of TRPV2 in astrocytes, the expression was determined by histological and physiological methods. Interestingly, TRPV2 expression was detected in plasma membrane of astrocytes, and the astrocytic TRPV2 was activated by very high temperature (>50 °C) consistent with the reported characteristic. We revealed that the astrocytic TRPV2 was also activated by lysophosphatidylcholine, a known endogenous lipid ligand for TRPV2, suggesting that astrocytic TRPV2 might regulate neuronal activities in response to lipid metabolism. Thus, for the first time we revealed that TRPV2 is functionally expressed in astrocytes in addition to neurons. Copyright © 2013 Elsevier Inc. All rights reserved.

Genome-Scale Reconstruction of the Human Astrocyte Metabolic Network

PubMed Central

Martín-Jiménez, Cynthia A.; Salazar-Barreto, Diego; Barreto, George E.; González, Janneth

2017-01-01

Astrocytes are the most abundant cells of the central nervous system; they have a predominant role in maintaining brain metabolism. In this sense, abnormal metabolic states have been found in different neuropathological diseases. Determination of metabolic states of astrocytes is difficult to model using current experimental approaches given the high number of reactions and metabolites present. Thus, genome-scale metabolic networks derived from transcriptomic data can be used as a framework to elucidate how astrocytes modulate human brain metabolic states during normal conditions and in neurodegenerative diseases. We performed a Genome-Scale Reconstruction of the Human Astrocyte Metabolic Network with the purpose of elucidating a significant portion of the metabolic map of the astrocyte. This is the first global high-quality, manually curated metabolic reconstruction network of a human astrocyte. It includes 5,007 metabolites and 5,659 reactions distributed among 8 cell compartments, (extracellular, cytoplasm, mitochondria, endoplasmic reticle, Golgi apparatus, lysosome, peroxisome and nucleus). Using the reconstructed network, the metabolic capabilities of human astrocytes were calculated and compared both in normal and ischemic conditions. We identified reactions activated in these two states, which can be useful for understanding the astrocytic pathways that are affected during brain disease. Additionally, we also showed that the obtained flux distributions in the model, are in accordance with literature-based findings. Up to date, this is the most complete representation of the human astrocyte in terms of inclusion of genes, proteins, reactions and metabolic pathways, being a useful guide for in-silico analysis of several metabolic behaviors of the astrocyte during normal and pathologic states. PMID:28243200

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

PubMed Central

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

2013-01-01

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

SLAM- and nectin-4-independent noncytolytic spread of canine distemper virus in astrocytes.

PubMed

Alves, Lisa; Khosravi, Mojtaba; Avila, Mislay; Ader-Ebert, Nadine; Bringolf, Fanny; Zurbriggen, Andreas; Vandevelde, Marc; Plattet, Philippe

2015-05-01

Measles and canine distemper viruses (MeV and CDV, respectively) first replicate in lymphatic and epithelial tissues by using SLAM and nectin-4 as entry receptors, respectively. The viruses may also invade the brain to establish persistent infections, triggering fatal complications, such as subacute sclerosis pan-encephalitis (SSPE) in MeV infection or chronic, multiple sclerosis-like, multifocal demyelinating lesions in the case of CDV infection. In both diseases, persistence is mediated by viral nucleocapsids that do not require packaging into particles for infectivity but are directly transmitted from cell to cell (neurons in SSPE or astrocytes in distemper encephalitis), presumably by relying on restricted microfusion events. Indeed, although morphological evidence of fusion remained undetectable, viral fusion machineries and, thus, a putative cellular receptor, were shown to contribute to persistent infections. Here, we first showed that nectin-4-dependent cell-cell fusion in Vero cells, triggered by a demyelinating CDV strain, remained extremely limited, thereby supporting a potential role of nectin-4 in mediating persistent infections in astrocytes. However, nectin-4 could not be detected in either primary cultured astrocytes or the white matter of tissue sections. In addition, a bioengineered "nectin-4-blind" recombinant CDV retained full cell-to-cell transmission efficacy in primary astrocytes. Combined with our previous report demonstrating the absence of SLAM expression in astrocytes, these findings are suggestive for the existence of a hitherto unrecognized third CDV receptor expressed by glial cells that contributes to the induction of noncytolytic cell-to-cell viral transmission in astrocytes. While persistent measles virus (MeV) infection induces SSPE in humans, persistent canine distemper virus (CDV) infection causes chronic progressive or relapsing demyelination in carnivores. Common to both central nervous system (CNS) infections is that

Sodium signaling and astrocyte energy metabolism.

PubMed

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

2016-10-01

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

Neuronal activity determines distinct gliotransmitter release from a single astrocyte

PubMed Central

Covelo, Ana

2018-01-01

Accumulating evidence indicates that astrocytes are actively involved in brain function by regulating synaptic activity and plasticity. Different gliotransmitters, such as glutamate, ATP, GABA or D-serine, released form astrocytes have been shown to induce different forms of synaptic regulation. However, whether a single astrocyte may release different gliotransmitters is unknown. Here we show that mouse hippocampal astrocytes activated by endogenous (neuron-released endocannabinoids or GABA) or exogenous (single astrocyte Ca2+ uncaging) stimuli modulate putative single CA3-CA1 hippocampal synapses. The astrocyte-mediated synaptic modulation was biphasic and consisted of an initial glutamate-mediated potentiation followed by a purinergic-mediated depression of neurotransmitter release. The temporal dynamic properties of this biphasic synaptic regulation depended on the firing frequency and duration of the neuronal activity that stimulated astrocytes. Present results indicate that single astrocytes can decode neuronal activity and, in response, release distinct gliotransmitters to differentially regulate neurotransmission at putative single synapses. PMID:29380725

Hypoosmotic swelling modifies glutamate-glutamine cycle in the cerebral cortex and in astrocyte cultures

PubMed Central

Hyzinski-García, María C.; Vincent, Melanie Y.; Haskew-Layton, Renée E.; Dohare, Preeti; Keller, Richard W.; Mongin, Alexander A.

2011-01-01

In our previous work, we found that perfusion of the rat cerebral cortex with hypoosmotic medium triggers massive release of the excitatory amino acid L-glutamate but decreases extracellular levels of L-glutamine (R.E. Haskew-Layton et al., PLoS ONE, 3: e3543). The release of glutamate was linked to activation of volume-regulated anion channels (VRAC), while mechanism(s) responsible for alterations in extracellular glutamine remained unclear. When mannitol was added to the hypoosmotic medium in order to reverse reductions in osmolarity, changes in microdialysate levels of glutamine were prevented, indicating an involvement of cellular swelling. Since the main source of brain glutamine is astrocytic synthesis and export, we explored the impact of hypoosmotic medium on glutamine synthesis and transport in rat primary astrocyte cultures. In astrocytes, a 40% reduction in medium osmolarity moderately stimulated the release of L-[3H]glutamine by ~2-fold and produced no changes in L-[3H]glutamine uptake. In comparison, hypoosmotic medium stimulated the release of glutamate (traced with D[3H]aspartate) by more than 20-fold. In whole-cell enzymatic assays, we discovered that hypoosmotic medium caused a 20% inhibition of astrocytic conversion of L[3H]glutamate into L-[3H]glutamine by glutamine synthetase. Using an HPLC assay we further found a 35% reduction in intracellular levels of endogenous glutamine. Overall, our findings suggest that cellular swelling (1) inhibits astrocytic glutamine synthetase activity, and (2) reduces substrate availability for this enzyme due to the activation of VRAC. These combined effects likely lead to reductions in astrocytic glutamine export in vivo and may partially explain occurrence of hyperexcitability and seizures in human hyponatremia. PMID:21517854

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

PubMed

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

2011-09-08

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. Copyright © 2011 Elsevier Inc. All rights reserved.

Effect of long-term (2 years) exposure of mouse brains to global system for mobile communication (GSM) radiofrequency fields on astrocytic immunoreactivity.

PubMed

Court-Kowalski, Stefan; Finnie, John W; Manavis, Jim; Blumbergs, Peter C; Helps, Stephen C; Vink, Robert

2015-04-01

This study was designed to determine whether long-term (2 years) brain exposure to mobile telephone radiofrequency (RF) fields produces any astrocytic activation as these glia react to a wide range of neural perturbations by astrogliosis. Using a purpose-designed exposure system at 900 MHz, mice were given a single, far-field whole body exposure at a specific absorption rate of 4 W/kg on five successive days per week for 104 weeks. Control mice were sham-exposed or freely mobile in a cage to control any stress caused by immobilization in the exposure module. Brains were perfusion-fixed with 4% paraformaldehyde and three coronal levels immunostained for glial fibrillary acidic protein (GFAP). These brain slices were then examined by light microscopy and the amount of this immunomarker quantified using a color deconvolution method. There was no change in astrocytic GFAP immunostaining in brains after long-term exposure to mobile telephony microwaves compared to control (sham-exposed or freely moving caged mice). It was concluded that long-term (2 years) exposure of murine brains to mobile telephone RF fields did not produce any astrocytic reaction (astrogliosis) detectable by GFAP immunostaining. © 2015 Wiley Periodicals, Inc.

Targeting of astrocytic glucose metabolism by beta-hydroxybutyrate.

PubMed

Valdebenito, Rocío; Ruminot, Iván; Garrido-Gerter, Pamela; Fernández-Moncada, Ignacio; Forero-Quintero, Linda; Alegría, Karin; Becker, Holger M; Deitmer, Joachim W; Barros, L Felipe

2016-10-01

The effectiveness of ketogenic diets and intermittent fasting against neurological disorders has brought interest to the effects of ketone bodies on brain cells. These compounds are known to modify the metabolism of neurons, but little is known about their effect on astrocytes, cells that control the supply of glucose to neurons and also modulate neuronal excitability through the glycolytic production of lactate. Here we have used genetically-encoded Förster Resonance Energy Transfer nanosensors for glucose, pyruvate and ATP to characterize astrocytic energy metabolism at cellular resolution. Our results show that the ketone body beta-hydroxybutyrate strongly inhibited astrocytic glucose consumption in mouse astrocytes in mixed cultures, in organotypic hippocampal slices and in acute hippocampal slices prepared from ketotic mice, while blunting the stimulation of glycolysis by physiological and pathophysiological stimuli. The inhibition of glycolysis was paralleled by an increased ability of astrocytic mitochondria to metabolize pyruvate. These results support the emerging notion that astrocytes contribute to the neuroprotective effect of ketone bodies. © The Author(s) 2015.

Astrocyte truncated-TrkB mediates BDNF antiapoptotic effect leading to neuroprotection.

PubMed

Saba, Julieta; Turati, Juan; Ramírez, Delia; Carniglia, Lila; Durand, Daniela; Lasaga, Mercedes; Caruso, Carla

2018-05-31

Astrocytes are glial cells that help maintain brain homeostasis and become reactive in neurodegenerative processes releasing both harmful and beneficial factors. We have demonstrated that brain-derived neurotrophic factor (BDNF) expression is induced by melanocortins in astrocytes but BDNF actions in astrocytes are largely unknown. We hypothesize that BDNF may prevent astrocyte death resulting in neuroprotection. We found that BDNF increased astrocyte viability, preventing apoptosis induced by serum deprivation by decreasing active caspase-3 and p53 expression. The antiapoptotic action of BDNF was abolished by ANA-12 (a specific TrkB antagonist) and by K252a (a general Trk antagonist). Astrocytes only express the BDNF receptor TrkB truncated isoform 1, TrkB-T1. BDNF induced ERK, Akt and Src (a non-receptor tyrosine kinase) activation in astrocytes. Blocking ERK and Akt pathways abolished BDNF protection in serum deprivation-induced cell death. Moreover, BDNF protected astrocytes from death by 3-nitropropionic acid (3-NP), an effect also blocked by ANA-12, K252a, and inhibitors of ERK, calcium and Src. BDNF reduced reactive oxygen species (ROS) levels induced in astrocytes by 3-NP and increased xCT expression and glutathione levels. Astrocyte conditioned media (ACM) from untreated astrocytes partially protected PC12 neurons whereas ACM from BDNF-treated astrocytes completely protected PC12 neurons from 3-NP-induced apoptosis. Both ACM from control and BDNF-treated astrocytes markedly reduced ROS levels induced by 3-NP in PC12 cells. Our results demonstrate that BDNF protects astrocytes from cell death through TrkB-T1 signaling, exerts an antioxidant action, and induces release of neuroprotective factors from astrocytes. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

HIV-1 Nef is released in extracellular vesicles derived from astrocytes: evidence for Nef-mediated neurotoxicity

PubMed Central

Sami Saribas, A; Cicalese, Stephanie; Ahooyi, Taha Mohseni; Khalili, Kamel; Amini, Shohreh; Sariyer, Ilker Kudret

2017-01-01

Human immunodeficiency virus-associated neurological disorders (HANDs) affect the majority of AIDS patients and are a significant problem among HIV-1-infected individuals who live longer because of combined anti-retroviral therapies. HIV-1 utilizes a number of viral proteins and subsequent cytokine inductions to unleash its toxicity on neurons. Among HIV-1 viral proteins, Nef is a small protein expressed abundantly in astrocytes of HIV-1-infected brains and has been suggested to have a role in the pathogenesis of HAND. In order to explore its effect in the central nervous system, HIV-1 Nef was expressed in primary human fetal astrocytes (PHFAs) using an adenovirus. Our results revealed that HIV-1 Nef is released in extracellular vesicles (EVs) derived from PHFA cells expressing the protein. Interestingly, HIV-1 Nef release in EVs was enriched significantly when the cells were treated with autophagy activators perifosine, tomaxifen, MG-132, and autophagy inhibitors LY294002 and wortmannin suggesting a novel role of autophagy signaling in HIV-1 Nef release from astrocytes. Next, Nef-carrying EVs were purified from astrocyte cultures and neurotoxic effects on neurons were analyzed. We observed that HIV-1 Nef-containing EVs were readily taken up by neurons as demonstrated by immunocytochemistry and immunoblotting. Furthermore, treatment of neurons with Nef-carrying EVs induced oxidative stress as evidenced by a decrease in glutathione levels. To further investigate its neurotoxic effects, we expressed HIV-1 Nef in primary neurons by adenoviral transduction. Intracellular expression of HIV-1 Nef caused axonal and neurite degeneration of neurons. Furthermore, expression of HIV-1 Nef decreased the levels of phospho-tau while enhancing total tau in primary neurons. In addition, treatment of primary neurons with Nef-carrying EVs suppressed functional neuronal action potential assessed by multielectrode array studies. Collectively, these data suggested that HIV-1 Nef can be

Genes Involved in the Astrocyte-Neuron Lactate Shuttle (ANLS) Are Specifically Regulated in Cortical Astrocytes Following Sleep Deprivation in Mice

PubMed Central

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

2013-01-01

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

Stretch-induced Ca2+ independent ATP release in hippocampal astrocytes.

PubMed

Xiong, Yingfei; Teng, Sasa; Zheng, Lianghong; Sun, Suhua; Li, Jie; Guo, Ning; Li, Mingli; Wang, Li; Zhu, Feipeng; Wang, Changhe; Rao, Zhiren; Zhou, Zhuan

2018-02-28

Similar to neurons, astrocytes actively participate in synaptic transmission via releasing gliotransmitters. The Ca 2+ -dependent release of gliotransmitters includes glutamate and ATP. Following an 'on-cell-like' mechanical stimulus to a single astrocyte, Ca 2+ independent single, large, non-quantal, ATP release occurs. Astrocytic ATP release is inhibited by either selective antagonist treatment or genetic knockdown of P2X7 receptor channels. Our work suggests that ATP can be released from astrocytes via two independent pathways in hippocampal astrocytes; in addition to the known Ca 2+ -dependent vesicular release, larger non-quantal ATP release depends on P2X7 channels following mechanical stretch. Astrocytic ATP release is essential for brain functions such as synaptic long-term potentiation for learning and memory. However, whether and how ATP is released via exocytosis remains hotly debated. All previous studies of non-vesicular ATP release have used indirect assays. By contrast, two recent studies report vesicular ATP release using more direct assays. In the present study, using patch clamped 'ATP-sniffer cells', we re-investigated astrocytic ATP release at single-vesicle resolution in hippocampal astrocytes. Following an 'on-cell-like' mechanical stimulus of a single astrocyte, a Ca 2+ independent single large non-quantal ATP release occurred, in contrast to the Ca 2+ -dependent multiple small quantal ATP release in a chromaffin cell. The mechanical stimulation-induced ATP release from an astrocyte was inhibited by either exposure to a selective antagonist or genetic knockdown of P2X7 receptor channels. Functional P2X7 channels were expressed in astrocytes in hippocampal brain slices. Thus, in addition to small quantal ATP release, larger non-quantal ATP release depends on P2X7 channels in astrocytes. © 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.

Astrocytic Activation Generates De Novo Neuronal Potentiation and Memory Enhancement.

PubMed

Adamsky, Adar; Kol, Adi; Kreisel, Tirzah; Doron, Adi; Ozeri-Engelhard, Nofar; Melcer, Talia; Refaeli, Ron; Horn, Henrike; Regev, Limor; Groysman, Maya; London, Michael; Goshen, Inbal

2018-05-18

Astrocytes respond to neuronal activity and were shown to be necessary for plasticity and memory. To test whether astrocytic activity is also sufficient to generate synaptic potentiation and enhance memory, we expressed the Gq-coupled receptor hM3Dq in CA1 astrocytes, allowing their activation by a designer drug. We discovered that astrocytic activation is not only necessary for synaptic plasticity, but also sufficient to induce NMDA-dependent de novo long-term potentiation in the hippocampus that persisted after astrocytic activation ceased. In vivo, astrocytic activation enhanced memory allocation; i.e., it increased neuronal activity in a task-specific way only when coupled with learning, but not in home-caged mice. Furthermore, astrocytic activation using either a chemogenetic or an optogenetic tool during acquisition resulted in memory recall enhancement on the following day. Conversely, directly increasing neuronal activity resulted in dramatic memory impairment. Our findings that astrocytes induce plasticity and enhance memory may have important clinical implications for cognitive augmentation treatments. Copyright © 2018 Elsevier Inc. All rights reserved.

Astrocytes Regulate GLP-1 Receptor-Mediated Effects on Energy Balance

PubMed Central

Reiner, David J.; Mietlicki-Baase, Elizabeth G.; McGrath, Lauren E.; Zimmer, Derek J.; Bence, Kendra K.; Sousa, Gregory L.; Konanur, Vaibhav R.; Krawczyk, Joanna; Burk, David H.; Kanoski, Scott E.; Hermann, Gerlinda E.; Rogers, Richard C.

2016-01-01

Astrocytes are well established modulators of extracellular glutamate, but their direct influence on energy balance-relevant behaviors is largely understudied. As the anorectic effects of glucagon-like peptide-1 receptor (GLP-1R) agonists are partly mediated by central modulation of glutamatergic signaling, we tested the hypothesis that astrocytic GLP-1R signaling regulates energy balance in rats. Central or peripheral administration of a fluorophore-labeled GLP-1R agonist, exendin-4, localizes within astrocytes and neurons in the nucleus tractus solitarius (NTS), a hindbrain nucleus critical for energy balance control. This effect is mediated by GLP-1R, as the uptake of systemically administered fluorophore-tagged exendin-4 was blocked by central pretreatment with the competitive GLP-1R antagonist exendin-(9–39). Ex vivo analyses show prolonged exendin-4-induced activation (live cell calcium signaling) of NTS astrocytes and neurons; these effects are also attenuated by exendin-(9–39), indicating mediation by the GLP-1R. In vitro analyses show that the application of GLP-1R agonists increases cAMP levels in astrocytes. Immunohistochemical analyses reveal that endogenous GLP-1 axons form close synaptic apposition with NTS astrocytes. Finally, pharmacological inhibition of NTS astrocytes attenuates the anorectic and body weight-suppressive effects of intra-NTS GLP-1R activation. Collectively, data demonstrate a role for NTS astrocytic GLP-1R signaling in energy balance control. SIGNIFICANCE STATEMENT Glucagon-like peptide-1 receptor (GLP-1R) agonists reduce food intake and are approved by the Food and Drug Administration for the treatment of obesity, but the cellular mechanisms underlying the anorectic effects of GLP-1 require further investigation. Astrocytes represent a major cellular population in the CNS that regulates neurotransmission, yet the role of astrocytes in mediating energy balance is largely unstudied. The current data provide novel evidence that

Manganese inhibits the ability of astrocytes to promote neuronal differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giordano, Gennaro; Pizzurro, Daniella; VanDeMark, Kathryn

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

Astrocytes and Developmental White Matter Disorders

ERIC Educational Resources Information Center

Sen, Ellora; Levison, Steven W.

2006-01-01

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

Astrocytes regulate heterogeneity of presynaptic strengths in hippocampal networks

PubMed Central

Letellier, Mathieu; Park, Yun Kyung; Chater, Thomas E.; Chipman, Peter H.; Gautam, Sunita Ghimire; Oshima-Takago, Tomoko; Goda, Yukiko

2016-01-01

Dendrites are neuronal structures specialized for receiving and processing information through their many synaptic inputs. How input strengths are modified across dendrites in ways that are crucial for synaptic integration and plasticity remains unclear. We examined in single hippocampal neurons the mechanism of heterosynaptic interactions and the heterogeneity of synaptic strengths of pyramidal cell inputs. Heterosynaptic presynaptic plasticity that counterbalances input strengths requires N-methyl-d-aspartate receptors (NMDARs) and astrocytes. Importantly, this mechanism is shared with the mechanism for maintaining highly heterogeneous basal presynaptic strengths, which requires astrocyte Ca2+ signaling involving NMDAR activation, astrocyte membrane depolarization, and L-type Ca2+ channels. Intracellular infusion of NMDARs or Ca2+-channel blockers into astrocytes, conditionally ablating the GluN1 NMDAR subunit, or optogenetically hyperpolarizing astrocytes with archaerhodopsin promotes homogenization of convergent presynaptic inputs. Our findings support the presence of an astrocyte-dependent cellular mechanism that enhances the heterogeneity of presynaptic strengths of convergent connections, which may help boost the computational power of dendrites. PMID:27118849

Glutaric aciduria type 1 metabolites impair the succinate transport from astrocytic to neuronal cells.

PubMed

Lamp, Jessica; Keyser, Britta; Koeller, David M; Ullrich, Kurt; Braulke, Thomas; Mühlhausen, Chris

2011-05-20

The inherited neurodegenerative disorder glutaric aciduria type 1 (GA1) results from mutations in the gene for the mitochondrial matrix enzyme glutaryl-CoA dehydrogenase (GCDH), which leads to elevations of the dicarboxylates glutaric acid (GA) and 3-hydroxyglutaric acid (3OHGA) in brain and blood. The characteristic clinical presentation of GA1 is a sudden onset of dystonia during catabolic situations, resulting from acute striatal injury. The underlying mechanisms are poorly understood, but the high levels of GA and 3OHGA that accumulate during catabolic illnesses are believed to play a primary role. Both GA and 3OHGA are known to be substrates for Na(+)-coupled dicarboxylate transporters, which are required for the anaplerotic transfer of the tricarboxylic acid cycle (TCA) intermediate succinate between astrocytes and neurons. We hypothesized that GA and 3OHGA inhibit the transfer of succinate from astrocytes to neurons, leading to reduced TCA cycle activity and cellular injury. Here, we show that both GA and 3OHGA inhibit the uptake of [(14)C]succinate by Na(+)-coupled dicarboxylate transporters in cultured astrocytic and neuronal cells of wild-type and Gcdh(-/-) mice. In addition, we demonstrate that the efflux of [(14)C]succinate from Gcdh(-/-) astrocytic cells mediated by a not yet identified transporter is strongly reduced. This is the first experimental evidence that GA and 3OHGA interfere with two essential anaplerotic transport processes: astrocytic efflux and neuronal uptake of TCA cycle intermediates, which occur between neurons and astrocytes. These results suggest that elevated levels of GA and 3OHGA may lead to neuronal injury and cell death via disruption of TCA cycle activity. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Development of a Novel Method for the Purification and Culture of Rodent Astrocytes

PubMed Central

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.

2011-01-01

Summary 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. PMID:21903074

Pazopanib Inhibits the Activation of PDGFRβ-Expressing Astrocytes in the Brain Metastatic Microenvironment of Breast Cancer Cells

PubMed Central

Gril, Brunilde; Palmieri, Diane; Qian, Yongzhen; Anwar, Talha; Liewehr, David J.; Steinberg, Seth M.; Andreu, Zoraida; Masana, Daniel; Fernández, Paloma; Steeg, Patricia S.; Vidal-Vanaclocha, Fernando

2014-01-01

Brain metastases occur in more than one-third of metastatic breast cancer patients whose tumors overexpress HER2 or are triple negative. Brain colonization of cancer cells occurs in a unique environment, containing microglia, oligodendrocytes, astrocytes, and neurons. Although a neuroinflammatory response has been documented in brain metastasis, its contribution to cancer progression and therapy remains poorly understood. Using an experimental brain metastasis model, we characterized the brain metastatic microenvironment of brain tropic, HER2-transfected MDA-MB-231 human breast carcinoma cells (231-BR-HER2). A previously unidentified subpopulation of metastasis-associated astrocytes expressing phosphorylated platelet-derived growth factor receptor β (at tyrosine 751; p751-PDGFRβ) was identified around perivascular brain micrometastases. p751-PDGFRβ+ astrocytes were also identified in human brain metastases from eight craniotomy specimens and in primary cultures of astrocyte-enriched glial cells. Previously, we reported that pazopanib, a multispecific tyrosine kinase inhibitor, prevented the outgrowth of 231-BR-HER2 large brain metastases by 73%. Here, we evaluated the effect of pazopanib on the brain neuroinflammatory microenvironment. Pazopanib treatment resulted in 70% (P = 0.023) decrease of the p751-PDGFRβ+ astrocyte population, at the lowest dose of 30 mg/kg, twice daily. Collectively, the data identify a subpopulation of activated astrocytes in the subclinical perivascular stage of brain metastases and show that they are inhibitable by pazopanib, suggesting its potential to prevent the development of brain micrometastases in breast cancer patients. PMID:23583652

(13)C heteronuclear NMR studies of the interaction of cultured neurons and astrocytes and aluminum blockade of the preferential release of citrate from astrocytes.

PubMed

Meshitsuka, Shunsuke; Aremu, David A

2008-02-01

Citrate has been identified as a major tricarboxylic acid (TCA) cycle constituent preferentially released by astrocytes. We undertook the present study to examine further the nature of metabolic compartmentation in central nervous system tissues using (13)C-labeled glucose and to provide new information on the influence of aluminum on the metabolic interaction between neurons and astrocytes. Metabolites released into the culture medium from astrocytes and neuron-astrocyte coculture, as well as the perchloric acid extracts of the cells were analyzed using 2D (1)H and (13)C NMR spectroscopy. Astrocytes released citrate into the culture medium and the released citrate was consumed by neurons in coculture. Citrate release by astrocytes was blocked in the presence of aluminum, with progressive accumulation of citrate within the cells. We propose citrate supply is a more efficient energy source than lactate for neurons to produce ATP, especially in the hypoglycemic state on account of it being a direct component of the TCA cycle. Astrocytes may be the cellular compartment for aluminum accumulation as a citrate complex in the brain.

Astrocytes, therapeutic targets for neuroprotection and neurorestoration in ischemic stroke

PubMed Central

Liu, Zhongwu; Chopp, Michael

2015-01-01

Astrocytes are the most abundant cell type within the central nervous system. They play essential roles in maintaining normal brain function, as they are a critical structural and functional part of the tripartite synapses and the neurovascular unit, and communicate with neurons, oligodendrocytes and endothelial cells. After an ischemic stroke, astrocytes perform multiple functions both detrimental and beneficial, for neuronal survival during the acute phase. Aspects of the astrocytic inflammatory response to stroke may aggravate the ischemic lesion, but astrocytes also provide benefit for neuroprotection, by limiting lesion extension via anti-excitotoxicity effects and releasing neurotrophins. Similarly, during the late recovery phase after stroke, the glial scar may obstruct axonal regeneration and subsequently reduce the functional outcome; however, astrocytes also contribute to angiogenesis, neurogenesis, synaptogenesis, and axonal remodeling, and thereby promote neurological recovery. Thus, the pivotal involvement of astrocytes in normal brain function and responses to an ischemic lesion designates them as excellent therapeutic targets to improve functional outcome following stroke. In this review, we will focus on functions of astrocytes and astrocyte-mediated events during stroke and recovery. We will provide an overview of approaches on how to reduce the detrimental effects and amplify the beneficial effects of astrocytes on neuroprotection and on neurorestoration post stroke, which may lead to novel and clinically relevant therapies for stroke. PMID:26455456

Insulin and heparin-binding epidermal growth factor-like growth factor synergistically promote astrocyte survival and proliferation in serum-free medium.

PubMed

Jia, Mei; Shi, Zhongfang; Yan, Xu; Xu, Lixin; Dong, Liping; Li, Jiaxin; Wang, Yujiao; Yang, Shaohua; Yuan, Fang

2018-06-08

In vitro systems allowing maintenance and experimentation on primary astrocyte cultures have been used for decades. Astrocyte cultures are most maintained in serum-containing medium which has been found to alter the morphology and gene profiles of astrocytes. Here, we reported a new serum-free medium for astrocyte culture, which consisted of DMEM and NB media supplemented with insulin and heparin-binding epidermal growth factor-like growth factor (HB-EGF) (SF-I-H medium). Meanwhile FBS-containing (FBS) medium composed of DMEM medium containing 10% FBS were used for comparison study. Cerebral cortex was harvested from postnatal day 1 Wistar rats and brain cells were isolated and seeded to poly-L-lysine coated culture dishes after 15 min differential velocity adherence. Compared with FBS medium, astrocytes in SF-I-H medium are smaller and exhibited process bearing morphologies. MTT assays showed that cell density and proliferation rate were higher in SF-I-H medium than in FBS medium all the time, and flow cytometry analysis revealed that SF-I-H medium promoted cell mitosis in a manner comparable to FBS medium. Consistently, western blot analysis further revealed that insulin and HB-EGF synergistically activated the PI3K-AKT and MAPK-ERK1/2 signaling cascades as FBS. Astrocytes cultured in SF-I-H medium grow faster than FBS medium. Taken together, our results indicated that SF-I-H medium, in which cell morphology was similar with astrocytes in brain, was more effective for astrocyte survival and proliferation than FBS medium, providing a new cell model to study astrocyte functions without the interference of serum. Copyright © 2018. Published by Elsevier B.V.

In Vivo Evidence for a Lactate Gradient from Astrocytes to Neurons.

PubMed

Mächler, Philipp; Wyss, Matthias T; Elsayed, Maha; Stobart, Jillian; Gutierrez, Robin; von Faber-Castell, Alexandra; Kaelin, Vincens; Zuend, Marc; San Martín, Alejandro; Romero-Gómez, Ignacio; Baeza-Lehnert, Felipe; Lengacher, Sylvain; Schneider, Bernard L; Aebischer, Patrick; Magistretti, Pierre J; Barros, L Felipe; Weber, Bruno

2016-01-12

Investigating lactate dynamics in brain tissue is challenging, partly because in vivo data at cellular resolution are not available. We monitored lactate in cortical astrocytes and neurons of mice using the genetically encoded FRET sensor Laconic in combination with two-photon microscopy. An intravenous lactate injection rapidly increased the Laconic signal in both astrocytes and neurons, demonstrating high lactate permeability across tissue. The signal increase was significantly smaller in astrocytes, pointing to higher basal lactate levels in these cells, confirmed by a one-point calibration protocol. Trans-acceleration of the monocarboxylate transporter with pyruvate was able to reduce intracellular lactate in astrocytes but not in neurons. Collectively, these data provide in vivo evidence for a lactate gradient from astrocytes to neurons. This gradient is a prerequisite for a carrier-mediated lactate flux from astrocytes to neurons and thus supports the astrocyte-neuron lactate shuttle model, in which astrocyte-derived lactate acts as an energy substrate for neurons. Copyright © 2016 Elsevier Inc. All rights reserved.

Astrocytes, Synapses and Brain Function: A Computational Approach

NASA Astrophysics Data System (ADS)

Nadkarni, Suhita

2006-03-01

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

Leptin receptor mRNA in rat brain astrocytes

PubMed Central

Hsuchou, Hung; Pan, Weihong; Barnes, Maria J.; Kastin, Abba J.

2009-01-01

We recently reported that mouse astrocytes express leptin receptors (ObR), and that obesity induces upregulation of astrocytic ObR. To provide further evidence of the importance of astrocytic ObR expression, we performed double-labeling fluorescent in-situ hybridization (FISH) and immunohistochemistry in the rat hypothalamus. Laser confocal microscopic image analysis showed that ObR mRNA was present in glial fibrillary acidic protein (+) cells that show distinctive astrocytic morphology as well as in neurons. In addition to the presence of ObR mRNA, ObR protein was shown in both astrocytes and neurons in the rat hypothalamus by double-labeling immunohistochemistry. In cultured rat C6 astrocytoma cells treated with different doses of lipopolysaccharide for 6 h, the mRNA for ObRa or ObRb did not show significant changes, as measured by quantitative RT-PCR. However, the protein expression of both ObRa and ObRb, determined by western blotting, was increased after the C6 cells were treated with either lipopolysaccharide or tumor necrosis factor-α. The results indicate that astrocytic ObR expression is present in rats as well as mice, and that it probably plays a role in the neuroinflammatory response. PMID:19747514

Modulation of Astrocyte Activity by Cannabidiol, a Nonpsychoactive Cannabinoid

PubMed Central

Kozela, Ewa; Juknat, Ana; Vogel, Zvi

2017-01-01

The astrocytes have gained in recent decades an enormous interest as a potential target for neurotherapies, due to their essential and pleiotropic roles in brain physiology and pathology. Their precise regulation is still far from understood, although several candidate molecules/systems arise as promising targets for astrocyte-mediated neuroregulation and/or neuroprotection. The cannabinoid system and its ligands have been shown to interact and affect activities of astrocytes. Cannabidiol (CBD) is the main non-psychotomimetic cannabinoid derived from Cannabis. CBD is devoid of direct CB1 and CB2 receptor activity, but exerts a number of important effects in the brain. Here, we attempt to sum up the current findings on the effects of CBD on astrocyte activity, and in this way on central nervous system (CNS) functions, across various tested models and neuropathologies. The collected data shows that increased astrocyte activity is suppressed in the presence of CBD in models of ischemia, Alzheimer-like and Multiple-Sclerosis-like neurodegenerations, sciatic nerve injury, epilepsy, and schizophrenia. Moreover, CBD has been shown to decrease proinflammatory functions and signaling in astrocytes. PMID:28788104

Modulation of Astrocyte Activity by Cannabidiol, a Nonpsychoactive Cannabinoid.

PubMed

Kozela, Ewa; Juknat, Ana; Vogel, Zvi

2017-07-31

The astrocytes have gained in recent decades an enormous interest as a potential target for neurotherapies, due to their essential and pleiotropic roles in brain physiology and pathology. Their precise regulation is still far from understood, although several candidate molecules/systems arise as promising targets for astrocyte-mediated neuroregulation and/or neuroprotection. The cannabinoid system and its ligands have been shown to interact and affect activities of astrocytes. Cannabidiol (CBD) is the main non-psychotomimetic cannabinoid derived from Cannabis . CBD is devoid of direct CB1 and CB2 receptor activity, but exerts a number of important effects in the brain. Here, we attempt to sum up the current findings on the effects of CBD on astrocyte activity, and in this way on central nervous system (CNS) functions, across various tested models and neuropathologies. The collected data shows that increased astrocyte activity is suppressed in the presence of CBD in models of ischemia, Alzheimer-like and Multiple-Sclerosis-like neurodegenerations, sciatic nerve injury, epilepsy, and schizophrenia. Moreover, CBD has been shown to decrease proinflammatory functions and signaling in astrocytes.

Astrocyte pathology in the ventral prefrontal white matter in depression.

PubMed

Rajkowska, Grazyna; Legutko, Beata; Moulana, Mohadetheh; Syed, Maryam; Romero, Damian G; Stockmeier, Craig A; Miguel-Hidalgo, Jose Javier

2018-04-07

Astrocyte functions in white matter are less well understood than in gray matter. Our recent study of white matter in ventral prefrontal cortex (vPFC) revealed alterations in expression of myelin-related genes in major depressive disorder (MDD). Since white matter astrocytes maintain myelin, we hypothesized that morphometry of these cells will be altered in MDD in the same prefrontal white matter region in which myelin-related genes are altered. White matter adjacent to vPFC was examined in 25 MDD and 21 control subjects. Density and size of GFAP-immunoreactive (-ir) astrocyte cell bodies was measured. The area fraction of GFAP-ir astrocytes (cell bodies + processes) was also estimated. GFAP mRNA expression was determined using qRT-PCR. The density of GFAP-ir astrocytes was also measured in vPFC white matter of rats subjected to chronic unpredictable stress (CUS) and control animals. Fibrous and smooth GFAP-ir astrocytes were distinguished in human white matter. The density of both types of astrocytes was significantly decreased in MDD. Area fraction of GFAP immunoreactivity was significantly decreased in MDD, but mean soma size remained unchanged. Expression of GFAP mRNA was significantly decreased in MDD. In CUS rats there was a significant decrease in astrocyte density in prefrontal white matter. The decrease in density and area fraction of white matter astrocytes and GFAP mRNA in MDD may be linked to myelin pathology previously noted in these subjects. Astrocyte pathology may contribute to axon disturbances in axon integrity reported by neuroimaging studies in MDD and interfere with signal conduction in the white matter. Copyright © 2018. Published by Elsevier Ltd.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bender, A.S.; Hertz, L.

1988-01-01

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

Activity-dependent switch of GABAergic inhibition into glutamatergic excitation in astrocyte-neuron networks.

PubMed

Perea, Gertrudis; Gómez, Ricardo; Mederos, Sara; Covelo, Ana; Ballesteros, Jesús J; Schlosser, Laura; Hernández-Vivanco, Alicia; Martín-Fernández, Mario; Quintana, Ruth; Rayan, Abdelrahman; Díez, Adolfo; Fuenzalida, Marco; Agarwal, Amit; Bergles, Dwight E; Bettler, Bernhard; Manahan-Vaughan, Denise; Martín, Eduardo D; Kirchhoff, Frank; Araque, Alfonso

2016-12-24

Interneurons are critical for proper neural network function and can activate Ca 2+ signaling in astrocytes. However, the impact of the interneuron-astrocyte signaling into neuronal network operation remains unknown. Using the simplest hippocampal Astrocyte-Neuron network, i.e., GABAergic interneuron, pyramidal neuron, single CA3-CA1 glutamatergic synapse, and astrocytes, we found that interneuron-astrocyte signaling dynamically affected excitatory neurotransmission in an activity- and time-dependent manner, and determined the sign (inhibition vs potentiation) of the GABA-mediated effects. While synaptic inhibition was mediated by GABA A receptors, potentiation involved astrocyte GABA B receptors, astrocytic glutamate release, and presynaptic metabotropic glutamate receptors. Using conditional astrocyte-specific GABA B receptor ( Gabbr1 ) knockout mice, we confirmed the glial source of the interneuron-induced potentiation, and demonstrated the involvement of astrocytes in hippocampal theta and gamma oscillations in vivo. Therefore, astrocytes decode interneuron activity and transform inhibitory into excitatory signals, contributing to the emergence of novel network properties resulting from the interneuron-astrocyte interplay.

Astrocytes in physiological aging and Alzheimer's disease.

PubMed

Rodríguez-Arellano, J J; Parpura, V; Zorec, R; Verkhratsky, A

2016-05-26

Astrocytes are fundamental for homoeostasis, defence and regeneration of the central nervous system. Loss of astroglial function and astroglial reactivity contributes to the aging of the brain and to neurodegenerative diseases. Changes in astroglia in aging and neurodegeneration are highly heterogeneous and region-specific. In animal models of Alzheimer's disease (AD) astrocytes undergo degeneration and atrophy at the early stages of pathological progression, which possibly may alter the homeostatic reserve of the brain and contribute to early cognitive deficits. At later stages of AD reactive astrocytes are associated with neurite plaques, the feature commonly found in animal models and in human diseased tissue. In animal models of the AD reactive astrogliosis develops in some (e.g. in the hippocampus) but not in all regions of the brain. For instance, in entorhinal and prefrontal cortices astrocytes do not mount gliotic response to emerging β-amyloid deposits. These deficits in reactivity coincide with higher vulnerability of these regions to AD-type pathology. Astroglial morphology and function can be regulated through environmental stimulation and/or medication suggesting that astrocytes can be regarded as a target for therapies aimed at the prevention and cure of neurodegenerative disorders. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

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

PubMed

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

2013-10-01

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

Pazopanib inhibits the activation of PDGFRβ-expressing astrocytes in the brain metastatic microenvironment of breast cancer cells.

PubMed

Gril, Brunilde; Palmieri, Diane; Qian, Yongzhen; Anwar, Talha; Liewehr, David J; Steinberg, Seth M; Andreu, Zoraida; Masana, Daniel; Fernández, Paloma; Steeg, Patricia S; Vidal-Vanaclocha, Fernando

2013-06-01

Brain metastases occur in more than one-third of metastatic breast cancer patients whose tumors overexpress HER2 or are triple negative. Brain colonization of cancer cells occurs in a unique environment, containing microglia, oligodendrocytes, astrocytes, and neurons. Although a neuroinflammatory response has been documented in brain metastasis, its contribution to cancer progression and therapy remains poorly understood. Using an experimental brain metastasis model, we characterized the brain metastatic microenvironment of brain tropic, HER2-transfected MDA-MB-231 human breast carcinoma cells (231-BR-HER2). A previously unidentified subpopulation of metastasis-associated astrocytes expressing phosphorylated platelet-derived growth factor receptor β (at tyrosine 751; p751-PDGFRβ) was identified around perivascular brain micrometastases. p751-PDGFRβ(+) astrocytes were also identified in human brain metastases from eight craniotomy specimens and in primary cultures of astrocyte-enriched glial cells. Previously, we reported that pazopanib, a multispecific tyrosine kinase inhibitor, prevented the outgrowth of 231-BR-HER2 large brain metastases by 73%. Here, we evaluated the effect of pazopanib on the brain neuroinflammatory microenvironment. Pazopanib treatment resulted in 70% (P = 0.023) decrease of the p751-PDGFRβ(+) astrocyte population, at the lowest dose of 30 mg/kg, twice daily. Collectively, the data identify a subpopulation of activated astrocytes in the subclinical perivascular stage of brain metastases and show that they are inhibitable by pazopanib, suggesting its potential to prevent the development of brain micrometastases in breast cancer patients. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Activity-dependent switch of GABAergic inhibition into glutamatergic excitation in astrocyte-neuron networks

PubMed Central

Perea, Gertrudis; Gómez, Ricardo; Mederos, Sara; Covelo, Ana; Ballesteros, Jesús J; Schlosser, Laura; Hernández-Vivanco, Alicia; Martín-Fernández, Mario; Quintana, Ruth; Rayan, Abdelrahman; Díez, Adolfo; Fuenzalida, Marco; Agarwal, Amit; Bergles, Dwight E; Bettler, Bernhard; Manahan-Vaughan, Denise; Martín, Eduardo D; Kirchhoff, Frank; Araque, Alfonso

2016-01-01

Interneurons are critical for proper neural network function and can activate Ca2+ signaling in astrocytes. However, the impact of the interneuron-astrocyte signaling into neuronal network operation remains unknown. Using the simplest hippocampal Astrocyte-Neuron network, i.e., GABAergic interneuron, pyramidal neuron, single CA3-CA1 glutamatergic synapse, and astrocytes, we found that interneuron-astrocyte signaling dynamically affected excitatory neurotransmission in an activity- and time-dependent manner, and determined the sign (inhibition vs potentiation) of the GABA-mediated effects. While synaptic inhibition was mediated by GABAA receptors, potentiation involved astrocyte GABAB receptors, astrocytic glutamate release, and presynaptic metabotropic glutamate receptors. Using conditional astrocyte-specific GABAB receptor (Gabbr1) knockout mice, we confirmed the glial source of the interneuron-induced potentiation, and demonstrated the involvement of astrocytes in hippocampal theta and gamma oscillations in vivo. Therefore, astrocytes decode interneuron activity and transform inhibitory into excitatory signals, contributing to the emergence of novel network properties resulting from the interneuron-astrocyte interplay. DOI: http://dx.doi.org/10.7554/eLife.20362.001 PMID:28012274

Establishment and characterization of a new conditionally immortalized human astrocyte cell line.

PubMed

Furihata, Tomomi; Ito, Ryo; Kamiichi, Atsuko; Saito, Kosuke; Chiba, Kan

2016-01-01

Astrocytes are the most abundant cell types in mammalian brains, within which they participate in various neuronal activities, partly by utilizing the numerous transporters expressed at their plasma membranes. Accordingly, detailed characterization of astrocytic functions, including transporters, are essential for understanding of mechanistic basis of normal brain functions, as well as the pathogenesis and treatment of various brain diseases. As a part of overall efforts to facilitate such studies, this study reports on the establishment of a new human astrocyte cell line, which is hereafter referred to as human astrocyte/conditionally immortalized, clone 35 (HASTR/ci35). This line, which was developed utilizing a cell immortalization method, showed excellent proliferative ability and expressed various astrocyte markers, including glial fibrillary acidic protein. When co-cultured with neuronal cells, HASTR/ci35 cells could facilitate their dendritic network formation. Furthermore, HASTR/ci35 cells not only possessed significant glutamate and adenosine transporter activities but also exhibited organic ion transporter activities. To summarize, HASTR/ci35 cells possess several key astrocytic characteristics, including various transporter functions, while simultaneously showing infinite proliferation and scalability. Based on these findings, HASTR/ci35 cells can be expected to contribute significantly to various human astrocyte study fields. In vitro astrocyte models are valuable experimental tools in various astrocyte studies. Here, we report the establishment of a new human astrocyte cell line, HASTR/ci35, which show various key astrocyte properties, including astrocytic transporter activities, glycogen storage and facilitation of neuronal cell differentiation. Thus, HASTR/ci35 is expected to significantly contribute to advances toward detailed understanding of human astrocyte functions. © 2015 International Society for Neurochemistry.

Activation of MAPK and FoxO by Manganese (Mn) in Rat Neonatal Primary Astrocyte Cultures

PubMed Central

Exil, Vernat; Ping, Li; Yu, Yingchun; Chakraborty, Sudipta; Caito, Samuel W.; Wells, K. Sam; Karki, Pratap; Lee, Eunsook; Aschner, Michael

2014-01-01

Environmental exposure to manganese (Mn) leads to a neurodegenerative disease that has shared clinical characteristics with Parkinson's disease (PD). Mn-induced neurotoxicity is time- and dose-dependent, due in part to oxidative stress. We ascertained the molecular targets involved in Mn-induced neurodegeneration using astrocyte culture as: (1) Astrocytes are vital for information processing within the brain, (2) their redox potential is essential in mitigating reactive oxygen species (ROS) levels, and (3) they are targeted early in the course of Mn toxicity. We first tested protein levels of Mn superoxide dismutase -2 (SOD-2) and glutathione peroxidase (GPx-1) as surrogates of astrocytic oxidative stress response. We assessed levels of the forkhead winged-helix transcription factor O (FoxO) in response to Mn exposure. FoxO is highly regulated by the insulin-signaling pathway. FoxO mediates cellular responses to toxic stress and modulates adaptive responses. We hypothesized that FoxO is fundamental in mediating oxidative stress response upon Mn treatment, and may be a biomarker of Mn-induced neurodegeneration. Our results indicate that 100 or 500 µM of MnCl2 led to increased levels of FoxO (dephosphorylated and phosphorylated) compared with control cells (P<0.01). p-FoxO disappeared from the cytosol upon Mn exposure. Pre-treatment of cultured cells with (R)-(−)-2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine analog rescued the cytosolic FoxO. At these concentrations, MAPK phosphorylation, in particular p38 and ERK, and PPAR gamma coactivator-1 (PGC-1) levels were increased, while AKT phosphorylation remained unchanged. FoxO phosphorylation level was markedly reduced with the use of SB203580 (a p38 MAPK inhibitor) and PD98059 (an ERK inhibitor). We conclude that FoxO phosphorylation after Mn exposure occurs in parallel with, and independent of the insulin-signaling pathway. FoxO levels and its translocation into the nucleus are part of early events

Transglutaminase 2: Friend or foe? The discordant role in neurons and astrocytes.

PubMed

Quinn, Breandan R; Yunes-Medina, Laura; Johnson, Gail V W

2018-03-23

Members of the transglutaminase family catalyze the formation of isopeptide bonds between a polypeptide-bound glutamine and a low molecular weight amine (e.g., spermidine) or the ɛ-amino group of a polypeptide-bound lysine. Transglutaminase 2 (TG2), a prominent member of this family, is unique because in addition to being a transamidating enzyme, it exhibits numerous other activities. As a result, TG2 plays a role in many physiological processes, and its function is highly cell type specific and relies upon a number of factors, including conformation, cellular compartment location, and local concentrations of Ca 2+ and guanine nucleotides. TG2 is the most abundant transglutaminase in the central nervous system (CNS) and plays a pivotal role in the CNS injury response. How TG2 affects the cell in response to an insult is strikingly different in astrocytes and neurons. In neurons, TG2 supports survival. Overexpression of TG2 in primary neurons protects against oxygen and glucose deprivation (OGD)-induced cell death and in vivo results in a reduction in infarct volume subsequent to a stroke. Knockdown of TG2 in primary neurons results in a loss of viability. In contrast, deletion of TG2 from astrocytes results in increased survival following OGD and improved ability to protect neurons from injury. Here, a brief overview of TG2 is provided, followed by a discussion of the role of TG2 in transcriptional regulation, cellular dynamics, and cell death. The differing roles TG2 plays in neurons and astrocytes are highlighted and compared to how TG2 functions in other cell types. © 2018 Wiley Periodicals, Inc.

Involvement of astrocyte metabolic coupling in Tourette syndrome pathogenesis.

PubMed

de Leeuw, Christiaan; Goudriaan, Andrea; Smit, August B; Yu, Dongmei; Mathews, Carol A; Scharf, Jeremiah M; Verheijen, Mark H G; Posthuma, Danielle

2015-11-01

Tourette syndrome is a heritable neurodevelopmental disorder whose pathophysiology remains unknown. Recent genome-wide association studies suggest that it is a polygenic disorder influenced by many genes of small effect. We tested whether these genes cluster in cellular function by applying gene-set analysis using expert curated sets of brain-expressed genes in the current largest available Tourette syndrome genome-wide association data set, involving 1285 cases and 4964 controls. The gene sets included specific synaptic, astrocytic, oligodendrocyte and microglial functions. We report association of Tourette syndrome with a set of genes involved in astrocyte function, specifically in astrocyte carbohydrate metabolism. This association is driven primarily by a subset of 33 genes involved in glycolysis and glutamate metabolism through which astrocytes support synaptic function. Our results indicate for the first time that the process of astrocyte-neuron metabolic coupling may be an important contributor to Tourette syndrome pathogenesis.

Involvement of astrocyte metabolic coupling in Tourette syndrome pathogenesis

PubMed Central

de Leeuw, Christiaan; Goudriaan, Andrea; Smit, August B; Yu, Dongmei; Mathews, Carol A; Scharf, Jeremiah M; Scharf, J M; Pauls, D L; Yu, D; Illmann, C; Osiecki, L; Neale, B M; Mathews, C A; Reus, V I; Lowe, T L; Freimer, N B; Cox, N J; Davis, L K; Rouleau, G A; Chouinard, S; Dion, Y; Girard, S; Cath, D C; Posthuma, D; Smit, J H; Heutink, P; King, R A; Fernandez, T; Leckman, J F; Sandor, P; Barr, C L; McMahon, W; Lyon, G; Leppert, M; Morgan, J; Weiss, R; Grados, M A; Singer, H; Jankovic, J; Tischfield, J A; Heiman, G A; Verheijen, Mark H G; Posthuma, Danielle

2015-01-01

Tourette syndrome is a heritable neurodevelopmental disorder whose pathophysiology remains unknown. Recent genome-wide association studies suggest that it is a polygenic disorder influenced by many genes of small effect. We tested whether these genes cluster in cellular function by applying gene-set analysis using expert curated sets of brain-expressed genes in the current largest available Tourette syndrome genome-wide association data set, involving 1285 cases and 4964 controls. The gene sets included specific synaptic, astrocytic, oligodendrocyte and microglial functions. We report association of Tourette syndrome with a set of genes involved in astrocyte function, specifically in astrocyte carbohydrate metabolism. This association is driven primarily by a subset of 33 genes involved in glycolysis and glutamate metabolism through which astrocytes support synaptic function. Our results indicate for the first time that the process of astrocyte-neuron metabolic coupling may be an important contributor to Tourette syndrome pathogenesis. PMID:25735483

Endothelial-astrocytic interactions in acute liver failure.

PubMed

Jayakumar, A R; Norenberg, M D

2013-06-01

Brain edema and the subsequent increase in intracranial pressure are major neurological complications of acute liver failure (ALF), and swelling of astrocytes (cytotoxic brain edema) is the most prominent neuropathological abnormality in ALF. Recent studies, however, have suggested the co-existence of cytotoxic and vasogenic mechanisms in the brain edema associated with ALF. This review 1) summarizes the nature of the brain edema in humans and experimental animals with ALF; 2) reviews in vitro studies supporting the presence of cytotoxic brain edema (cell swelling in cultured astrocytes); and 3) documents the role of brain endothelial cells in the development of astrocyte swelling/brain edema in ALF.

Interactions between Sirt1 and MAPKs regulate astrocyte activation induced by brain injury in vitro and in vivo.

PubMed

Li, Dan; Liu, Nan; Zhao, Hai-Hua; Zhang, Xu; Kawano, Hitoshi; Liu, Lu; Zhao, Liang; Li, Hong-Peng

2017-03-29

Astrocyte activation is a hallmark of traumatic brain injury resulting in neurological dysfunction or death for an overproduction of inflammatory cytokines and glial scar formation. Both the silent mating type information (Sirt1) expression and mitogen-activated protein kinase (MAPK) signal pathway activation represent a promising therapeutic target for several models of neurodegenerative diseases. We investigated the potential effects of Sirt1 upregulation and MAPK pathway pharmacological inhibition on astrocyte activation in vitro and in vivo. Moreover, we attempted to confirm the underlying interactions between Sirt1 and MAPK pathways in astrocyte activation after brain injury. The present study employs an interleukin-1β (IL-1β) stimulated primary cortical astrocyte model in vitro and a nigrostriatal pathway injury model in vivo to mimic the astrocyte activation induced by traumatic brain injury. The activation of GFAP, Sirt1, and MAPK pathways were detected by Western blot; astrocyte morphological hypertrophy was assessed using immunofluorescence staining; in order to explore the neuroprotective effect of regulation Sirt1 expression and MAPK pathway activation, the motor and neurological function tests were assessed after injury. GFAP level and morphological hypertrophy of astrocytes are elevated after injury in vitro or in vivo. Furthermore, the expressions of phosphorylated extracellular regulated protein kinases (p-ERK), phosphorylated c-Jun N-terminal kinase (p-JNK), and phosphorylated p38 activation (p-p38) are upregulated, but the Sirt1 expression is downregulated. Overexpression of Sirt1 significantly increases the p-ERK expression and reduces the p-JNK and p-p38 expressions. Inhibition of ERK, JNK, or p38 activation respectively with their inhibitors significantly elevated the Sirt1 expression and attenuated the astrocyte activation. Both the overproduction of Sirt1 and inhibition of ERK, JNK, or p38 activation can alleviate the astrocyte activation

Is there an astrocyte-neuron ketone body shuttle?

PubMed

Guzmán, M; Blázquez, C

2001-01-01

Ketone bodies can replace glucose as the major source of brain energy when glucose becomes scarce. Although it is generally assumed that the liver supplies extrahepatic tissues with ketone bodies, recent evidence shows that astrocytes are also ketogenic cells. Moreover, the partitioning of fatty acids between ketogenesis and ceramide synthesis de novo might control the survival/death decision of neural cells. These findings support the notion that astrocytes might supply neurons with ketone bodies in situ, and raise the possibility that astrocyte ketogenesis is a cytoprotective pathway.

Astrocyte and Neuronal Plasticity in the Somatosensory System

PubMed Central

Sims, Robert E.; Butcher, John B.; Parri, H. Rheinallt; Glazewski, Stanislaw

2015-01-01

Changing the whisker complement on a rodent's snout can lead to two forms of experience-dependent plasticity (EDP) in the neurons of the barrel cortex, where whiskers are somatotopically represented. One form, termed coding plasticity, concerns changes in synaptic transmission and connectivity between neurons. This is thought to underlie learning and memory processes and so adaptation to a changing environment. The second, called homeostatic plasticity, serves to maintain a restricted dynamic range of neuronal activity thus preventing its saturation or total downregulation. Current explanatory models of cortical EDP are almost exclusively neurocentric. However, in recent years, increasing evidence has emerged on the role of astrocytes in brain function, including plasticity. Indeed, astrocytes appear as necessary partners of neurons at the core of the mechanisms of coding and homeostatic plasticity recorded in neurons. In addition to neuronal plasticity, several different forms of astrocytic plasticity have recently been discovered. They extend from changes in receptor expression and dynamic changes in morphology to alteration in gliotransmitter release. It is however unclear how astrocytic plasticity contributes to the neuronal EDP. Here, we review the known and possible roles for astrocytes in the barrel cortex, including its plasticity. PMID:26345481

Immunocytochemical detection of the microsomal glucose-6-phosphatase in human brain astrocytes.

PubMed

Bell, J E; Hume, R; Busuttil, A; Burchell, A

1993-10-01

Using an antibody raised against the catalytic subunit of glucose-6-phosphatase, this enzyme was immunolocalized in many astrocytes in 20 normal human brains. Double immunofluorescence studies showed co-localization of glial fibrillary acidic protein (GFAP) with glucose-6-phosphatase in astrocytes. However, not all GFAP-positive cells were also glucose-6-phosphatase positive, indicating that some astrocytes do not contain demonstrable expression of this enzyme. Reactive astrocytes in a variety of abnormal brains were strongly glucose-6-phosphatase positive, but neoplastic astrocytes were often only weakly positive. Expression of the enzyme could not be demonstrated in radial glia, neurons or oligodendroglia. Astrocytes normally contain glycogen and the demonstration that some astrocytes also contain glucose-6-phosphatase indicates that they are competent for both glycogenolysis and gluconeogenesis, which may be critical for neuronal welfare.

Sulfatase-mediated manipulation of the astrocyte-Schwann cell interface.

PubMed

O'Neill, Paul; Lindsay, Susan L; Pantiru, Andreea; Guimond, Scott E; Fagoe, Nitish; Verhaagen, Joost; Turnbull, Jeremy E; Riddell, John S; Barnett, Susan C

2017-01-01

Schwann cell (SC) transplantation following spinal cord injury (SCI) may have therapeutic potential. Functional recovery is limited however, due to poor SC interactions with host astrocytes and the induction of astrogliosis. Olfactory ensheathing cells (OECs) are closely related to SCs, but intermix more readily with astrocytes in culture and induce less astrogliosis. We previously demonstrated that OECs express higher levels of sulfatases, enzymes that remove 6-O-sulfate groups from heparan sulphate proteoglycans, than SCs and that RNAi knockdown of sulfatase prevented OEC-astrocyte mixing in vitro. As human OECs are difficult to culture in large numbers we have genetically engineered SCs using lentiviral vectors to express sulfatase 1 and 2 (SC-S1S2) and assessed their ability to interact with astrocytes. We demonstrate that SC-S1S2s have increased integrin-dependent motility in the presence of astrocytes via modulation of NRG and FGF receptor-linked PI3K/AKT intracellular signaling and do not form boundaries with astrocytes in culture. SC-astrocyte mixing is dependent on local NRG concentration and we propose that sulfatase enzymes influence the bioavailability of NRG ligand and thus influence SC behavior. We further demonstrate that injection of sulfatase expressing SCs into spinal cord white matter results in less glial reactivity than control SC injections comparable to that of OEC injections. Our data indicate that sulfatase-mediated modification of the extracellular matrix can influence glial interactions with astrocytes, and that SCs engineered to express sulfatase may be more OEC-like in character. This approach may be beneficial for cell transplant-mediated spinal cord repair. GLIA 2016 GLIA 2017;65:19-33. © 2016 The Authors. Glia Published by Wiley Periodicals, Inc.

Kynurenine Pathway Metabolism is Involved in the Maintenance of the Intracellular NAD+ Concentration in Human Primary Astrocytes

PubMed Central

Grant, Ross; Nguyen, Susan; Guillemin, Gilles

2010-01-01

Efficient synthesis of NAD+ is critical to maintaining cell viability in all organs of the body. However, little is known of the pathway(s) by which cells of the central nervous system produce NAD+. The aim of this study was to investigate the relationship, between tryptophan degradation via the kynurenine pathway (KP) and de novo NAD+ synthesis in human astrocytes, a major cell type within the brain. In this study we observed that inhibition of single enzymes of the KP resulted in significant decreases in NAD+ levels in astroglial cells after a 24 hr period. We also observed that astrocytes cultured in media deficient in tryptophan, nicotinic acid and nicotinamide resulted in a 50% decrease in NAD+ levels after 24 hrs. This decrease in NAD+ was partially restored by supplementation of the culture media with either tryptophan or kynurenine, or nicotinic acid or with supply of the salvage pathway precursor nicotinamide. PMID:22084595

P2 receptor stimulation induces amyloid precursor protein production and secretion in rat cortical astrocytes.

PubMed

Tran, Minh D

2011-04-04

Amyloid precursor protein (APP) is ubiquitously expressed in a variety of tissues but is predominantly expressed in the brain. The expression of APP has been well studied in neurons but little is known about its presence in astrocytes. The study presented here shows that purinergic signaling is involved in the production and secretion of APP in primary cultures of rat cortical astrocytes. Extracellular ATP caused an increase in APP production and release in a time- and concentration-dependent manner and was inhibited by antagonists of P2 receptors. Further agonist and antagonist studies revealed involvement of P2Y2 and P2Y4 receptors in nucleotide-stimulated production and release of APP. In addition, signaling studies with various protein kinase inhibitors demonstrated that blockade of mitogen-activated protein kinases, but not Akt, inhibited nucleotide-stimulated APP expression and release. These results indicate that APP production and secretion can be regulated by activation of P2Y2/4 receptors coupled to protein kinase signaling pathways and suggest that astrocytes can be a potential source of APP. Published by Elsevier Ireland Ltd.

Fingolimod Phosphate Inhibits Astrocyte Inflammatory Activity in Mucolipidosis IV.

PubMed

Weinstock, Laura; Furness, Amanda M; Herron, Shawn; Smith, Sierra S; Sankar, Sitara; DeRosa, Samantha G; Gao, Dadi; Mepyans, Molly E; Scotto Rosato, Anna; Medina, Diego L; Vardi, Ayelet; Ferreira, Natalia S; Cho, Soo Min; Futerman, Anthony H; Slaugenhaupt, Susan A; Wood, Levi B; Grishchuk, Yulia

2018-05-16

Mucolipidosis IV (MLIV) is an orphan neurodevelopmental disease that causes severe neurologic dysfunction and loss of vision. Currently there is no therapy for MLIV. It is caused by loss of function of the lysosomal channel mucolipin-1, also known as TRPML1. Knockout of the Mcoln1 gene in a mouse model mirrors clinical and neuropathological signs in humans. Using this model, we previously observed robust activation of microglia and astrocytes in early symptomatic stages of disease. Here we investigate the consequence of mucolipin-1 loss on astrocyte inflammatory activation in vivo and in vitro and apply a pharmacological approach to restore Mcoln1-/- astrocyte homeostasis using a clinically approved immunomodulator, fingolimod. We found that Mcoln1-/- mice over-express numerous pro-inflammatory cytokines, some of which were also over-expressed in astrocyte cultures. Changes in the cytokine profile in Mcoln1-/- astrocytes are concomitant with changes in phospho-protein signaling, including activation of PI3K/Akt and MAPK pathways. Fingolimod promotes cytokine homeostasis, down-regulates signaling within the PI3K/Akt and MAPK pathways, and restores the lysosomal compartment in Mcoln1-/- astrocytes. These data suggest that fingolimod is a promising candidate for preclinical evaluation in our MLIV mouse model, which, in case of success, can be rapidly translated into clinical trial.

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

PubMed

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

2017-03-01

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

Disruption of astrocyte-neuron cholesterol cross talk affects neuronal function in Huntington's disease.

PubMed

Valenza, M; Marullo, M; Di Paolo, E; Cesana, E; Zuccato, C; Biella, G; Cattaneo, E

2015-04-01

In the adult brain, neurons require local cholesterol production, which is supplied by astrocytes through apoE-containing lipoproteins. In Huntington's disease (HD), such cholesterol biosynthesis in the brain is severely reduced. Here we show that this defect, occurring in astrocytes, is detrimental for HD neurons. Astrocytes bearing the huntingtin protein containing increasing CAG repeats secreted less apoE-lipoprotein-bound cholesterol in the medium. Conditioned media from HD astrocytes and lipoprotein-depleted conditioned media from wild-type (wt) astrocytes were equally detrimental in a neurite outgrowth assay and did not support synaptic activity in HD neurons, compared with conditions of cholesterol supplementation or conditioned media from wt astrocytes. Molecular perturbation of cholesterol biosynthesis and efflux in astrocytes caused similarly altered astrocyte-neuron cross talk, whereas enhancement of glial SREBP2 and ABCA1 function reversed the aspects of neuronal dysfunction in HD. These findings indicate that astrocyte-mediated cholesterol homeostasis could be a potential therapeutic target to ameliorate neuronal dysfunction in HD.

Hippocampal astrocytes are necessary for antidepressant treatment of learned helplessness rats.

PubMed

Iwata, Masaaki; Shirayama, Yukihiko; Ishida, Hisahito; Hazama, Gen-i; Nakagome, Kazuyuki

2011-08-01

The astrocyte is a major component of the neural network and plays a role in brain function. Previous studies demonstrated changes in the number of astrocytes in depression. In this study, we examined alterations in the number of astrocytes in the learned helplessness (LH) rat, an animal model of depression. The numbers of activated and nonactivated astrocytes in the dentate gyrus (molecular layer, subgranular zone, and hilus), and CA1 and CA3 regions of the hippocampus were significantly increased 2 and 8 days after attainment of LH. Subchronic treatment with imipramine showed a tendency (although not statistically significant) to decrease the LH-induced increment of activated astrocytes in the CA3 region and dentate gyrus. Furthermore, subchronic treatment of naïve rats with imipramine did not alter the numbers of activated and nonactivated astrocytes. However, the antidepressant-like effects of imipramine in the LH paradigm were blocked when fluorocitrate (a reversible inhibitor of astrocyte function) was injected into the dentate gyrus or CA3 region. Injection of fluorocitrate into naive rats failed to induce behavioral deficits in the conditioned avoidance test. These results indicate that astrocytes are responsive to the antidepressant-like effect of imipramine in the dentate gyrus and CA3 region of the hippocampus. Copyright © 2010 Wiley-Liss, Inc.

ASTROCYTE PATHOLOGY IN MAJOR DEPRESSIVE DISORDER: INSIGHTS FROM HUMAN POSTMORTEM BRAIN TISSUE

PubMed Central

Rajkowska, Grazyna; Stockmeier, Craig A.

2013-01-01

The present paper reviews astrocyte pathology in major depressive disorder (MDD) and proposes that reductions in astrocytes and related markers are key features in the pathology of MDD. Astrocytes are the most numerous and versatile of all types of glial cells. They are crucial to the neuronal microenvironment by regulating glucose metabolism, neurotransmitter uptake (particularly for glutamate), synaptic development and maturation and the blood brain barrier. Pathology of astrocytes has been consistently noted in MDD as well as in rodent models of depressive-like behavior. This review summarizes evidence from human postmortem tissue showing alterations in the expression of protein and mRNA for astrocyte markers such as glial fibrillary acidic protein (GFAP), gap junction proteins (connexin 40 and 43), the water channel aquaporin-4 (AQP4), a calcium-binding protein S100B and glutamatergic markers including the excitatory amino acid transporters 1 and 2 (EAAT1, EAAT2) and glutamine synthetase. Moreover, preclinical studies are presented that demonstrate the involvement of GFAP and astrocytes in animal models of stress and depressive-like behavior and the influence of different classes of antidepressant medications on astrocytes. In light of the various astrocyte deficits noted in MDD, astrocytes may be novel targets for the action of antidepressant medications. Possible functional consequences of altered expression of astrocytic markers in MDD are also discussed. Finally, the unique pattern of cell pathology in MDD, characterized by prominent reductions in the density of astrocytes and in the expression of their markers without obvious neuronal loss, is contrasted with that found in other neuropsychiatric and neurodegenerative disorders. PMID:23469922

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

PubMed

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

2016-09-02

Glycolysis is the primary step for major energy production in the cell. There is strong evidence suggesting that glucose consumption and rate of glycolysis are highly modulated by cytosolic pH/[H(+)], but those can also be stimulated by an increase in the intracellular [HCO3 (-)]. Because proton and bicarbonate shift concomitantly, it remained unclear whether enhanced glucose consumption and glycolytic rate were mediated by the changes in intracellular [H(+)] or [HCO3 (-)]. We have asked whether glucose metabolism is enhanced by either a fall in intracellular [H(+)] or a rise in intracellular [HCO3 (-)], or by both, in mammalian astrocytes. We have recorded intracellular glucose in mouse astrocytes using a FRET-based nanosensor, while imposing different intracellular [H(+)] and [CO2]/[HCO3 (-)]. Glucose consumption and glycolytic rate were augmented by a fall in intracellular [H(+)], irrespective of a concomitant rise or fall in intracellular [HCO3 (-)]. Transport of HCO3 (-) into and out of astrocytes by the electrogenic sodium bicarbonate cotransporter (NBCe1) played a crucial role in causing changes in intracellular pH and [HCO3 (-)], but was not obligatory for the pH-dependent changes in glucose metabolism. Our results clearly show that it is the cytosolic pH that modulates glucose metabolism in cortical astrocytes, and possibly also in other cell types. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Ras Family GTPases Control Growth of Astrocyte Processes

PubMed Central

Kalman, Daniel; Gomperts, Stephen N.; Hardy, Stephen; Kitamura, Marina; Bishop, J. Michael

1999-01-01

Astrocytes in neuron-free cultures typically lack processes, although they are highly process-bearing in vivo. We show that basic fibroblast growth factor (bFGF) induces cultured astrocytes to grow processes and that Ras family GTPases mediate these morphological changes. Activated alleles of rac1 and rhoA blocked and reversed bFGF effects when introduced into astrocytes in dissociated culture and in brain slices using recombinant adenoviruses. By contrast, dominant negative (DN) alleles of both GTPases mimicked bFGF effects. A DN allele of Ha-ras blocked bFGF effects but not those of Rac1-DN or RhoA-DN. Our results show that bFGF acting through c-Ha-Ras inhibits endogenous Rac1 and RhoA GTPases thereby triggering astrocyte process growth, and they provide evidence for the regulation of this cascade in vivo by a yet undetermined neuron-derived factor. PMID:10233170

Formate generated by cellular oxidation of formaldehyde accelerates the glycolytic flux in cultured astrocytes.

PubMed

Tulpule, Ketki; Dringen, Ralf

2012-04-01

Formaldehyde is a neurotoxic compound that can be endogenously generated in the brain. Because astrocytes play a key role in metabolism and detoxification processes in brain, we have investigated the capacity of these cells to metabolize formaldehyde using primary astrocyte-rich cultures as a model system. Application of formaldehyde to these cultures resulted in the appearance of formate in cells and in a time-, concentration- and temperature-dependent disappearance of formaldehyde from the medium that was accompanied by a matching extracellular accumulation of formate. This formaldehyde-oxidizing capacity of astrocyte cultures is likely to be catalyzed by alcohol dehydrogenase 3 and aldehyde dehydrogenase 2, because the cells of the cultures contain the mRNAs of these formaldehyde-oxidizing enzymes. In addition, exposure to formaldehyde increased both glucose consumption and lactate production by the cells. Both the strong increase in the cellular formate content and the increase in glycolytic flux were only observed after application of formaldehyde to the cells, but not after treatment with exogenous methanol or formate. The accelerated lactate production was not additive to that obtained for azide, a known inhibitor of complex IV of the respiratory chain, and persisted after removal of formaldehyde after a formaldehyde exposure for 1.5 h. These data demonstrate that cultured astrocytes efficiently oxidize formaldehyde to formate, which subsequently enhances glycolytic flux, most likely by inhibition of mitochondrial respiration. Copyright © 2012 Wiley Periodicals, Inc.

Astrocytes Pathology in ALS: A Potential Therapeutic Target?

PubMed

Johann, Sonja

2017-01-01

The mechanisms underlying neurodegeneration in amyotrophic lateral sclerosis (ALS) are multifactorial and include genetic and environmental factors. Nowadays, it is well accepted that neuronal loss is driven by non-cell autonomous toxicity. Non-neuronal cells, such as astrocytes, have been described to significantly contribute to motoneuron cell death and disease progression in cell culture experiments and animal models of ALS. Astrocytes are essential for neuronal survival and function by regulating neurotransmitter and ion homeostasis, immune response, blood flow and glucose uptake, antioxidant defence and growth factor release. Based on their significant functions in "housekeeping" the central nervous system (CNS), they are no longer thought to be passive bystanders but rather contributors to ALS pathogenesis. Findings from animal models have broadened our knowledge about different pathomechanisms in ALS, but therapeutic approaches to impede disease progression failed. So far, there is no cure for ALS and effective medication to slow down disease progression is limited. Targeting only a single aspect of this multifactorial disease may exhibit therapeutic limitations. Hence, novel cellular targets must be defined and new pharmaceutical strategies, such as combinatorial drug therapies are urgently needed. The present review discusses the physiological role of astrocytes and current hypotheses of astrocyte pathology in ALS. Furthermore, recent investigation of potential drug candidates in astrocyte cell culture systems and animal models, as well as data obtained from clinical trials, will be addressed. The central role of astrocytes in ALS pathogenesis makes them a promising target for pharmaceutical interventions. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol.

PubMed

Wolfes, Anne C; Dean, Camin

2018-01-10

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca 2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca 2+ signaling similar to astrocytes in vivo.

Differential activation of catalase expression and activity by PPAR agonists: Implications for astrocyte protection in anti-glioma therapy☆

PubMed Central

Khoo, Nicholas K.H.; Hebbar, Sachin; Zhao, Weiling; Moore, Steven A.; Domann, Frederick E.; Robbins, Mike E.

2013-01-01

Glioma survival is dismal, in part, due to an imbalance in antioxidant expression and activity. Peroxisome proliferator-activated receptor (PPAR) agonists have antineoplastic properties which present new redox-dependent targets for glioma anticancer therapies. Herein, we demonstrate that treatment of primary cultures of normal rat astrocytes with PPAR agonists increased the expression of catalase mRNA protein, and enzymatic activity. In contrast, these same agonists had no effect on catalase expression and activity in malignant rat glioma cells. The increase in steady-state catalase mRNA observed in normal rat astrocytes was due, in part, to de novo mRNA synthesis as opposed to increased catalase mRNA stability. Moreover, pioglitazone-mediated induction of catalase activity in normal rat astrocytes was completely blocked by transfection with a PPARγ-dominant negative plasmid. These data suggest that defects in PPAR-mediated signaling and gene expression may represent a block to normal catalase expression and induction in malignant glioma. The ability of PPAR agonists to differentially increase catalase expression and activity in normal astrocytes but not glioma cells suggests that these compounds might represent novel adjuvant therapeutic agents for the treatment of gliomas. PMID:24024139

General anesthetics have differential inhibitory effects on gap junction channels and hemichannels in astrocytes and neurons.

PubMed

Liu, Xinhe; Gangoso, Ester; Yi, Chenju; Jeanson, Tiffany; Kandelman, Stanislas; Mantz, Jean; Giaume, Christian

2016-04-01

Astrocytes represent a major non-neuronal cell population actively involved in brain functions and pathologies. They express a large amount of gap junction proteins that allow communication between adjacent glial cells and the formation of glial networks. In addition, these membrane proteins can also operate as hemichannels, through which "gliotransmitters" are released, and thus contribute to neuroglial interaction. There are now reports demonstrating that alterations of astroglial gap junction communication and/or hemichannel activity impact neuronal and synaptic activity. Two decades ago we reported that several general anesthetics inhibited gap junctions in primary cultures of astrocytes (Mantz et al., (1993) Anesthesiology 78(5):892-901). As there are increasing studies investigating neuroglial interactions in anesthetized mice, we here updated this previous study by employing acute cortical slices and by characterizing the effects of general anesthetics on both astroglial gap junctions and hemichannels. As hemichannel activity is not detected in cortical astrocytes under basal conditions, we treated acute slices with the endotoxin LPS or proinflammatory cytokines to induce hemichannel activity in astrocytes, which in turn activated neuronal hemichannels. We studied two extensively used anesthetics, propofol and ketamine, and the more recently developed dexmedetomidine. We report that these drugs have differential inhibitory effects on gap junctional communication and hemichannel activity in astrocytes when used in their respective, clinically relevant concentrations, and that dexmedetomidine appears to be the least effective on both channel functions. In addition, the three anesthetics have similar effects on neuronal hemichannels. Altogether, our observations may contribute to optimizing the selection of anesthetics for in vivo animal studies. © 2015 Wiley Periodicals, Inc.

Bi-directionally protective communication between neurons and astrocytes under ischemia.

PubMed

Wu, Xiao-Mei; Qian, Christopher; Zhou, Yu-Fu; Yan, Yick-Chun; Luo, Qian-Qian; Yung, Wing-Ho; Zhang, Fa-Li; Jiang, Li-Rong; Qian, Zhong Ming; Ke, Ya

2017-10-01

The extensive existing knowledge on bi-directional communication between astrocytes and neurons led us to hypothesize that not only ischemia-preconditioned (IP) astrocytes can protect neurons but also IP neurons protect astrocytes from lethal ischemic injury. Here, we demonstrated for the first time that neurons have a significant role in protecting astrocytes from ischemic injury. The cultured medium from IP neurons (IPcNCM) induced a remarkable reduction in LDH and an increase in cell viability in ischemic astrocytes in vitro. Selective neuronal loss by kainic acid injection induced a significant increase in apoptotic astrocyte numbers in the brain of ischemic rats in vivo. Furthermore, TUNEL analysis, DNA ladder assay, and the measurements of ROS, GSH, pro- and anti-apoptotic factors, anti-oxidant enzymes and signal molecules in vitro and/or in vivo demonstrated that IP neurons protect astrocytes by an EPO-mediated inhibition of pro-apoptotic signals, activation of anti-apoptotic proteins via the P13K/ERK/STAT5 pathways and activation of anti-oxidant proteins via up-regulation of anti-oxidant enzymes. We demonstrated the existence of astro-protection by IP neurons under ischemia and proposed that the bi-directionally protective communications between cells might be a common activity in the brain or peripheral organs under most if not all pathological conditions. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

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

DTIC Science & Technology

2012-10-01

Pluripotent Stem Cell -Derived Astrocyte Transplantation in ALS PRINCIPAL INVESTIGATOR: Nicholas J. Maragakis, M.D...Pluripotent Stem Cell -Derived Astrocyte Transplantation in ALS 5b. GRANT NUMBER W81XWH-10-1-0520 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d...into astrocytes following transplantation. 15. SUBJECT TERMS Stem Cells , iPS cells, astrocytes, familial ALS 16. SECURITY CLASSIFICATION OF

Effects of aspartame metabolites on astrocytes and neurons.

PubMed

Rycerz, Karol; Jaworska-Adamu, Jadwiga Elżbieta

2013-01-01

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

AXL promotes Zika virus infection in astrocytes by antagonizing type I interferon signalling.

PubMed

Chen, Jian; Yang, Yi-Feng; Yang, Yu; Zou, Peng; Chen, Jun; He, Yongquan; Shui, Sai-Lan; Cui, Yan-Ru; Bai, Ru; Liang, Ya-Jun; Hu, Yunwen; Jiang, Biao; Lu, Lu; Zhang, Xiaoyan; Liu, Jia; Xu, Jianqing

2018-03-01

Zika virus (ZIKV) is associated with neonatal microcephaly and Guillain-Barré syndrome 1,2 . While progress has been made in understanding the causal link between ZIKV infection and microcephaly 3-9 , the life cycle and pathogenesis of ZIKV are less well understood. In particular, there are conflicting reports on the role of AXL, a TAM family kinase receptor that was initially described as the entry receptor for ZIKV 10-22 . Here, we show that while genetic ablation of AXL protected primary human astrocytes and astrocytoma cell lines from ZIKV infection, AXL knockout did not block the entry of ZIKV. We found, instead, that the presence of AXL attenuated the ZIKV-induced activation of type I interferon (IFN) signalling genes, including several type I IFNs and IFN-stimulating genes. Knocking out type I IFN receptor α chain (IFNAR1) restored the vulnerability of AXL knockout astrocytes to ZIKV infection. Further experiments suggested that AXL regulates the expression of SOCS1, a known type I IFN signalling suppressor, in a STAT1/STAT2-dependent manner. Collectively, our results demonstrate that AXL is unlikely to function as an entry receptor for ZIKV and may instead promote ZIKV infection in human astrocytes by antagonizing type I IFN signalling.

Astrocyte structural reactivity and plasticity in models of retinal detachment.

PubMed

Luna, Gabriel; Keeley, Patrick W; Reese, Benjamin E; Linberg, Kenneth A; Lewis, Geoffrey P; Fisher, Steven K

2016-09-01

Although retinal neurodegenerative conditions such as age-related macular degeneration, glaucoma, diabetic retinopathy, retinitis pigmentosa, and retinal detachment have different etiologies and pathological characteristics, they also have many responses in common at the cellular level, including neural and glial remodeling. Structural changes in Müller cells, the large radial glia of the retina in retinal disease and injury have been well described, that of the retinal astrocytes remains less so. Using modern imaging technology to describe the structural remodeling of retinal astrocytes after retinal detachment is the focus of this paper. We present both a review of critical literature as well as novel work focusing on the responses of astrocytes following rhegmatogenous and serous retinal detachment. The mouse presents a convenient model system in which to study astrocyte reactivity since the Mϋller cell response is muted in comparison to other species thereby allowing better visualization of the astrocytes. We also show data from rat, cat, squirrel, and human retina demonstrating similarities and differences across species. Our data from immunolabeling and dye-filling experiments demonstrate previously undescribed morphological characteristics of normal astrocytes and changes induced by detachment. Astrocytes not only upregulate GFAP, but structurally remodel, becoming increasingly irregular in appearance, and often penetrating deep into neural retina. Understanding these responses, their consequences, and what drives them may prove to be an important component in improving visual outcome in a variety of therapeutic situations. Our data further supports the concept that astrocytes are important players in the retina's overall response to injury and disease. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Erk1/2 and Akt kinases are involved in the protective effect of aniracetam in astrocytes subjected to simulated ischemia in vitro.

PubMed

Gabryel, Bozena; Pudelko, Anna; Malecki, Andrzej

2004-06-28

The present study focused on the mechanism of cytoprotective effect of aniracetam on the primary rat astrocyte cultures exposed to simulated ischemia conditions in vitro. To study these mechanisms, the aniracetam-mediated modulation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/Akt kinase pathways was determined. Simulated in vitro ischemia caused death of approximately 35% of astrocytes via apoptosis and decreased cell viability about 50% at 8 h. Exposure to aniracetam at concentrations of 0.1-10 microM in these conditions significantly decreased the number of apoptotic cells. Moreover, the intensification of 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolinum bromide (MTT) conversion and the decrease of lactate dehydrogenase (LDH) release after 1 and 10 microM aniracetam treatment were observed indicating a significant increase in cell viability. When cultured astrocytes were incubated during 8 h simulated ischemia with [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] (U0126), an extracellular regulated kinase 1 and 2 (Erk1/2) inhibitor or wortmannin, a phosphatidylinositol 3-kinase (PI3 kinase)/Akt inhibitor, the cell apoptosis was accelerated. These effects of used kinase inhibitors (both U0126 and wortmannin) were antagonized by adding 1 and 10 microM aniracetam to the culture medium. In addition, aniracetam significantly stimulated of phospho-Erk1/2 kinase and phospho-Akt expression. Maximum levels of Erk1/2 and Akt activation were observed as a result of treatment with 10 microM aniracetam. U0126 and wortmannin markedly attenuated the effects of aniracetam on expression of activated kinases. Results of the present study indicate that both Erk1/2 and PI 3-K/Akt kinase pathways are vital for cytoprotective effect of aniracetam.

The role of astrocytes in amyloid production and Alzheimer's disease

PubMed Central

Frost, Georgia R.

2017-01-01

Alzheimer's disease (AD) is marked by the presence of extracellular amyloid beta (Aβ) plaques, intracellular neurofibrillary tangles (NFTs) and gliosis, activated glial cells, in the brain. It is thought that Aβ plaques trigger NFT formation, neuronal cell death, neuroinflammation and gliosis and, ultimately, cognitive impairment. There are increased numbers of reactive astrocytes in AD, which surround amyloid plaques and secrete proinflammatory factors and can phagocytize and break down Aβ. It was thought that neuronal cells were the major source of Aβ. However, mounting evidence suggests that astrocytes may play an additional role in AD by secreting significant quantities of Aβ and contributing to overall amyloid burden in the brain. Astrocytes are the most numerous cell type in the brain, and therefore even minor quantities of amyloid secretion from individual astrocytes could prove to be substantial when taken across the whole brain. Reactive astrocytes have increased levels of the three necessary components for Aβ production: amyloid precursor protein, β-secretase (BACE1) and γ-secretase. The identification of environmental factors, such as neuroinflammation, that promote astrocytic Aβ production, could redefine how we think about developing therapeutics for AD. PMID:29237809

Matrix metalloproteinase-3 is a possible mediator of neurodevelopmental impairment due to polyI:C-induced innate immune activation of astrocytes.

PubMed

Yamada, Shinnosuke; Nagai, Taku; Nakai, Tsuyoshi; Ibi, Daisuke; Nakajima, Akira; Yamada, Kiyofumi

2014-05-01

Increasing epidemiological evidence indicates that prenatal infection and childhood central nervous system infection with various viral pathogens enhance the risk for several neuropsychiatric disorders. Polyriboinosinic-polyribocytidilic acid (polyI:C) is known to induce strong innate immune responses that mimic immune activation by viral infections. Our previous findings suggested that activation of the innate immune system in astrocytes results in impairments of neurite outgrowth and spine formation, which lead to behavioral abnormalities in adulthood. To identify candidates of astrocyte-derived humoral factors that affect neuronal development, we analyzed astrocyte-conditioned medium (ACM) from murine astrocyte cultures treated with polyI:C (polyI:C-ACM) by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Through a quantitative proteomic screen, we found that 13 protein spots were differentially expressed compared with ACM from vehicle-treated astrocytes (control-ACM), and characterized one of the candidates, matrix metalloproteinase-3 (Mmp3). PolyI:C treatment significantly increased the expression levels of Mmp3 mRNA and protein in astrocytes, but not microglia. PolyI:C-ACM was associated with significantly higher Mmp3 protein level and enzyme activity than control-ACM. The addition of recombinant Mmp3 into control-ACM impaired dendritic elongation of primary cultured hippocampal neurons, while the deleterious effect of polyI:C-ACM on neurite elongation was attenuated by knockdown of Mmp3 in astrocytes. These results suggest that Mmp3 is a possible mediator of polyI:C-ACM-induced neurodevelopmental impairment. Copyright © 2014. Published by Elsevier Inc.

Phenotypic Heterogeneity and Plasticity of Isocortical and Hippocampal Astrocytes in the Human Brain

PubMed Central

Sosunov, Alexander A.; Wu, Xiaoping; Tsankova, Nadejda M.; Guilfoyle, Eileen; McKhann, Guy M.

2014-01-01

To examine the diversity of astrocytes in the human brain, we immunostained surgical specimens of temporal cortex and hippocampus and autopsy brains for CD44, a plasma membrane protein and extracellular matrix receptor. CD44 antibodies outline the details of astrocyte morphology to a degree not possible with glial fibrillary acidic protein (GFAP) antibodies. CD44+ astrocytes could be subdivided into two groups. First, CD44+ astrocytes with long processes were consistently found in the subpial area (“interlaminar” astrocytes), the deep isocortical layers, and the hippocampus. Many of these processes ended on blood vessels. Some were also found adjacent to large blood vessels, from which they extended long processes. We observed these CD44+, long-process astrocytes in every brain we examined, from fetal to adult. These astrocytes generally displayed high immunostaining for GFAP, S100β, and CD44, but low immunostaining for glutamine synthetase, excitatory amino-acid transporter 1 (EAAT1), and EAAT2. Aquaporin 4 (AQP4) appeared distributed all over the cell bodies and processes of the CD44+ astrocytes, while, in contrast, AQP4 localized to perivascular end feet in the CD44− protoplasmic astrocytes. Second, there were CD44+ astrocytes without long processes in the cortex. These were not present during gestation or at birth, and in adult brains varied substantially in number, shape, and immunohistochemical phenotype. Many of these displayed a “mixed” morphological and immunocytochemical phenotype between protoplasmic and fibrous astrocytes. We conclude that the diversity of astrocyte populations in the isocortex and archicortex in the human brain reflects both intrinsic and acquired phenotypes, the latter perhaps representing a shift from CD44− “protoplasmic” to CD44+ “fibrous”-like astrocytes. PMID:24501367

Bicarbonate sensing in mouse cortical astrocytes during extracellular acid/base disturbances.

PubMed

Theparambil, Shefeeq M; Naoshin, Zinnia; Defren, Sabrina; Schmaelzle, Jana; Weber, Tobias; Schneider, Hans-Peter; Deitmer, Joachim W

2017-04-15

The present study suggests that the electrogenic sodium-bicarbonate cotransporter, NBCe1, supported by carbonic anhydrase II, CAII, provides an efficient mechanism of bicarbonate sensing in cortical astrocytes. This mechanism is proposed to play a major role in setting the pH i responses to extracellular acid/base challenges in astrocytes. A decrease in extracellular [HCO 3 - ] during isocapnic acidosis and isohydric hypocapnia, or an increase in intracellular [HCO 3 - ] during hypercapnic acidosis, was effectively sensed by NBCe1, which carried bicarbonate out of the cells under these conditions, and caused an acidification and sodium fall in WT astrocytes, but not in NBCe1-knockout astrocytes. Isocapnic acidosis, hypercapnic acidosis and isohydric hypocapnia evoked inward currents in NBCe1- and CAII-expressing Xenopus laevis oocytes, but not in native oocytes, suggesting that NBCe1 operates in the outwardly directed mode under these conditions consistent with our findings in astrocytes. We propose that bicarbonate sensing of astrocytes may have functional significance during extracellular acid/base disturbances in the brain, as it not only alters intracellular pH/[HCO 3 - ]-dependent functions of astrocytes, but also modulates the extracellular pH/[HCO 3 - ] in brain tissue. Extracellular acid/base status of the mammalian brain undergoes dynamic changes during many physiological and pathological events. Although intracellular pH (pH i ) of astrocytes responds to extracellular acid/base changes, the mechanisms mediating these changes have remained unresolved. We have previously shown that the electrogenic sodium-bicarbonate cotransporter, NBCe1, is a high-affinity bicarbonate carrier in cortical astrocytes. In the present study, we investigated whether NBCe1 plays a role in bicarbonate sensing in astrocytes, and in determining the pH i responses to extracellular acid/base challenges. We measured changes in intracellular H + and Na + in astrocytes from wild

Bicarbonate sensing in mouse cortical astrocytes during extracellular acid/base disturbances

PubMed Central

Naoshin, Zinnia; Defren, Sabrina; Schmaelzle, Jana; Weber, Tobias; Schneider, Hans‐Peter

2017-01-01

Key points The present study suggests that the electrogenic sodium–bicarbonate cotransporter, NBCe1, supported by carbonic anhydrase II, CAII, provides an efficient mechanism of bicarbonate sensing in cortical astrocytes. This mechanism is proposed to play a major role in setting the pHi responses to extracellular acid/base challenges in astrocytes.A decrease in extracellular [HCO3 −] during isocapnic acidosis and isohydric hypocapnia, or an increase in intracellular [HCO3 −] during hypercapnic acidosis, was effectively sensed by NBCe1, which carried bicarbonate out of the cells under these conditions, and caused an acidification and sodium fall in WT astrocytes, but not in NBCe1‐knockout astrocytes.Isocapnic acidosis, hypercapnic acidosis and isohydric hypocapnia evoked inward currents in NBCe1‐ and CAII‐expressing Xenopus laevis oocytes, but not in native oocytes, suggesting that NBCe1 operates in the outwardly directed mode under these conditions consistent with our findings in astrocytes.We propose that bicarbonate sensing of astrocytes may have functional significance during extracellular acid/base disturbances in the brain, as it not only alters intracellular pH/[HCO3 −]‐dependent functions of astrocytes, but also modulates the extracellular pH/[HCO3 −] in brain tissue. Abstract Extracellular acid/base status of the mammalian brain undergoes dynamic changes during many physiological and pathological events. Although intracellular pH (pHi) of astrocytes responds to extracellular acid/base changes, the mechanisms mediating these changes have remained unresolved. We have previously shown that the electrogenic sodium–bicarbonate cotransporter, NBCe1, is a high‐affinity bicarbonate carrier in cortical astrocytes. In the present study, we investigated whether NBCe1 plays a role in bicarbonate sensing in astrocytes, and in determining the pHi responses to extracellular acid/base challenges. We measured changes in intracellular H+ and Na+ in

Conditions and constraints for astrocyte calcium signaling in the hippocampal mossy fiber pathway

PubMed Central

Haustein, Martin D.; Kracun, Sebastian; Lu, Xiao-Hong; Shih, Tiffany; Jackson-Weaver, Olan; Tong, Xiaoping; Xu, Ji; Yang, X. William; O'Dell, Thomas J.; Marvin, Jonathan S.; Ellisman, Mark H.; Bushong, Eric A.; Looger, Loren L.; Khakh, Baljit S.

2014-01-01

Summary The spatiotemporal activities of astrocyte Ca2+ signaling in mature neuronal circuits remain unclear. We used genetically encoded Ca2+ and glutamate indicators as well as pharmacogenetic and electrical control of neurotransmitter release to explore astrocyte activity in the hippocampal mossy fiber pathway. Our data revealed numerous localised spontaneous Ca2+ signals in astrocyte branches and territories, but these were not driven by neuronal activity or glutamate. Moreover, evoked astrocyte Ca2+ signaling changed linearly with the number of mossy fiber action potentials. Under these settings astrocyte responses were global, suppressed by neurotransmitter clearance and mediated by glutamate and GABA. Thus, astrocyte engagement in the fully developed mossy fiber pathway was slow and territorial, contrary to that frequently proposed for astrocytes within microcircuits. We show that astrocyte Ca2+ signaling functionally segregates large volumes of neuropil and that these transients are not suited for responding to, or regulating, single synapses in the mossy fiber pathway. PMID:24742463

A comparative transcriptomic analysis of astrocytes differentiation from human neural progenitor cells.

PubMed

Magistri, Marco; Khoury, Nathalie; Mazza, Emilia Maria Cristina; Velmeshev, Dmitry; Lee, Jae K; Bicciato, Silvio; Tsoulfas, Pantelis; Faghihi, Mohammad Ali

2016-11-01

Astrocytes are a morphologically and functionally heterogeneous population of cells that play critical roles in neurodevelopment and in the regulation of central nervous system homeostasis. Studies of human astrocytes have been hampered by the lack of specific molecular markers and by the difficulties associated with purifying and culturing astrocytes from adult human brains. Human neural progenitor cells (NPCs) with self-renewal and multipotent properties represent an appealing model system to gain insight into the developmental genetics and function of human astrocytes, but a comprehensive molecular characterization that confirms the validity of this cellular system is still missing. Here we used an unbiased transcriptomic analysis to characterize in vitro culture of human NPCs and to define the gene expression programs activated during the differentiation of these cells into astrocytes using FBS or the combination of CNTF and BMP4. Our results demonstrate that in vitro cultures of human NPCs isolated during the gliogenic phase of neurodevelopment mainly consist of radial glial cells (RGCs) and glia-restricted progenitor cells. In these cells the combination of CNTF and BMP4 activates the JAK/STAT and SMAD signaling cascades, leading to the inhibition of oligodendrocytes lineage commitment and activation of astrocytes differentiation. On the other hand, FBS-derived astrocytes have properties of reactive astrocytes. Our work suggests that in vitro culture of human NPCs represents a valuable cellular system to study human disorders characterized by impairment of astrocytes development and function. Our datasets represent an important resource for researchers studying human astrocytes development and might set the basis for the discovery of novel human-specific astrocyte markers. © 2016 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Astrocyte-neuronal interactions in epileptogenesis.

PubMed

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

2015-07-01

Pentylenetetrazol, kainic acid, or pilocarpine can be used to induce seizures in animal models of epilepsy. The present Review describes disturbances in astrocyte-neuron interactions in the acute, latent, and chronic phases analyzed by magnetic resonance spectroscopy of brain tissue extracts from rats injected with [1-(13)C]glucose and [1,2-(13)C]acetate. The most consistent change after onset of seizures was the decrease in (13)C labeling of glutamate (GLU) from [1-(13) C]glucose regardless of brain area, severity, or duration of the period with seizures and toxin used. In most cases this decrease was accompanied by a reduction in glutamine (GLN) labeling from [1-(13)C]glucose, presumably as a direct consequence of the reduction in labeling of GLU and the GLU-GLN cycle. Amounts of GLN were never changed. Reduction in the content of N-acetyl aspartate (NAA) was first detectable some time after status epilepticus but before the occurrence of spontaneous seizures. This decrease can be an indication of neuronal death and/or mitochondrial impairment and might indicate beginning gliosis. It is known that gliosis occurs in the chronic phase of temporal lobe epilepsy in hippocampus, but astrocyte metabolism appears normal in this phase, indicating that the gliotic astrocytes have a somewhat reduced metabolism per volume. A decrease in (13)C labeling of GLU from [1-(13)C]glucose is a very sensitive measure for the onset of epileptogenesis, whereas reduction of NAA is first detectable later. In the chronic phases of the hippocampal formation, astrocyte metabolism is upregulated given that the number of neurons is reduced. © 2015 Wiley Periodicals, Inc.

Sex Differences and Laterality in Astrocyte Number and Complexity in the Adult Rat Medial Amygdala

PubMed Central

JOHNSON, RYAN T.; BREEDLOVE, S. MARC; JORDAN, CYNTHIA L.

2008-01-01

The posterodorsal portion of the medial amygdala (MePD) is sexually dimorphic in several rodent species. In several other brain nuclei, astrocytes change morphology in response to steroid hormones. We visualized MePD astrocytes using glial-fibrillary acidic protein (GFAP) immunocytochemistry. We compared the number and process complexity of MePD astrocytes in adult wildtype male and female rats and testicular feminized mutant (TFM) male rats that lack functional androgen receptors (ARs) to determine whether MePD astrocytes are sexually differentiated and whether ARs have a role. Unbiased stereological methods revealed laterality and sex differences in MePD astrocyte number and complexity. The right MePD contained more astrocytes than the left in all three genotypes, and the number of astrocytes was also sexually differentiated in the right MePD, with males having more astrocytes than females. In contrast, the left MePD contained more complex astrocytes than did the right MePD in all three genotypes, and males had more complex astrocytes than females in this hemisphere. TFM males were comparable to wildtype females, having fewer astrocytes on the right and simpler astrocytes on the left than do wildtype males. Taken together, these results demonstrate that astrocytes are sexually dimorphic in the adult MePD and that the nature of the sex difference is hemisphere-dependent: a sex difference in astrocyte number in the right MePD and a sex difference in astrocyte complexity in the left MePD. Moreover, functional ARs appear to be critical in establishing these sex differences in MePD astrocyte morphology. PMID:18853427

Mutant Huntingtin Inhibits αB-Crystallin Expression and Impairs Exosome Secretion from Astrocytes

PubMed Central

2017-01-01

In the brain, astrocytes secrete diverse substances that regulate neuronal function and viability. Exosomes, which are vesicles produced through the formation of multivesicular bodies and their subsequent fusion with the plasma membrane, are also released from astrocytes via exocytotic secretion. Astrocytic exosomes carry heat shock proteins that can reduce the cellular toxicity of misfolded proteins and prevent neurodegeneration. Although mutant huntingtin (mHtt) affects multiple functions of astrocytes, it remains unknown whether mHtt impairs the production of exosomes from astrocytes. We found that mHtt is not present in astrocytic exosomes, but can decrease exosome secretion from astrocytes in HD140Q knock-in (KI) mice. N-terminal mHtt accumulates in the nuclei and forms aggregates, causing decreased secretion of exosomes from cultured astrocytes. Consistently, there is a significant decrease in secreted exosomes in both female and male HD KI mouse striatum in which abundant nuclear mHtt aggregates are present. Conversely, injection of astrocytic exosomes into the striatum of HD140Q KI mice reduces the density of mHtt aggregates. Further, mHtt in astrocytes decreased the expression of αB-crystallin, a small heat shock protein that is enriched in astrocytes and mediates exosome secretion, by reducing the association of Sp1 with the enhancer of the αB-crystallin gene. Importantly, overexpression of αB-crystallin rescues defective exosome release from HD astrocytes as well as mHtt aggregates in the striatum of HD140Q KI mice. Our results demonstrate that mHtt reduces the expression of αB-crystallin in astrocytes to decrease exosome secretion in the HD brains, contributing to non–cell-autonomous neurotoxicity in HD. SIGNIFICANCE STATEMENT Huntington's disease (HD) is characterized by selective neurodegeneration that preferentially occurs in the striatal medium spiny neurons. Recent studies in different HD mouse models demonstrated that dysfunction of

Effect of NDP-α-MSH on PPAR-γ and –β Expression and Anti-Inflammatory Cytokine Release in Rat Astrocytes and Microglia

PubMed Central

Carniglia, Lila; Durand, Daniela; Caruso, Carla; Lasaga, Mercedes

2013-01-01

Brain inflammation plays a central role in numerous brain pathologies. Microglia and astrocytes are the main effector cells that become activated when an inflammatory process takes place within the central nervous system. α-melanocyte-stimulating hormone (α-MSH) is a neuropeptide with proven anti-inflammatory properties. It binds with highest affinity to the melanocortin receptor 4 (MC4R), which is present in astrocytes and upon activation triggers anti-inflammatory pathways. The aim of this research was to identify anti-inflammatory mediators that may participate in the immunomodulatory effects of melanocortins in glial cells. Since peroxisome proliferator-activated receptors (PPARs) have recently been implicated in the modulation of inflammation, we investigated the effect of an α-MSH analog, [Nle4, D-Phe7]-α-MSH (NDP-α-MSH), on PPAR-β and PPAR-γ gene and protein expression in rat primary astrocytes and microglia. We initially demonstrated that rat primary microglia express MC4R and showed that treatment with NDP-α-MSH increases PPAR-γ protein levels and strongly decreases PPAR-β levels in both astrocytes and microglia. We also showed that extracellular signal-regulated kinase 1/2 (ERK1/2)–mediated signaling is partially involved in these effects in a cell-specific fashion. Finally, we showed that NDP-α-MSH stimulates the release of the anti-inflammatory cytokines IL-10 and TGF-β from microglia and astrocytes, respectively. The presented data suggest a role for IL-10 and TGF-β in the protective action of melanocortins and a connection between MC4R pathway and that of the nuclear receptor PPAR-γ. This is the first report providing evidence that MC4R is expressed in rat primary microglia and that melanocortins modulate PPAR levels in glial cells. Our findings provide new insights into the mechanisms underlying the activation of glial MC4R and open perspectives for new therapeutic strategies for the treatment of inflammation-mediated brain diseases

Morphometric analysis of astrocytes in brainstem respiratory regions.

PubMed

Sheikhbahaei, Shahriar; Morris, Brian; Collina, Jared; Anjum, Sommer; Znati, Sami; Gamarra, Julio; Zhang, Ruli; Gourine, Alexander V; Smith, Jeffrey C

2018-06-11

Astrocytes, the most abundant and structurally complex glial cells of the central nervous system, are proposed to play an important role in modulating the activities of neuronal networks, including respiratory rhythm-generating circuits of the preBötzinger complex (preBötC) located in the ventrolateral medulla of the brainstem. However, structural properties of astrocytes residing within different brainstem regions are unknown. In this study astrocytes in the preBötC, an intermediate reticular formation (IRF) region with respiratory-related function, and a region of the nucleus tractus solitarius (NTS) in adult rats were reconstructed and their morphological features were compared. Detailed morphological analysis revealed that preBötC astrocytes are structurally more complex than those residing within the functionally distinct neighboring IRF region, or the NTS, located at the dorsal aspect of the medulla oblongata. Structural analyses of the brainstem microvasculature indicated no significant regional differences in vascular properties. We hypothesize that high morphological complexity of preBötC astrocytes reflects their functional role in providing structural/metabolic support and modulation of the key neuronal circuits essential for breathing, as well as constraints imposed by arrangements of associated neurons and/or other local structural features of the brainstem parenchyma. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Investigation on the suitable pressure for the preservation of astrocyte

NASA Astrophysics Data System (ADS)

Sotome, S.; Nakajima, K.; Yoshimura, Y.; Shimizu, A.

2010-03-01

The effects of pressure on the survival rate of astrocytes in growth medium (DMEM) were investigated at room temperature and at 4°C, in an effort to establish the best conditions for the preservation. Survival rate at 4°C was found to be higher than that at room temperature. The survival rate of astrocytes preserved for 4 days at 4°C increased with increasing pressure up to 1.6 MPa, but decreased with increasing pressure above 1.6 MPa. At 10 MPa, all astrocytes died. The survival rate of cultured astrocytes decreased significantly following pressurization for 2 hours and the subsequent preservation for 2 days at atmospheric pressure. Therefore, it is necessary to maintain pressure when preserving astrocytes. These results indicate that the cells can be stored at 4°C under pressurization without freezing and without adding cryoprotective agents. Moreover, it may be possible to use this procedure as a new preservation method when cryopreservation is impractical.

THE ENZYMATIC RESPONSE OF ASTROCYTES TO VARIOUS IONS IN VITRO

PubMed Central

Friede, Reinhard L.

1964-01-01

The effect of environmental ion concentration on the enzyme activity of astrocytes was investigated in tissue cultures of rat cerebral cortex. It was found that the oxidative enzymatic activity (succinic dehydrogenase, DPN-diaphorase, and several other enzymes) of astrocytes depended on the concentration of NaCl in the environment. This response was not specific for NaCl, but was also elicited by MgCl2 and LiCl; the response was less consistent, and often questionable for KCl. However, only NaCl could elicit enzymatic changes in astrocytes at concentrations known to be present in a living organism. Astrocytes were the only cells which responded this way; it appeared that the foot-plates were particularly involved in the response since increase of enzyme activity occurred earlier in the foot-plates than in the perikarya. It was concluded that astrocytes are metabolically involved in the maintenance of the ionic and osmotic environment of the central nervous system, particularly in regard to the active transport of sodium. PMID:14105217

Mechanisms of Endogenous Neuroprotective Effects of Astrocytes in Brain Injury

PubMed Central

2018-01-01

Astrocytes, once believed to serve only as “glue” for the structural support of neurons, have been demonstrated to serve critical functions for the maintenance and protection of neurons, especially under conditions of acute or chronic injury. There are at least seven distinct mechanisms by which astrocytes protect neurons from damage; these are (1) protection against glutamate toxicity, (2) protection against redox stress, (3) mediation of mitochondrial repair mechanisms, (4) protection against glucose-induced metabolic stress, (5) protection against iron toxicity, (6) modulation of the immune response in the brain, and (7) maintenance of tissue homeostasis in the presence of DNA damage. Astrocytes support these critical functions through specialized responses to stress or toxic conditions. The detoxifying activities of astrocytes are essential for maintenance of the microenvironment surrounding neurons and in whole tissue homeostasis. Improved understanding of the mechanisms by which astrocytes protect the brain could lead to the development of novel targets for the development of neuroprotective strategies.

Differential signaling mechanism for HIV-1 Nef-mediated production of IL-6 and IL-8 in human astrocytes.

PubMed

Liu, Xun; Kumar, Anil

2015-06-15

Variety of HIV-1 viral proteins including HIV-1 Nef are known to activate astrocytes and microglia in the brain and cause the release of pro-inflammatory cytokines, which is thought to be one of the mechanisms leading to HIV-1- mediated neurotoxicity. IL-6 and IL-8 have been found in the CSF of patients with HIV-1 associated dementia (HAD), suggesting that they might play important roles in HIV-1 neuropathology. In the present study we examined the effects of HIV-1 Nef on IL-6 and IL-8 induction in astrocytes. The results demonstrate that both IL-6 and IL-8 are significantly induced in HIV-1 Nef-transfected SVGA astrocytes and HIV-1 Nef-treated primary fetal astrocytes. We also determined the molecular mechanisms responsible for the HIV-1 Nef-induced increased IL-6 and IL-8 by using chemical inhibitors and siRNAs against PI3K/Akt/PKC, p38 MAPK, NF-κB, CEBP and AP-1. Our results clearly demonstrate that the PI3K/PKC, p38 MAPK, NF-κB and AP-1 pathways are involved in HIV-1 Nef-induced IL-6 production in astrocytes, while PI3K/PKC and NF-κB pathways are involved in HIV-1 Nef-induced IL-8 production. These results offer new potential targets to develop therapeutic strategy for treatment of HIV-1 associated neurological disorders, prevalent in > 40% of individuals infected with HIV-1.

Electrodiffusive Model for Astrocytic and Neuronal Ion Concentration Dynamics

PubMed Central

Halnes, Geir; Østby, Ivar; Pettersen, Klas H.; Omholt, Stig W.; Einevoll, Gaute T.

2013-01-01

The cable equation is a proper framework for modeling electrical neural signalling that takes place at a timescale at which the ionic concentrations vary little. However, in neural tissue there are also key dynamic processes that occur at longer timescales. For example, endured periods of intense neural signaling may cause the local extracellular K+-concentration to increase by several millimolars. The clearance of this excess K+ depends partly on diffusion in the extracellular space, partly on local uptake by astrocytes, and partly on intracellular transport (spatial buffering) within astrocytes. These processes, that take place at the time scale of seconds, demand a mathematical description able to account for the spatiotemporal variations in ion concentrations as well as the subsequent effects of these variations on the membrane potential. Here, we present a general electrodiffusive formalism for modeling of ion concentration dynamics in a one-dimensional geometry, including both the intra- and extracellular domains. Based on the Nernst-Planck equations, this formalism ensures that the membrane potential and ion concentrations are in consistency, it ensures global particle/charge conservation and it accounts for diffusion and concentration dependent variations in resistivity. We apply the formalism to a model of astrocytes exchanging ions with the extracellular space. The simulations show that K+-removal from high-concentration regions is driven by a local depolarization of the astrocyte membrane, which concertedly (i) increases the local astrocytic uptake of K+, (ii) suppresses extracellular transport of K+, (iii) increases axial transport of K+ within astrocytes, and (iv) facilitates astrocytic relase of K+ in regions where the extracellular concentration is low. Together, these mechanisms seem to provide a robust regulatory scheme for shielding the extracellular space from excess K+. PMID:24367247

K(+)- and temperature-evoked taurine efflux from hypothalamic astrocytes.

PubMed

Tigges, G A; Philibert, R A; Dutton, G R

1990-10-30

Hypothalamic astrocytes in culture released taurine, a suspected inhibitory amino acid neurotransmitter/neuromodulator/osmoregulator, in response to isoosmotically increasing extracellular K+ in a dose-dependent fashion. In the absence of added Ca2+, basal release levels rose to approach those obtained after exposure to 60 mM K+ in the presence of 2.5 mM Ca2+, and were only partially lowered by the addition of 10 mM Mg2+. Stimulation with K+ (60 mM) did not further increase taurine efflux above the high basal levels seen in the absence of Ca2+. Under standard conditions complete replacement of Na+ with choline Cl had little effect on basal taurine release, but reduced K(+)-evoked (60 mM) efflux by 60%. The temperature dependence of the basal levels of taurine released from hypothalamic astrocytes was similar to that seen for cultured cerebellar astrocytes and neurons over the range 5-50 degrees C. Taurine release increased from 5 to 15 degrees C, remained constant between 15 and 33 degrees C, decreased between 33 and 37 degrees C and increased thereafter. The infection point of increased basal taurine release seen around 37 degrees C (most prominent in astrocytes), may be of physiological significance. Results presented also show that the ion (Na+, Ca2+ and K+) sensitivities of taurine efflux for cultured hypothalamic astrocytes are similar to those previously reported for cultured astrocytes from the cerebellum.

Huperzine A inhibits CCL2 production in experimental autoimmune encephalomyelitis mice and in cultured astrocyte.

PubMed

Tian, G X; Zhu, X Q; Chen, Y; Wu, G C; Wang, J

2013-01-01

The active role of chemokines and inflammatory cytokines in the central nervous system (CNS) during the pathogenesis of experimental autoimmune encephalomyelitis (EAE) has been clearly established. Recent studies from our laboratory reported that Huperzine A (HupA) can attenuate the disease process in EAE by the inhibition of inflammation, demyelination, and axonal injury in the spinal cord as well as encephalomyelitic T-cell proliferation. In this study, the effects of low dose HupA on CCL2, TNF-alpha, IL-6, and IL-1beta expression were evaluated in EAE. The effect of HupA on lipopolysachharide (LPS)-induced inflammatory molecule secretion was investigated in cultured-astrocytes in vitro. In MOG35-55-induced EAE mice, intraperitoneal injections of HupA (0.1 mg/kg•d−1) significantly suppressed the expression of CCL2, IL-6, TNF-alpha, and IL-1beta in the spinal cord. HupA also repressed LPS-induced CCL2 production, but with little influence on pro-inflammatory cytokines in primary cultured astrocytes. The inhibition effect of HupA on CCL2 is PPARgamma-dependent and nicotine receptor-independent. Conditioned culture media from HupA-treated astrocyte decreased PBMC migration in vitro. Collectively, these results suggest that HupA can ameliorate EAE by inhibiting CCL2 production in astrocyte, which may consequently decrease inflammatory cell infiltration in the spinal cord. HupA may have a potential therapeutic value for the treatment of MS and other neuroinflammatory diseases.

Astrocyte lipid metabolism is critical for synapse development and function in vivo.

PubMed

van Deijk, Anne-Lieke F; Camargo, Nutabi; Timmerman, Jaap; Heistek, Tim; Brouwers, Jos F; Mogavero, Floriana; Mansvelder, Huibert D; Smit, August B; Verheijen, Mark H G

2017-04-01

The brain is considered to be autonomous in lipid synthesis with astrocytes producing lipids far more efficiently than neurons. Accordingly, it is generally assumed that astrocyte-derived lipids are taken up by neurons to support synapse formation and function. Initial confirmation of this assumption has been obtained in cell cultures, but whether astrocyte-derived lipids support synapses in vivo is not known. Here, we address this issue and determined the role of astrocyte lipid metabolism in hippocampal synapse formation and function in vivo. Hippocampal protein expression for the sterol regulatory element-binding protein (SREBP) and its target gene fatty acid synthase (Fasn) was found in astrocytes but not in neurons. Diminishing SREBP activity in astrocytes using mice in which the SREBP cleavage-activating protein (SCAP) was deleted from GFAP-expressing cells resulted in decreased cholesterol and phospholipid secretion by astrocytes. Interestingly, SCAP mutant mice showed more immature synapses, lower presynaptic protein SNAP-25 levels as well as reduced numbers of synaptic vesicles, indicating impaired development of the presynaptic terminal. Accordingly, hippocampal short-term and long-term synaptic plasticity were defective in mutant mice. These findings establish a critical role for astrocyte lipid metabolism in presynaptic terminal development and function in vivo. GLIA 2017;65:670-682. © 2017 Wiley Periodicals, Inc.

Identification and staining of distinct populations of secretory organelles in astrocytes.

PubMed

Bezzi, Paola; Volterra, Andrea

2014-05-01

Increasing evidence indicates that astrocytes, the most abundant glial cell type in the brain, respond to an elevation in cytoplasmic calcium concentration ([Ca(2+)]i) by releasing chemical transmitters (also called gliotransmitters) via regulated exocytosis of heterogeneous classes of organelles. By this process, astrocytes exert modulatory influences on neighboring cells and are thought to participate in the control of synaptic circuits and cerebral blood flow. Studying the properties of exocytosis in astrocytes is a challenge, because the cell biological basis of this process is incompletely defined. Astrocytic exocytosis involves multiple populations of secretory vesicles, including synaptic-like microvesicles (SLMVs), dense-core granules (DCGs), and lysosomes. Here we summarize the available information for identifying individual populations of secretory organelles in astrocytes, including DCGs, SLMVs, and lysosomes, and present experimental procedures for specifically staining such populations.

Astrocyte glycogen and lactate: New insights into learning and memory mechanisms.

PubMed

Alberini, Cristina M; Cruz, Emmanuel; Descalzi, Giannina; Bessières, Benjamin; Gao, Virginia

2018-06-01

Memory, the ability to retain learned information, is necessary for survival. Thus far, molecular and cellular investigations of memory formation and storage have mainly focused on neuronal mechanisms. In addition to neurons, however, the brain comprises other types of cells and systems, including glia and vasculature. Accordingly, recent experimental work has begun to ask questions about the roles of non-neuronal cells in memory formation. These studies provide evidence that all types of glial cells (astrocytes, oligodendrocytes, and microglia) make important contributions to the processing of encoded information and storing memories. In this review, we summarize and discuss recent findings on the critical role of astrocytes as providers of energy for the long-lasting neuronal changes that are necessary for long-term memory formation. We focus on three main findings: first, the role of glucose metabolism and the learning- and activity-dependent metabolic coupling between astrocytes and neurons in the service of long-term memory formation; second, the role of astrocytic glucose metabolism in arousal, a state that contributes to the formation of very long-lasting and detailed memories; and finally, in light of the high energy demands of the brain during early development, we will discuss the possible role of astrocytic and neuronal glucose metabolisms in the formation of early-life memories. We conclude by proposing future directions and discussing the implications of these findings for brain health and disease. Astrocyte glycogenolysis and lactate play a critical role in memory formation. Emotionally salient experiences form strong memories by recruiting astrocytic β2 adrenergic receptors and astrocyte-generated lactate. Glycogenolysis and astrocyte-neuron metabolic coupling may also play critical roles in memory formation during development, when the energy requirements of brain metabolism are at their peak. © 2017 Wiley Periodicals, Inc.

Conditions and constraints for astrocyte calcium signaling in the hippocampal mossy fiber pathway.

PubMed

Haustein, Martin D; Kracun, Sebastian; Lu, Xiao-Hong; Shih, Tiffany; Jackson-Weaver, Olan; Tong, Xiaoping; Xu, Ji; Yang, X William; O'Dell, Thomas J; Marvin, Jonathan S; Ellisman, Mark H; Bushong, Eric A; Looger, Loren L; Khakh, Baljit S

2014-04-16

The spatiotemporal activities of astrocyte Ca²⁺ signaling in mature neuronal circuits remain unclear. We used genetically encoded Ca²⁺ and glutamate indicators as well as pharmacogenetic and electrical control of neurotransmitter release to explore astrocyte activity in the hippocampal mossy fiber pathway. Our data revealed numerous localized, spontaneous Ca²⁺ signals in astrocyte branches and territories, but these were not driven by neuronal activity or glutamate. Moreover, evoked astrocyte Ca²⁺ signaling changed linearly with the number of mossy fiber action potentials. Under these settings, astrocyte responses were global, suppressed by neurotransmitter clearance, and mediated by glutamate and GABA. Thus, astrocyte engagement in the fully developed mossy fiber pathway was slow and territorial, contrary to that frequently proposed for astrocytes within microcircuits. We show that astrocyte Ca²⁺ signaling functionally segregates large volumes of neuropil and that these transients are not suited for responding to, or regulating, single synapses in the mossy fiber pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

Prostaglandin E(2) stimulates glutamate receptor-dependent astrocyte neuromodulation in cultured hippocampal cells.

PubMed

Sanzgiri, R P; Araque, A; Haydon, P G

1999-11-05

Recent Ca(2+) imaging studies in cell culture and in situ have shown that Ca(2+) elevations in astrocytes stimulate glutamate release and increase neuronal Ca(2+) levels, and that this astrocyte-neuron signaling can be stimulated by prostaglandin E(2) (PGE(2)). We investigated the electrophysiological consequences of the PGE(2)-mediated astrocyte-neuron signaling using whole-cell recordings on cultured rat hippocampal cells. Focal application of PGE(2) to astrocytes evoked a Ca(2+) elevation in the stimulated cell by mobilizing internal Ca(2+) stores, which further propagated as a Ca(2+) wave to neighboring astrocytes. Whole-cell recordings from neurons revealed that PGE(2) evoked a slow inward current in neurons adjacent to astrocytes. This neuronal response required the presence of an astrocyte Ca(2+) wave and was mediated through both N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors. Taken together with previous studies, these data demonstrate that PGE(2)-evoked Ca(2+) elevations in astrocyte cause the release of glutamate which activates neuronal ionotropic receptors. Copyright 1999 John Wiley & Sons, Inc.

Extracellular microvesicles from astrocytes contain functional glutamate transporters: regulation by protein kinase C and cell activation

PubMed Central

Gosselin, Romain-Daniel; Meylan, Patrick; Decosterd, Isabelle

2013-01-01

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. PMID:24368897

Astrocyte-neuron crosstalk regulates the expression and subcellular localization of carbohydrate metabolism enzymes.

PubMed

Mamczur, Piotr; Borsuk, Borys; Paszko, Jadwiga; Sas, Zuzanna; Mozrzymas, Jerzy; Wiśniewski, Jacek R; Gizak, Agnieszka; Rakus, Dariusz

2015-02-01

Astrocytes releasing glucose- and/or glycogen-derived lactate and glutamine play a crucial role in shaping neuronal function and plasticity. Little is known, however, how metabolic functions of astrocytes, e.g., their ability to degrade glucosyl units, are affected by the presence of neurons. To address this issue we carried out experiments which demonstrated that co-culturing of rat hippocampal astrocytes with neurons significantly elevates the level of mRNA and protein for crucial enzymes of glycolysis (phosphofructokinase, aldolase, and pyruvate kinase), glycogen metabolism (glycogen synthase and glycogen phosphorylase), and glutamine synthetase in astrocytes. Simultaneously, the decrease of the capability of neurons to metabolize glucose and glutamine is observed. We provide evidence that neurons alter the expression of astrocytic enzymes by secretion of as yet unknown molecule(s) into the extracellular fluid. Moreover, our data demonstrate that almost all studied enzymes may localize in astrocytic nuclei and this localization is affected by the co-culturing with neurons which also reduces proliferative activity of astrocytes. Our results provide the first experimental evidence that the astrocyte-neuron crosstalk substantially affects the expression of basal metabolic enzymes in the both types of cells and influences their subcellular localization in astrocytes. © 2014 Wiley Periodicals, Inc.

Traumatically injured astrocytes release a proteomic signature modulated by STAT3 dependent cell survival

PubMed Central

Levine, Jaclynn; Kwon, Eunice; Paez, Pablo; Yan, Weihong; Czerwieniec, Gregg; Loo, Joseph A.; Sofroniew, Michael V.; Wanner, Ina-Beate

2015-01-01

Molecular markers associated with CNS injury are of diagnostic interest. Mechanical trauma generates cellular deformation associated with membrane permeability with unknown molecular consequences. We used an in vitro model of stretch-injury and proteomic analyses to determine protein changes in murine astrocytes and their surrounding fluids. Abrupt pressure-pulse stretching resulted in the rapid release of 59 astrocytic proteins with profiles reflecting cell injury and cell death, i.e. mechanoporation and cell lysis. This acute trauma-release proteome was overrepresented with metabolic proteins compared to the uninjured cellular proteome, bearing relevance for post-traumatic metabolic depression. Astrocyte-specific deletion of signal transducer and activator of transcription 3 (STAT3-CKO) resulted in reduced stretch-injury tolerance, elevated necrosis and increased protein release. Consistent with more lysed cells, more protein complexes, nuclear and transport proteins were released from STAT3-CKO versus non-transgenic astrocytes. STAT3-CKO astrocytes had reduced basal expression of GFAP, lactate dehydrogenase B (LDHB), aldolase C (ALDOC) and astrocytic phosphoprotein 15 (PEA15), and elevated levels of tropomyosin (TPM4) and α actinin 4 (ACTN4). Stretching caused STAT3 dependent cellular depletion of PEA15 and GFAP, and its filament disassembly in subpopulations of injured astrocytes. PEA15 and ALDOC signals were low in injured astrocytes acutely after mouse spinal cord crush injury and robustly expressed in reactive astrocytes one day post-injury. In contrast, α crystallin (CRYAB) was present in acutely injured astrocytes, and absent from uninjured and reactive astrocytes, demonstrating novel marker differences among post-injury astrocytes. These findings reveal a proteomic signature of traumatically-injured astrocytes reflecting STAT3-dependent cellular survival with potential diagnostic value. PMID:26683444

Role of astrocytes in reproduction and neuroprotection.

PubMed

Mahesh, Virendra B; Dhandapani, Krishnan M; Brann, Darrell W

2006-02-26

Hypothalamic astrocytes secrete TGF-beta and 3 alpha,5 alpha-tetrahydro progesterone (3 alpha,5 alpha-THP) in culture. When the astrocyte-conditioned medium (ACM) was incubated with the hypothalamic cell line GT1-7, it resulted in the secretion of GnRH. Immunoneutralization with TGF-beta antibody or ultra-filteration with a 10 kDa cut off filter resulted in attenuation of the GnRH releasing ability of ACM, indicating that TGF-beta was a major factor involved with GnRH release. Treatment with estrogens increases TGF-beta secretion. These observations indicate a significant role of astrocytes in GnRH secretion. Serum-deprivation results in the death of GT1-7 neurons in culture and addition of ACM or TGF-beta to the culture, attenuates cell death. The mechanism of protection from cell death appears to involve phosphorylation of MKK4, JNK, c-Jun(Ser63), and enhancement of AP-1 binding. Co-administration of JNK inhibitors, but not MEK inhibitors attenuated ACM or TGF-beta-induced c-Jun(Ser63) phosphorylation and their neuroprotective effects. These studies suggest that astrocytes can protect neurons, at least in part, by the release of TGF-beta and activation of a c-Jun/AP-1 protective pathway.

Age-Dependent Neurochemical Remodeling of Hypothalamic Astrocytes.

PubMed

Santos, Camila Leite; Roppa, Paola Haack Amaral; Truccolo, Pedro; Fontella, Fernanda Urruth; Souza, Diogo Onofre; Bobermin, Larissa Daniele; Quincozes-Santos, André

2017-10-04

The hypothalamus is a crucial integrative center in the central nervous system, responsible for the regulation of homeostatic activities, including systemic energy balance. Increasing evidence has highlighted a critical role of astrocytes in orchestrating hypothalamic functions; they participate in the modulation of synaptic transmission, metabolic and trophic support to neurons, immune defense, and nutrient sensing. In this context, disturbance of systemic energy homeostasis, which is a common feature of obesity and the aging process, involves inflammatory responses. This may be related to dysfunction of hypothalamic astrocytes. In this regard, the aim of this study was to evaluate the neurochemical properties of hypothalamic astrocyte cultures from newborn, adult, and aged Wistar rats. Age-dependent changes in the regulation of glutamatergic homeostasis, glutathione biosynthesis, amino acid profile, glucose metabolism, trophic support, and inflammatory response were observed. Additionally, signaling pathways including nuclear factor erythroid-derived 2-like 2/heme oxygenase-1 p38 mitogen-activated protein kinase, nuclear factor kappa B, phosphatidylinositide 3-kinase/Akt, and leptin receptor expression may represent putative mechanisms associated with the cellular alterations. In summary, our findings indicate that as age increases, hypothalamic astrocytes remodel and exhibit changes in their neurochemical properties. This process may play a role in the onset and/or progression of metabolic disorders.

Insufficient Astrocyte-Derived Brain-Derived Neurotrophic Factor Contributes to Propofol-Induced Neuron Death Through Akt/Glycogen Synthase Kinase 3β/Mitochondrial Fission Pathway.

PubMed

Liu, Yanan; Yan, Yasheng; Inagaki, Yasuyoshi; Logan, Sarah; Bosnjak, Zeljko J; Bai, Xiaowen

2017-07-01

Growing animal evidence demonstrates that prolonged exposure to propofol during brain development induces widespread neuronal cell death, but there is little information on the role of astrocytes. Astrocytes can release neurotrophic growth factors such as brain-derived neurotrophic factor (BDNF), which can exert the protective effect on neurons in paracrine fashion. We hypothesize that during propofol anesthesia, BDNF released from developing astrocytes may not be sufficient to prevent propofol-induced neurotoxicity. Hippocampal astrocytes and neurons isolated from neonatal Sprague Dawley rats were exposed to propofol at a clinically relevant dose of 30 μM or dimethyl sulfoxide as control for 6 hours. Propofol-induced cell death was determined by propidium iodide (PI) staining in astrocyte-alone cultures, neuron-alone cultures, or cocultures containing either low or high density of astrocytes (1:9 or 1:1 ratio of astrocytes to neurons ratio [ANR], respectively). The astrocyte-conditioned medium was collected 12 hours after propofol exposure and measured by protein array assay. BDNF concentration in astrocyte-conditioned medium was quantified using enzyme-linked immunosorbent assay. Neuron-alone cultures were treated with BDNF, tyrosine receptor kinase B inhibitor cyclotraxin-B, glycogen synthase kinase 3β (GSK3β) inhibitor CHIR99021, or mitochondrial fission inhibitor Mdivi-1 before propofol exposure. Western blot was performed for quantification of the level of protein kinase B and GSK3β. Mitochondrial shape was visualized through translocase of the outer membrane 20 staining. Propofol increased cell death in neurons by 1.8-fold (% of PI-positive cells [PI%] = 18.6; 95% confidence interval [CI], 15.2-21.9, P < .05) but did not influence astrocyte viability. The neuronal death was attenuated by a high ANR (1:1 cocultures; fold change [FC] = 1.17, 95% CI, 0.96-1.38, P < .05), but not with a low ANR [1:9 cocultures; FC = 1.87, 95% CI, 1.48-2.26, P > .05

Mutant Huntingtin Inhibits αB-Crystallin Expression and Impairs Exosome Secretion from Astrocytes.

PubMed

Hong, Yan; Zhao, Ting; Li, Xiao-Jiang; Li, Shihua

2017-09-27

In the brain, astrocytes secrete diverse substances that regulate neuronal function and viability. Exosomes, which are vesicles produced through the formation of multivesicular bodies and their subsequent fusion with the plasma membrane, are also released from astrocytes via exocytotic secretion. Astrocytic exosomes carry heat shock proteins that can reduce the cellular toxicity of misfolded proteins and prevent neurodegeneration. Although mutant huntingtin (mHtt) affects multiple functions of astrocytes, it remains unknown whether mHtt impairs the production of exosomes from astrocytes. We found that mHtt is not present in astrocytic exosomes, but can decrease exosome secretion from astrocytes in HD140Q knock-in (KI) mice. N-terminal mHtt accumulates in the nuclei and forms aggregates, causing decreased secretion of exosomes from cultured astrocytes. Consistently, there is a significant decrease in secreted exosomes in both female and male HD KI mouse striatum in which abundant nuclear mHtt aggregates are present. Conversely, injection of astrocytic exosomes into the striatum of HD140Q KI mice reduces the density of mHtt aggregates. Further, mHtt in astrocytes decreased the expression of αB-crystallin, a small heat shock protein that is enriched in astrocytes and mediates exosome secretion, by reducing the association of Sp1 with the enhancer of the α B-crystallin gene. Importantly, overexpression of αB-crystallin rescues defective exosome release from HD astrocytes as well as mHtt aggregates in the striatum of HD140Q KI mice. Our results demonstrate that mHtt reduces the expression of αB-crystallin in astrocytes to decrease exosome secretion in the HD brains, contributing to non-cell-autonomous neurotoxicity in HD. SIGNIFICANCE STATEMENT Huntington's disease (HD) is characterized by selective neurodegeneration that preferentially occurs in the striatal medium spiny neurons. Recent studies in different HD mouse models demonstrated that dysfunction of

HIV-tat alters Connexin43 expression and trafficking in human astrocytes: role in NeuroAIDS.

PubMed

Berman, Joan W; Carvallo, Loreto; Buckner, Clarisa M; Luers, Aimée; Prevedel, Lisa; Bennett, Michael V; Eugenin, Eliseo A

2016-03-02

HIV-associated neurocognitive disorders (HAND) are a major complication in at least half of the infected population despite effective antiretroviral treatment and immune reconstitution. HIV-associated CNS damage is not correlated with active viral replication but instead is associated with mechanisms that regulate inflammation and neuronal compromise. Our data indicate that one of these mechanisms is mediated by gap junction channels and/or hemichannels. Normally, gap junction channels shutdown under inflammatory conditions, including viral diseases. However, HIV infection upregulates Connexin43 (Cx43) expression and maintains gap junctional communication by unknown mechanism(s). Human primary astrocytes were exposed to several HIV proteins as well as to HIV, and expression and function of Connexin43- and Connexin30-containing channels were determined by western blot, immunofluorescence, microinjection of a fluorescent tracer and chromatin immunoprecipitation (ChIP). Here, we demonstrate that HIV infection increases Cx43 expression in vivo. HIV-tat, the transactivator of the virus, and no other HIV proteins tested, increases Cx43 expression and maintains functional gap junctional communication in human astrocytes. Cx43 upregulation is mediated by binding of the HIV-tat protein to the Cx43 promoter, but not to the Cx30 promoter, resulting in increased Cx43 messenger RNA (mRNA) and protein as well as gap junctional communication. We propose that HIV-tat contributes to the spread of intracellular toxic signals generated in a few HIV-infected cells into surrounding uninfected cells by upregulating gap junctional communication. In the current antiretroviral era, where HIV replication is often completely suppressed, viral factors such as HIV-tat are still produced and released from infected cells. Thus, blocking the effects of HIV-tat could result in new strategies to reduce the damaging consequences of HIV infection of the CNS.

Neuroprotective Role of Gap Junctions in a Neuron Astrocyte Network Model.

PubMed

Huguet, Gemma; Joglekar, Anoushka; Messi, Leopold Matamba; Buckalew, Richard; Wong, Sarah; Terman, David

2016-07-26

A detailed biophysical model for a neuron/astrocyte network is developed to explore mechanisms responsible for the initiation and propagation of cortical spreading depolarizations and the role of astrocytes in maintaining ion homeostasis, thereby preventing these pathological waves. Simulations of the model illustrate how properties of spreading depolarizations, such as wave speed and duration of depolarization, depend on several factors, including the neuron and astrocyte Na(+)-K(+) ATPase pump strengths. In particular, we consider the neuroprotective role of astrocyte gap junction coupling. The model demonstrates that a syncytium of electrically coupled astrocytes can maintain a physiological membrane potential in the presence of an elevated extracellular K(+) concentration and efficiently distribute the excess K(+) across the syncytium. This provides an effective neuroprotective mechanism for delaying or preventing the initiation of spreading depolarizations. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Mechanisms of astrocytic K(+) clearance and swelling under high extracellular K(+) concentrations.

PubMed

Murakami, Shingo; Kurachi, Yoshihisa

2016-03-01

In response to the elevation of extracellular K(+) concentration ([K(+)]out), astrocytes clear excessive K(+) to maintain conditions necessary for neural activity. K(+) clearance in astrocytes occurs via two processes: K(+) uptake and K(+) spatial buffering. High [K(+)]out also induces swelling in astrocytes, leading to edema and cell death in the brain. Despite the importance of astrocytic K(+) clearance and swelling, the underlying mechanisms remain unclear. Here, we report results from a simulation analysis of astrocytic K(+) clearance and swelling. Astrocyte models were constructed by incorporating various mechanisms such as intra/extracellular ion concentrations of Na(+), K(+), and Cl(-), cell volume, and models of Na,K-ATPase, Na-K-Cl cotransporter (NKCC), K-Cl cotransporter, inwardly-rectifying K(+) (KIR) channel, passive Cl(-) current, and aquaporin channel. The simulated response of astrocyte models under the uniform distribution of high [K(+)]out revealed significant contributions of NKCC and Na,K-ATPase to increases of intracellular K(+) and Cl(-) concentrations, and swelling. Moreover, we found that, under the non-uniform distribution of high [K(+)]out, KIR channels localized at synaptic clefts absorbed excess K(+) by depolarizing the equivalent potential of K(+) (E K) above membrane potential, while K(+) released through perivascular KIR channels was enhanced by hyperpolarizing E K and depolarizing membrane potential. Further analysis of simulated drug effects revealed that astrocyte swelling was modulated by blocking each of the ion channels and transporters. Our simulation analysis revealed controversial mechanisms of astrocytic K(+) clearance and swelling resulting from complex interactions among ion channels and transporters.

MicroRNA profiling reveals new aspects of HIV neurodegeneration: caspase-6 regulates astrocyte survival.

PubMed

Noorbakhsh, Farshid; Ramachandran, Rithwik; Barsby, Nicola; Ellestad, Kristofor K; LeBlanc, Andrea; Dickie, Peter; Baker, Glen; Hollenberg, Morley D; Cohen, Eric A; Power, Christopher

2010-06-01

MicroRNAs (miRNAs) are small noncoding RNA molecules, which are known to regulate gene expression in physiological and pathological conditions. miRNA profiling was performed using brain tissue from patients with HIV encephalitis (HIVE), a neuroinflammatory/degenerative disorder caused by HIV infection of the brain. Microarray analysis showed differential expression of multiple miRNAs in HIVE compared to control brains. Target prediction and gene ontology enrichment analysis disclosed targeting of several gene families/biological processes by differentially expressed miRNAs (DEMs), with cell death-related genes, including caspase-6, showing a bias toward down-regulated DEMs. Consistent with the miRNA data, HIVE brains exhibited higher levels of caspase-6 transcripts compared with control patients. Immunohistochemical analysis showed localization of the cleaved form of caspase-6 in astrocytes in HIVE brain sections. Exposure of cultured human primary astrocytes to HIV viral protein R (Vpr) induced p53 up-regulation, loss of mitochondrial membrane potential, and caspase-6 activation followed by cell injury. Transgenic mice, expressing Vpr in microglial cells, demonstrated astrocyte apoptosis in brain, which was associated with caspase-6 activation and neurobehavioral abnormalities. Overall, these data point to previously unrecognized alterations in miRNA profile in the brain during HIV infection, which contribute to cell death through dysregulation of cell death machinery.

Contributions of glycogen to astrocytic energetics during brain activation.

PubMed

Dienel, Gerald A; Cruz, Nancy F

2015-02-01

Glycogen is the major store of glucose in brain and is mainly in astrocytes. Brain glycogen levels in unstimulated, carefully-handled rats are 10-12 μmol/g, and assuming that astrocytes account for half the brain mass, astrocytic glycogen content is twice as high. Glycogen turnover is slow under basal conditions, but it is mobilized during activation. There is no net increase in incorporation of label from glucose during activation, whereas label release from pre-labeled glycogen exceeds net glycogen consumption, which increases during stronger stimuli. Because glycogen level is restored by non-oxidative metabolism, astrocytes can influence the global ratio of oxygen to glucose utilization. Compensatory increases in utilization of blood glucose during inhibition of glycogen phosphorylase are large and approximate glycogenolysis rates during sensory stimulation. In contrast, glycogenolysis rates during hypoglycemia are low due to continued glucose delivery and oxidation of endogenous substrates; rates that preserve neuronal function in the absence of glucose are also low, probably due to metabolite oxidation. Modeling studies predict that glycogenolysis maintains a high level of glucose-6-phosphate in astrocytes to maintain feedback inhibition of hexokinase, thereby diverting glucose for use by neurons. The fate of glycogen carbon in vivo is not known, but lactate efflux from brain best accounts for the major metabolic characteristics during activation of living brain. Substantial shuttling coupled with oxidation of glycogen-derived lactate is inconsistent with available evidence. Glycogen has important roles in astrocytic energetics, including glucose sparing, control of extracellular K(+) level, oxidative stress management, and memory consolidation; it is a multi-functional compound.

Contributions of Glycogen to Astrocytic Energetics during Brain Activation

PubMed Central

Dienel, Gerald A.; Cruz, Nancy F.

2014-01-01

Glycogen is the major store of glucose in brain and is mainly in astrocytes. Brain glycogen levels in unstimulated, carefully-handled rats are 10-12 mol/g, and assuming that astrocytes account for half the brain mass, astrocytic glycogen content is twice as high. Glycogen turnover is slow under basal conditions, but it is mobilized during activation. There is no net increase in incorporation of label from glucose during activation, whereas label release from pre-labeled glycogen exceeds net glycogen consumption, which increases during stronger stimuli. Because glycogen level is restored by non-oxidative metabolism, astrocytes can influence the global ratio of oxygen to glucose utilization. Compensatory increases in utilization of blood glucose during inhibition of glycogen phosphorylase are large and approximate glycogenolysis rates during sensory stimulation. In contrast, glycogenolysis rates during hypoglycemia are low due to continued glucose delivery and oxidation of endogenous substrates; rates that preserve neuronal function in the absence of glucose are also low, probably due to metabolite oxidation. Modeling studies predict that glycogenolysis maintains a high level of glucose-6-phosphate in astrocytes to maintain feedback inhibition of hexokinase, thereby diverting glucose for use by neurons. The fate of glycogen carbon in vivo is not known, but lactate efflux from brain best accounts for the major metabolic characteristics during activation of living brain. Substantial shuttling coupled with oxidation of glycogen-derived lactate is inconsistent with available evidence. Glycogen has important roles in astrocytic energetics, including glucose sparing, control of extracellular K+ level, oxidative stress management, and memory consolidation; it is a multi-functional compound. PMID:24515302

Altered Astrocyte-Neuron Interactions and Epileptogenesis in Tuberous Sclerosis Complex Disorder

DTIC Science & Technology

2015-06-01

Tsc1-deficient astrocytes on neuronal morphology and neuronal activity associated with seizures . 2. KEY WORDS epilepsy , seizure , tuberous sclerosis...AWARD NUMBER: W81XWH-12-1-0196 TITLE: Altered Astrocyte-Neuron Interactions and Epileptogenesis in Tuberous Sclerosis Complex Disorder PRINCIPAL...TITLE AND SUBTITLE Altered Astrocyte-Neuron Interactions and Epileptogenesis in Tuberous Sclerosis Complex Disorder 5a. CONTRACT NUMBER 5b. GRANT

Acute stress enhances adult rat hippocampal neurogenesis and activation of newborn neurons via secreted astrocytic FGF2

PubMed Central

Kirby, Elizabeth D; Muroy, Sandra E; Sun, Wayne G; Covarrubias, David; Leong, Megan J; Barchas, Laurel A; Kaufer, Daniela

2013-01-01

Stress is a potent modulator of the mammalian brain. The highly conserved stress hormone response influences many brain regions, particularly the hippocampus, a region important for memory function. The effect of acute stress on the unique population of adult neural stem/progenitor cells (NPCs) that resides in the adult hippocampus is unclear. We found that acute stress increased hippocampal cell proliferation and astrocytic fibroblast growth factor 2 (FGF2) expression. The effect of acute stress occurred independent of basolateral amygdala neural input and was mimicked by treating isolated NPCs with conditioned media from corticosterone-treated primary astrocytes. Neutralization of FGF2 revealed that astrocyte-secreted FGF2 mediated stress-hormone-induced NPC proliferation. 2 weeks, but not 2 days, after acute stress, rats also showed enhanced fear extinction memory coincident with enhanced activation of newborn neurons. Our findings suggest a beneficial role for brief stress on the hippocampus and improve understanding of the adaptive capacity of the brain. DOI: http://dx.doi.org/10.7554/eLife.00362.001 PMID:23599891

Astrocytes from adult Wistar rats aged in vitro show changes in glial functions.

PubMed

Souza, Débora Guerini; Bellaver, Bruna; Raupp, Gustavo Santos; Souza, Diogo Onofre; Quincozes-Santos, André

2015-11-01

Astrocytes, the most versatile cells of the central nervous system, play an important role in the regulation of neurotransmitter homeostasis, energy metabolism, antioxidant defenses and the anti-inflammatory response. Recently, our group characterized cortical astrocyte cultures from adult Wistar rats. In line with that work, we studied glial function using an experimental in vitro model of aging astrocytes (30 days in vitro after reaching confluence) from newborn (NB), adult (AD) and aged (AG) Wistar rats. We evaluated metabolic parameters, such as the glucose uptake, glutamine synthetase (GS) activity, and glutathione (GSH) content, as well as the GFAP, GLUT-1 and xCT expression. AD and AG astrocytes take up less glucose than NB astrocytes and had decreased GLUT1 expression levels. Furthermore, AD and AG astrocytes exhibited decreased GS activity compared to NB cells. Simultaneously, AD and AG astrocytes showed an increase in GSH levels, along with an increase in xCT expression. NB, AD and AG astrocytes presented similar morphology; however, differences in GFAP levels were observed. Taken together, these results improve the knowledge of cerebral senescence and represent an innovative tool for brain studies of aging. Copyright © 2015 Elsevier Ltd. All rights reserved.

Brain energy metabolism: focus on astrocyte-neuron metabolic cooperation.

PubMed

Bélanger, Mireille; Allaman, Igor; Magistretti, Pierre J

2011-12-07

The energy requirements of the brain are very high, and tight regulatory mechanisms operate to ensure adequate spatial and temporal delivery of energy substrates in register with neuronal activity. Astrocytes-a type of glial cell-have emerged as active players in brain energy delivery, production, utilization, and storage. Our understanding of neuroenergetics is rapidly evolving from a "neurocentric" view to a more integrated picture involving an intense cooperativity between astrocytes and neurons. This review focuses on the cellular aspects of brain energy metabolism, with a particular emphasis on the metabolic interactions between neurons and astrocytes. Copyright © 2011 Elsevier Inc. All rights reserved.

The phosphorylation status and cytoskeletal remodeling of striatal astrocytes treated with quinolinic acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pierozan, Paula; Ferreira, Fernanda; Ortiz de Lima, Bárbara

2014-04-01

Quinolinic acid (QUIN) is a glutamate agonist which markedly enhances the vulnerability of neural cells to excitotoxicity. QUIN is produced from the amino acid tryptophan through the kynurenine pathway (KP). Dysregulation of this pathway is associated with neurodegenerative conditions. In this study we treated striatal astrocytes in culture with QUIN and assayed the endogenous phosphorylating system associated with glial fibrillary acidic protein (GFAP) and vimentin as well as cytoskeletal remodeling. After 24 h incubation with 100 µM QUIN, cells were exposed to {sup 32}P-orthophosphate and/or protein kinase A (PKA), protein kinase dependent of Ca{sup 2+}/calmodulin II (PKCaMII) or protein kinasemore » C (PKC) inhibitors, H89 (20 μM), KN93 (10 μM) and staurosporin (10 nM), respectively. Results showed that hyperphosphorylation was abrogated by PKA and PKC inhibitors but not by the PKCaMII inhibitor. The specific antagonists to ionotropic NMDA and non-NMDA (50 µM DL-AP5 and CNQX, respectively) glutamate receptors as well as to metabotropic glutamate receptor (mGLUR; 50 µM MCPG), mGLUR1 (100 µM MPEP) and mGLUR5 (10 µM 4C3HPG) prevented the hyperphosphorylation provoked by QUIN. Also, intra and extracellular Ca{sup 2+} quelators (1 mM EGTA; 10 µM BAPTA-AM, respectively) prevented QUIN-mediated effect, while Ca{sup 2+} influx through voltage-dependent Ca{sup 2+} channel type L (L-VDCC) (blocker: 10 µM verapamil) is not implicated in this effect. Morphological analysis showed dramatically altered actin cytoskeleton with concomitant change of morphology to fusiform and/or flattened cells with retracted cytoplasm and disruption of the GFAP meshwork, supporting misregulation of actin cytoskeleton. Both hyperphosphorylation and cytoskeletal remodeling were reversed 24 h after QUIN removal. Astrocytes are highly plastic cells and the vulnerability of astrocyte cytoskeleton may have important implications for understanding the neurotoxicity of QUIN in

The role of organic osmolytes in the response of cultured astrocytes to hyperosmolarity.

PubMed

Hijab, Samina; Havalad, Suresh; Snyder, Ann K

2011-09-01

Idiogenic osmoles are volume-regulatory organic solutes that accumulate within a cell in response to hyperosmolar conditions such as those found in diabetic ketoacidosis or hypernatremic dehydration in infants. Intracellular metabolites known to play this role include certain amino acids and taurine, polyols, and trimethylamines. In this study, in vitro astrocyte cultures prepared from the cerebral cortices of 1-day-old Sprague-Dawley rats were exposed to graded conditions of hypernatremia (325-375 mOsm/kg), a range that can be observed in vivo, for 24, 48, and 72 hours. Cell survival and generation of idiogenic osmoles were determined. Next, we assessed the ability of selected exogenous osmoles to protect the cultured cells from the effects of hypernatremia. Significant cell loss occurred after 48 to 72 hours of exposure and was proportional to the degree of hyperosmolarity. Addition of myoinositol (1 mM) to the cultures reduced cell loss resulting from hypernatremia by approximately 50%. In agreement with previous studies, intracellular levels of myoinositol and taurine correlated with the degree of in vitro hypernatremic exposure and play a significant role in increasing survival of astrocytes subjected to hypertonic insult. (C) 2011 Lippincott Williams & Wilkins, Inc.

Cerebral blood flow modulation by Basal forebrain or whisker stimulation can occur independently of large cytosolic Ca2+ signaling in astrocytes.

PubMed

Takata, Norio; Nagai, Terumi; Ozawa, Katsuya; Oe, Yuki; Mikoshiba, Katsuhiko; Hirase, Hajime

2013-01-01

We report that a brief electrical stimulation of the nucleus basalis of Meynert (NBM), the primary source of cholinergic projection to the cerebral cortex, induces a biphasic cerebral cortical blood flow (CBF) response in the somatosensory cortex of C57BL/6J mice. This CBF response, measured by laser Doppler flowmetry, was attenuated by the muscarinic type acetylcholine receptor antagonist atropine, suggesting a possible involvement of astrocytes in this type of CBF modulation. However, we find that IP3R2 knockout mice, which lack cytosolic Ca2+ surges in astrocytes, show similar CBF changes. Moreover, whisker stimulation resulted in similar degrees of CBF increase in IP3R2 knockout mice and the background strain C57BL/6J. Our results show that neural activity-driven CBF modulation could occur without large cytosolic increases of Ca2+ in astrocytes.

Dual patch voltage clamp study of low membrane resistance astrocytes in situ.

PubMed

Ma, Baofeng; Xu, Guangjin; Wang, Wei; Enyeart, John J; Zhou, Min

2014-03-17

Whole-cell patch clamp recording has been successfully used in identifying the voltage-dependent gating and conductance properties of ion channels in a variety of cells. However, this powerful technique is of limited value in studying low membrane resistance cells, such as astrocytes in situ, because of the inability to control or accurately measure the real amplitude of command voltages. To facilitate the study of ionic conductances of astrocytes, we have developed a dual patch recording method which permits membrane current and membrane potential to be simultaneously recorded from astrocytes in spite of their extraordinarily low membrane resistance. The utility of this technique is demonstrated by measuring the voltage-dependent activation of the inwardly rectifying K+ current abundantly expressed in astrocytes and multiple ionic events associated with astrocytic GABAA receptor activation. This protocol can be performed routinely in the study of astrocytes. This method will be valuable for identifying and characterizing the individual ion channels that orchestrate the electrical activity of low membrane resistance cells.

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

PubMed

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

2017-09-01

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.

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

PubMed

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

2014-01-01

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

Attenuating astrocyte activation accelerates plaque pathogenesis in APP/PS1 mice

PubMed Central

Kraft, Andrew W.; Hu, Xiaoyan; Yoon, Hyejin; Yan, Ping; Xiao, Qingli; Wang, Yan; Gil, So Chon; Brown, Jennifer; Wilhelmsson, Ulrika; Restivo, Jessica L.; Cirrito, John R.; Holtzman, David M.; Kim, Jungsu; Pekny, Milos; Lee, Jin-Moo

2013-01-01

The accumulation of aggregated amyloid-β (Aβ) in amyloid plaques is a neuropathological hallmark of Alzheimer's disease (AD). Reactive astrocytes are intimately associated with amyloid plaques; however, their role in AD pathogenesis is unclear. We deleted the genes encoding two intermediate filament proteins required for astrocyte activation—glial fibrillary acid protein (Gfap) and vimentin (Vim)—in transgenic mice expressing mutant human amyloid precursor protein and presenilin-1 (APP/PS1). The gene deletions increased amyloid plaque load: APP/PS1 Gfap−/−Vim−/− mice had twice the plaque load of APP/PS1 Gfap+/+Vim+/+ mice at 8 and 12 mo of age. APP expression and soluble and interstitial fluid Aβ levels were unchanged, suggesting that the deletions had no effect on APP processing or Aβ generation. Astrocyte morphology was markedly altered by the deletions: wild-type astrocytes had hypertrophied processes that surrounded and infiltrated plaques, whereas Gfap−/−Vim−/− astrocytes had little process hypertrophy and lacked contact with adjacent plaques. Moreover, Gfap and Vim gene deletion resulted in a marked increase in dystrophic neurites (2- to 3-fold higher than APP/PS1 Gfap+/+Vim+/+ mice), even after normalization for amyloid load. These results suggest that astrocyte activation limits plaque growth and attenuates plaque-related dystrophic neurites. These activities may require intimate contact between astrocyte and plaque.—Kraft, A. W., Hu, X., Yoon, H., Yan, P., Xiao, Q., Wang, Y., Gil, S. C., Brown, J., Wilhelmsson, U., Restivo, J. L., Cirrito, J. R., Holtzman, D. M., Kim, J., Pekny, M., Lee, J.-M. Attenuating astrocyte activation accelerates plaque pathogenesis in APP/PS1 mice. PMID:23038755

Carbon monoxide stimulates astrocytic mitochondrial biogenesis via L-type Ca{sup 2+} channel-mediated PGC-1α/ERRα activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Yoon Kyung; Park, Joon Ha; Baek, Yi-Yong

Carbon monoxide (CO), derived by the enzymatic reaction of heme oxygenase (HO), is a cellular regulator of energy metabolism and cytoprotection; however, its underlying mechanism has not been clearly elucidated. Astrocytes pre-exposed to the CO-releasing compound CORM-2 increased mitochondrial biogenesis, mitochondrial electron transport components (cytochrome c, Cyt c; cytochrome c oxidase subunit 2, COX2), and ATP synthesis. The increased mitochondrial function was correlated with activation of AMP-activated protein kinase α and upregulation of HO-1, peroxisome proliferators-activated receptor γ-coactivator-1α (PGC-1α), and estrogen-related receptor α (ERRα). These events elicited by CORM-2 were suppressed by Ca{sup 2+} chelators, a HO inhibitor, and anmore » L-type Ca{sup 2+} channel blocker, but not other Ca{sup 2+} channel inhibitors. Among the HO byproducts, combined CORM-2 and bilirubin treatment effectively increased PGC-1α, Cyt c and COX2 expression, mitochondrial biogenesis, and ATP synthesis, and these increases were blocked by Ca{sup 2+} chelators. Moreover, cerebral ischemia significantly increased HO-1, PGC-1α, and ERRα levels, subsequently increasing Cyt c and COX2 expression, in wild-type mice, compared with HO-1{sup +/−} mice. These results suggest that HO-1-derived CO enhances mitochondrial biogenesis in astrocytes by activating L-type Ca{sup 2+} channel-mediated PGC-1α/ERRα axis, leading to maintenance of astrocyte function and neuroprotection/recovery against damage of brain function. - Highlights: • CORM-pretreated astrocytes induces mitochondrial biogenesis by activating L-type Ca{sup 2+} channel-mediated PGC-1α stabilization. • Cerebral ischemia increased electron transport chain proteins (e.g. Cyt c and COX2), in WT mice, compared with HO-1{sup +/−} mice. • CO/HO-1 pathway increases astrocytic mitochondrial functions via a PGC-1α/ERRα axis.« less

Expression of Gls and Gls2 glutaminase isoforms in astrocytes.

PubMed

Cardona, Carolina; Sánchez-Mejías, Elisabeth; Dávila, José C; Martín-Rufián, Mercedes; Campos-Sandoval, José A; Vitorica, Javier; Alonso, Francisco J; Matés, José M; Segura, Juan A; Norenberg, Michael D; Rama Rao, Kakulavarapu V; Jayakumar, Arumugan R; Gutiérrez, Antonia; Márquez, Javier

2015-03-01

The expression of glutaminase in glial cells has been a controversial issue and matter of debate for many years. Actually, glutaminase is essentially considered as a neuronal marker in brain. Astrocytes are endowed with efficient and high capacity transport systems to recapture synaptic glutamate which seems to be consistent with the absence of glutaminase in these glial cells. In this work, a comprehensive study was devised to elucidate expression of glutaminase in neuroglia and, more concretely, in astrocytes. Immunocytochemistry in rat and human brain tissues employing isoform-specific antibodies revealed expression of both Gls and Gls2 glutaminase isozymes in glutamatergic and GABAergic neuronal populations as well as in astrocytes. Nevertheless, there was a different subcellular distribution: Gls isoform was always present in mitochondria while Gls2 appeared in two different locations, mitochondria and nucleus. Confocal microscopy and double immunofluorescence labeling in cultured astrocytes confirmed the same pattern previously seen in brain tissue samples. Astrocytic glutaminase expression was also assessed at the mRNA level, real-time quantitative RT-PCR detected transcripts of four glutaminase isozymes but with marked differences on their absolute copy number: the predominance of Gls isoforms over Gls2 transcripts was remarkable (ratio of 144:1). Finally, we proved that astrocytic glutaminase proteins possess enzymatic activity by in situ activity staining: concrete populations of astrocytes were labeled in the cortex, cerebellum and hippocampus of rat brain demonstrating functional catalytic activity. These results are relevant for the stoichiometry of the Glu/Gln cycle at the tripartite synapse and suggest novel functions for these classical metabolic enzymes. © 2014 Wiley Periodicals, Inc.

Anethole dithiolethione prevents oxidative damage in glutathione-depleted astrocytes.

PubMed

Drukarch, B; Schepens, E; Stoof, J C; Langeveld, C H

1997-06-25

Astrocytes protect neurons against reactive oxygen species such as hydrogen peroxide, a capacity which reportedly is abolished following loss of the antioxidant glutathione. Anethole dithiolethione, a sulfur-containing compound which is used in humans, is known to increase cellular glutathione levels and thought thereby to protect against oxidative damage. In the present study we found that anethole dithiolethione increased the glutathione content of cultured rat striatal astrocytes. This effect was abolished by coincubation with the glutathione synthesis inhibitor buthionine sulfoximine. Nevertheless, in the presence of buthionine sulfoximine, despite the lack of an increase in the lowered glutathione level, anethole dithiolethione fully protected the astrocytes against the enhanced toxicity of hydrogen peroxide. Thus, apparently other mechanisms than stimulation of glutathione synthesis are involved in the compound's protective action in astrocytes. Considering the occurrence of lowered glutathione levels in neurodegenerative syndromes, we conclude that further evaluation of the therapeutic potential of anethole dithiolethione is warranted.

Identification of diverse astrocyte populations and their malignant analogs.

PubMed

John Lin, Chia-Ching; Yu, Kwanha; Hatcher, Asante; Huang, Teng-Wei; Lee, Hyun Kyoung; Carlson, Jeffrey; Weston, Matthew C; Chen, Fengju; Zhang, Yiqun; Zhu, Wenyi; Mohila, Carrie A; Ahmed, Nabil; Patel, Akash J; Arenkiel, Benjamin R; Noebels, Jeffrey L; Creighton, Chad J; Deneen, Benjamin

2017-03-01

Astrocytes are the most abundant cell type in the brain, where they perform a wide array of functions, yet the nature of their cellular heterogeneity and how it oversees these diverse roles remains shrouded in mystery. Using an intersectional fluorescence-activated cell sorting-based strategy, we identified five distinct astrocyte subpopulations present across three brain regions that show extensive molecular diversity. Application of this molecular insight toward function revealed that these populations differentially support synaptogenesis between neurons. We identified correlative populations in mouse and human glioma and found that the emergence of specific subpopulations during tumor progression corresponded with the onset of seizures and tumor invasion. In sum, we have identified subpopulations of astrocytes in the adult brain and their correlates in glioma that are endowed with diverse cellular, molecular and functional properties. These populations selectively contribute to synaptogenesis and tumor pathophysiology, providing a blueprint for understanding diverse astrocyte contributions to neurological disease.

Astrocytic glycogen metabolism in the healthy and diseased brain.

PubMed

Bak, Lasse K; Walls, Anne B; Schousboe, Arne; Waagepetersen, Helle S

2018-05-11

The brain contains a fairly low amount of glycogen, mostly located in astrocytes, a fact that has prompted the suggestion that glycogen does not have a significant physiological role in the brain. However, glycogen metabolism in astrocytes is essential for several key physiological processes and is adversely affected in disease. For instance, diminished ability to break down glycogen impinges on learning, and epilepsy, Alzheimer's disease, and type 2 diabetes are all associated with abnormal astrocyte glycogen metabolism. Glycogen metabolism supports astrocytic K + and neurotransmitter glutamate uptake and subsequent glutamine synthesis-three fundamental steps in excitatory signaling at most brain synapses. Thus, there is abundant evidence for a key role of glycogen in brain function. Here, we summarize the physiological brain functions that depend on glycogen, discuss glycogen metabolism in disease, and investigate how glycogen breakdown is regulated at the cellular and molecular levels. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Layer-specific morphological and molecular differences in neocortical astrocytes and their dependence on neuronal layers.

PubMed

Lanjakornsiripan, Darin; Pior, Baek-Jun; Kawaguchi, Daichi; Furutachi, Shohei; Tahara, Tomoaki; Katsuyama, Yu; Suzuki, Yutaka; Fukazawa, Yugo; Gotoh, Yukiko

2018-04-24

Non-pial neocortical astrocytes have historically been thought to comprise largely a nondiverse population of protoplasmic astrocytes. Here we show that astrocytes of the mouse somatosensory cortex manifest layer-specific morphological and molecular differences. Two- and three-dimensional observations revealed that astrocytes in the different layers possess distinct morphologies as reflected by differences in cell orientation, territorial volume, and arborization. The extent of ensheathment of synaptic clefts by astrocytes in layer II/III was greater than that by those in layer VI. Moreover, differences in gene expression were observed between upper-layer and deep-layer astrocytes. Importantly, layer-specific differences in astrocyte properties were abrogated in reeler and Dab1 conditional knockout mice, in which neuronal layers are disturbed, suggesting that neuronal layers are a prerequisite for the observed morphological and molecular differences of neocortical astrocytes. This study thus demonstrates the existence of layer-specific interactions between neurons and astrocytes, which may underlie their layer-specific functions.

Both neurons and astrocytes exhibited tetrodotoxin-resistant metabotropic glutamate receptor-dependent spontaneous slow Ca2+ oscillations in striatum.

PubMed

Tamura, Atsushi; Yamada, Naohiro; Yaguchi, Yuichi; Machida, Yoshio; Mori, Issei; Osanai, Makoto

2014-01-01

The striatum plays an important role in linking cortical activity to basal ganglia outputs. Group I metabotropic glutamate receptors (mGluRs) are densely expressed in the medium spiny projection neurons and may be a therapeutic target for Parkinson's disease. The group I mGluRs are known to modulate the intracellular Ca(2+) signaling. To characterize Ca(2+) signaling in striatal cells, spontaneous cytoplasmic Ca(2+) transients were examined in acute slice preparations from transgenic mice expressing green fluorescent protein (GFP) in the astrocytes. In both the GFP-negative cells (putative-neurons) and astrocytes of the striatum, spontaneous slow and long-lasting intracellular Ca(2+) transients (referred to as slow Ca(2+) oscillations), which lasted up to approximately 200 s, were found. Neither the inhibition of action potentials nor ionotropic glutamate receptors blocked the slow Ca(2+) oscillation. Depletion of the intracellular Ca(2+) store and the blockade of inositol 1,4,5-trisphosphate receptors greatly reduced the transient rate of the slow Ca(2+) oscillation, and the application of an antagonist against mGluR5 also blocked the slow Ca(2+) oscillation in both putative-neurons and astrocytes. Thus, the mGluR5-inositol 1,4,5-trisphosphate signal cascade is the primary contributor to the slow Ca(2+) oscillation in both putative-neurons and astrocytes. The slow Ca(2+) oscillation features multicellular synchrony, and both putative-neurons and astrocytes participate in the synchronous activity. Therefore, the mGluR5-dependent slow Ca(2+) oscillation may involve in the neuron-glia interaction in the striatum.

Phagocytic clearance of presynaptic dystrophies by reactive astrocytes in Alzheimer's disease

PubMed Central

Gomez‐Arboledas, Angela; Davila, Jose C.; Sanchez‐Mejias, Elisabeth; Navarro, Victoria; Nuñez‐Diaz, Cristina; Sanchez‐Varo, Raquel; Sanchez‐Mico, Maria Virtudes; Trujillo‐Estrada, Laura; Fernandez‐Valenzuela, Juan Jose; Vizuete, Marisa; Comella, Joan X.; Galea, Elena

2017-01-01

Abstract Reactive astrogliosis, a complex process characterized by cell hypertrophy and upregulation of components of intermediate filaments, is a common feature in brains of Alzheimer's patients. Reactive astrocytes are found in close association with neuritic plaques; however, the precise role of these glial cells in disease pathogenesis is unknown. In this study, using immunohistochemical techniques and light and electron microscopy, we report that plaque‐associated reactive astrocytes enwrap, engulf and may digest presynaptic dystrophies in the hippocampus of amyloid precursor protein/presenilin‐1 (APP/PS1) mice. Microglia, the brain phagocytic population, was apparently not engaged in this clearance. Phagocytic reactive astrocytes were present in 35% and 67% of amyloid plaques at 6 and 12 months of age, respectively. The proportion of engulfed dystrophic neurites was low, around 7% of total dystrophies around plaques at both ages. This fact, along with the accumulation of dystrophic neurites during disease course, suggests that the efficiency of the astrocyte phagocytic process might be limited or impaired. Reactive astrocytes surrounding and engulfing dystrophic neurites were also detected in the hippocampus of Alzheimer's patients by confocal and ultrastructural analysis. We posit that the phagocytic activity of reactive astrocytes might contribute to clear dysfunctional synapses or synaptic debris, thereby restoring impaired neural circuits and reducing the inflammatory impact of damaged neuronal parts and/or limiting the amyloid pathology. Therefore, potentiation of the phagocytic properties of reactive astrocytes may represent a potential therapy in Alzheimer's disease. PMID:29178139

Astrocytes contribute to gamma oscillations and recognition memory.

PubMed

Lee, Hosuk Sean; Ghetti, Andrea; Pinto-Duarte, António; Wang, Xin; Dziewczapolski, Gustavo; Galimi, Francesco; Huitron-Resendiz, Salvador; Piña-Crespo, Juan C; Roberts, Amanda J; Verma, Inder M; Sejnowski, Terrence J; Heinemann, Stephen F

2014-08-12

Glial cells are an integral part of functional communication in the brain. Here we show that astrocytes contribute to the fast dynamics of neural circuits that underlie normal cognitive behaviors. In particular, we found that the selective expression of tetanus neurotoxin (TeNT) in astrocytes significantly reduced the duration of carbachol-induced gamma oscillations in hippocampal slices. These data prompted us to develop a novel transgenic mouse model, specifically with inducible tetanus toxin expression in astrocytes. In this in vivo model, we found evidence of a marked decrease in electroencephalographic (EEG) power in the gamma frequency range in awake-behaving mice, whereas neuronal synaptic activity remained intact. The reduction in cortical gamma oscillations was accompanied by impaired behavioral performance in the novel object recognition test, whereas other forms of memory, including working memory and fear conditioning, remained unchanged. These results support a key role for gamma oscillations in recognition memory. Both EEG alterations and behavioral deficits in novel object recognition were reversed by suppression of tetanus toxin expression. These data reveal an unexpected role for astrocytes as essential contributors to information processing and cognitive behavior.

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

PubMed

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

2007-01-01

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

Functional Characterization of Resting and Adenovirus-Induced Reactive Astrocytes in Three-Dimensional Culture

PubMed Central

Woo, Junsung; Im, Sun-Kyoung; Chun, Heejung; Jung, Soon-Young; Oh, Soo-Jin; Choi, Nakwon

2017-01-01

Brain is a rich environment where neurons and glia interact with neighboring cells as well as extracellular matrix in three-dimensional (3D) space. Astrocytes, which are the most abundant cells in the mammalian brain, reside in 3D space and extend highly branched processes that form microdomains and contact synapses. It has been suggested that astrocytes cultured in 3D might be maintained in a less reactive state as compared to those growing in a traditional, two-dimensional (2D) monolayer culture. However, the functional characterization of the astrocytes in 3D culture has been lacking. Here we cocultured neurons and astrocytes in 3D and examined the morphological, molecular biological, and electrophysiological properties of the 3D-cultured hippocampal astrocytes. In our 3D neuron-astrocyte coculture, astrocytes showed a typical morphology of a small soma with many branches and exhibited a unique membrane property of passive conductance, more closely resembling their native in vivo counterparts. Moreover, we also induced reactive astrocytosis in culture by infecting with high-titer adenovirus to mimic pathophysiological conditions in vivo. Adenoviral infection induced morphological changes in astrocytes, increased passive conductance, and increased GABA content as well as tonic GABA release, which are characteristics of reactive gliosis. Together, our study presents a powerful in vitro model resembling both physiological and pathophysiological conditions in vivo, and thereby provides a versatile experimental tool for studying various neurological diseases that accompany reactive astrocytes. PMID:28680301

Ca2+ Entry is Required for Mechanical Stimulation-induced ATP Release from Astrocyte

PubMed Central

Lee, Jaekwang; Chun, Ye-Eun; Han, Kyung-Seok; Lee, Jungmoo; Woo, Dong Ho

2015-01-01

Astrocytes and neurons are inseparable partners in the brain. Neurotransmitters released from neurons activate corresponding G protein-coupled receptors (GPCR) expressed in astrocytes, resulting in release of gliotransmitters such as glutamate, D-serine, and ATP. These gliotransmitters in turn influence neuronal excitability and synaptic activities. Among these gliotransmitters, ATP regulates the level of network excitability and is critically involved in sleep homeostasis and astrocytic Ca2+ oscillations. ATP is known to be released from astrocytes by Ca2+-dependent manner. However, the precise source of Ca2+, whether it is Ca2+ entry from outside of cell or from the intracellular store, is still not clear yet. Here, we performed sniffer patch to detect ATP release from astrocyte by using various stimulation. We found that ATP was not released from astrocyte when Ca2+ was released from intracellular stores by activation of Gαq-coupled GPCR including PAR1, P2YR, and B2R. More importantly, mechanical stimulation (MS)-induced ATP release from astrocyte was eliminated when external Ca2+ was omitted. Our results suggest that Ca2+ entry, but not release from intracellular Ca2+ store, is critical for MS-induced ATP release from astrocyte. PMID:25792866

Astrocyte dysfunction in temporal lobe epilepsy: K+ channels and gap junction coupling.

PubMed

Steinhäuser, Christian; Seifert, Gerald; Bedner, Peter

2012-08-01

Astrocytes are endowed with the machinery to sense and respond to neuronal activity. Recent work has demonstrated that astrocytes play important physiological roles in the CNS, e.g., they synchronize action potential firing, ensure ion homeostasis, transmitter clearance and glucose metabolism, and regulate the vascular tone. Astrocytes are abundantly coupled through gap junctions, which is a prerequisite to redistribute elevated K(+) from sites of excessive neuronal activity to sites of lower extracellular K(+) concentration. Recent studies identified dysfunctional astrocytes as crucial players in epilepsy. Investigation of specimens from patients with pharmacoresistant temporal lobe epilepsy and epilepsy models revealed alterations in expression, localization, and function of astroglial inwardly rectifying K(+) (Kir) channels, particularly Kir4.1, which is suspected to entail impaired K(+) buffering. Gap junctions in astrocytes appear to play a dual role: on the one hand they counteract the generation of hyperactivity by facilitating clearance of elevated extracellular K(+) levels while in contrast, they constitute a pathway for energetic substrate delivery to fuel neuronal (hyper)activity. Recent work suggests that astrocyte dysfunction is causative of the generation or spread of seizure activity. Thus, astrocytes should be considered as promising targets for alternative antiepileptic therapies. Copyright © 2012 Wiley Periodicals, Inc.

Dephosphorylation of 2-deoxyglucose 6-phosphate and 2-deoxyglucose export from cultured astrocytes.

PubMed

Forsyth, R J; Bartlett, K; Eyre, J

1996-03-01

Neurotransmitter-stimulated mobilization of astrocyte glycogen has been proposed as a basis for local energy homeostasis in brain. However, uncertainty remains over the fate of astrocyte glycogen. Upon transfer of cultured astrocytes pre-loaded with [2-3H]2-deoxyglucose 6-phosphate at non-tracer concentrations to a glucose-free, 2-deoxyglucose-free medium, rapid dephosphorylation of a proportion of the intracellular 2-deoxyglucose 6-phosphate pool and export of 2-deoxyglucose to the extracellular fluid occurs. Astrocytes show very low, basal rates of gluconeogenesis from pyruvate (approx. 1 nmol mg protein-1 h-1). Astrocytes in vivo may be capable of physiologically significant glucose export from glucose-6-phosphate. The low gluconeogenic activity in astrocytes suggests that the most likely source of glucose-6-phosphate may be glycogen. These findings support the hypothesis that export, as glucose, to adjacent neurons may be one of the possible fate(s) of astrocytic glycogen. Such export of glycogen as glucose occurring in response to increases in neuronal activity could contribute to energy homeostasis on a paracrine scale within brain.

Intracellular water preexchange lifetime in neurons and astrocytes.

PubMed

Yang, Donghan M; Huettner, James E; Bretthorst, G Larry; Neil, Jeffrey J; Garbow, Joel R; Ackerman, Joseph J H

2018-03-01

To determine the intracellular water preexchange lifetime, τ i , the "average residence time" of water, in the intracellular milieu of neurons and astrocytes. The preexchange lifetime is important for modeling a variety of MR data sets, including relaxation, diffusion-sensitive, and dynamic contrast-enhanced data sets. Herein, τ i in neurons and astrocytes is determined in a microbead-adherent, cultured cell system. In concert with thin-slice selection, rapid flow of extracellular media suppresses extracellular signal, allowing determination of the transcytolemmal-exchange-dominated, intracellular T 1 . With this knowledge, and that of the intracellular T 1 in the absence of exchange, τ i can be derived. Under normal culture conditions, τ i for neurons is 0.75 ± 0.05 s versus 0.57 ± 0.03 s for astrocytes. Both neuronal and astrocytic τ i s decrease within 30 min after the onset of oxygen-glucose deprivation, with the astrocytic τ i showing a substantially greater decrease than the neuronal τ i . Given an approximate intra- to extracellular volume ratio of 4:1 in the brain, these data imply that, under normal physiological conditions, an MR experimental characteristic time of less than 0.012 s is required for a nonexchanging, two-compartment (intra- and extracellular) model to be valid for MR studies. This characteristic time shortens significantly (i.e., 0.004 s) under injury conditions. Magn Reson Med 79:1616-1627, 2018. © 2017 International Society for Magnetic Resonance in Medicine. © 2017 International Society for Magnetic Resonance in Medicine.

Distinct functional states of astrocytes during sleep and wakefulness: Is norepinephrine the master regulator?

PubMed Central

O’Donnell, John; Ding, Fengfei; Nedergaard, Maiken

2015-01-01

Astrocytes are the chief supportive cells in the central nervous system, but work over the past 20 years have documented that astrocytes also contribute to complex neural processes, such as working memory. Recent discoveries of norepinephrine-mediated astrocytic Ca2+ responses have raised the possibility that astrocytic activity in the adult brain is driven by global responses to changes in behavioral state. Moreover, analysis of the interstitial space volume suggests that astrocytes may undergo changes in cell volume in response to activation of norepinephrine receptors. This review will focus on what is known about astrocytic functions within the nervous system, and how these functions interrelate with rapid changes in behavioral state mediated by norepinephrine signaling. PMID:26618103

Fingolimod induces neuroprotective factors in human astrocytes.

PubMed

Hoffmann, Franziska S; Hofereiter, Johann; Rübsamen, Heike; Melms, Johannes; Schwarz, Sigrid; Faber, Hans; Weber, Peter; Pütz, Benno; Loleit, Verena; Weber, Frank; Hohlfeld, Reinhard; Meinl, Edgar; Krumbholz, Markus

2015-09-30

Fingolimod (FTY720) is the first sphingosine-1-phosphate (S1P) receptor modulator approved for the treatment of multiple sclerosis. The phosphorylated active metabolite FTY720-phosphate (FTY-P) interferes with lymphocyte trafficking. In addition, it accumulates in the CNS and reduces brain atrophy in multiple sclerosis (MS), and neuroprotective effects are hypothesized. Human primary astrocytes as well as human astrocytoma cells were stimulated with FTY-P or S1P. We analyzed gene expression by a genome-wide microarray and validated induced candidate genes by quantitative PCR (qPCR) and ELISA. To identify the S1P-receptor subtypes involved, we applied a membrane-impermeable S1P analog (dihydro-S1P), receptor subtype specific agonists and antagonists, as well as RNAi silencing. FTY-P induced leukemia inhibitory factor (LIF), interleukin 11 (IL11), and heparin-binding EGF-like growth factor (HBEGF) mRNA, as well as secretion of LIF and IL11 protein. In order to mimic an inflammatory milieu as observed in active MS lesions, we combined FTY-P application with tumor necrosis factor (TNF). In the presence of this key inflammatory cytokine, FTY-P synergistically induced LIF, HBEGF, and IL11 mRNA, as well as secretion of LIF and IL11 protein. TNF itself induced inflammatory, B-cell promoting, and antiviral factors (CXCL10, BAFF, MX1, and OAS2). Their induction was blocked by FTY-P. After continuous exposure of cells to FTY-P or S1P for up to 7 days, the extent of induction of neurotrophic factors and the suppression of TNF-induced inflammatory genes declined but was still detectable. The induction of neurotrophic factors was mediated via surface S1P receptors 1 (S1PR1) and 3 (S1PR3). We identified effects of FTY-P on astrocytes, namely induction of neurotrophic mediators (LIF, HBEGF, and IL11) and inhibition of TNF-induced inflammatory genes (CXCL10, BAFF, MX1, and OAS2). This supports the view that a part of the effects of fingolimod may be mediated via astrocytes.

Hippocampal Astrocytes in Migrating and Wintering Semipalmated Sandpiper Calidris pusilla.

PubMed

Carvalho-Paulo, Dario; de Morais Magalhães, Nara G; de Almeida Miranda, Diego; Diniz, Daniel G; Henrique, Ediely P; Moraes, Isis A M; Pereira, Patrick D C; de Melo, Mauro A D; de Lima, Camila M; de Oliveira, Marcus A; Guerreiro-Diniz, Cristovam; Sherry, David F; Diniz, Cristovam W P

2017-01-01

Seasonal migratory birds return to the same breeding and wintering grounds year after year, and migratory long-distance shorebirds are good examples of this. These tasks require learning and long-term spatial memory abilities that are integrated into a navigational system for repeatedly locating breeding, wintering, and stopover sites. Previous investigations focused on the neurobiological basis of hippocampal plasticity and numerical estimates of hippocampal neurogenesis in birds but only a few studies investigated potential contributions of glial cells to hippocampal-dependent tasks related to migration. Here we hypothesized that the astrocytes of migrating and wintering birds may exhibit significant morphological and numerical differences connected to the long-distance flight. We used as a model the semipalmated sandpiper Calidris pusilla , that migrates from northern Canada and Alaska to South America. Before the transatlantic non-stop long-distance component of their flight, the birds make a stopover at the Bay of Fundy in Canada. To test our hypothesis, we estimated total numbers and compared the three-dimensional (3-D) morphological features of adult C. pusilla astrocytes captured in the Bay of Fundy ( n = 249 cells) with those from birds captured in the coastal region of Bragança, Brazil, during the wintering period ( n = 250 cells). Optical fractionator was used to estimate the number of astrocytes and for 3-D reconstructions we used hierarchical cluster analysis. Both morphological phenotypes showed reduced morphological complexity after the long-distance non-stop flight, but the reduction in complexity was much greater in Type I than in Type II astrocytes. Coherently, we also found a significant reduction in the total number of astrocytes after the transatlantic flight. Taken together these findings suggest that the long-distance non-stop flight altered significantly the astrocytes population and that morphologically distinct astrocytes may play

Hippocampal Astrocytes in Migrating and Wintering Semipalmated Sandpiper Calidris pusilla

PubMed Central

Carvalho-Paulo, Dario; de Morais Magalhães, Nara G.; de Almeida Miranda, Diego; Diniz, Daniel G.; Henrique, Ediely P.; Moraes, Isis A. M.; Pereira, Patrick D. C.; de Melo, Mauro A. D.; de Lima, Camila M.; de Oliveira, Marcus A.; Guerreiro-Diniz, Cristovam; Sherry, David F.; Diniz, Cristovam W. P.

2018-01-01

Seasonal migratory birds return to the same breeding and wintering grounds year after year, and migratory long-distance shorebirds are good examples of this. These tasks require learning and long-term spatial memory abilities that are integrated into a navigational system for repeatedly locating breeding, wintering, and stopover sites. Previous investigations focused on the neurobiological basis of hippocampal plasticity and numerical estimates of hippocampal neurogenesis in birds but only a few studies investigated potential contributions of glial cells to hippocampal-dependent tasks related to migration. Here we hypothesized that the astrocytes of migrating and wintering birds may exhibit significant morphological and numerical differences connected to the long-distance flight. We used as a model the semipalmated sandpiper Calidris pusilla, that migrates from northern Canada and Alaska to South America. Before the transatlantic non-stop long-distance component of their flight, the birds make a stopover at the Bay of Fundy in Canada. To test our hypothesis, we estimated total numbers and compared the three-dimensional (3-D) morphological features of adult C. pusilla astrocytes captured in the Bay of Fundy (n = 249 cells) with those from birds captured in the coastal region of Bragança, Brazil, during the wintering period (n = 250 cells). Optical fractionator was used to estimate the number of astrocytes and for 3-D reconstructions we used hierarchical cluster analysis. Both morphological phenotypes showed reduced morphological complexity after the long-distance non-stop flight, but the reduction in complexity was much greater in Type I than in Type II astrocytes. Coherently, we also found a significant reduction in the total number of astrocytes after the transatlantic flight. Taken together these findings suggest that the long-distance non-stop flight altered significantly the astrocytes population and that morphologically distinct astrocytes may play different

Deletion of Monoglyceride Lipase in Astrocytes Attenuates Lipopolysaccharide-induced Neuroinflammation*

PubMed Central

Grabner, Gernot F.; Eichmann, Thomas O.; Wagner, Bernhard; Gao, Yuanqing; Farzi, Aitak; Taschler, Ulrike; Radner, Franz P. W.; Schweiger, Martina; Lass, Achim; Holzer, Peter; Zinser, Erwin; Tschöp, Matthias H.; Yi, Chun-Xia; Zimmermann, Robert

2016-01-01

Monoglyceride lipase (MGL) is required for efficient hydrolysis of the endocannabinoid 2-arachidonoylglyerol (2-AG) in the brain generating arachidonic acid (AA) and glycerol. This metabolic function makes MGL an interesting target for the treatment of neuroinflammation, since 2-AG exhibits anti-inflammatory properties and AA is a precursor for pro-inflammatory prostaglandins. Astrocytes are an important source of AA and 2-AG, and highly express MGL. In the present study, we dissected the distinct contribution of MGL in astrocytes on brain 2-AG and AA metabolism by generating a mouse model with genetic deletion of MGL specifically in astrocytes (MKOGFAP). MKOGFAP mice exhibit moderately increased 2-AG and reduced AA levels in brain. Minor accumulation of 2-AG in the brain of MKOGFAP mice does not cause cannabinoid receptor desensitization as previously observed in mice globally lacking MGL. Importantly, MKOGFAP mice exhibit reduced brain prostaglandin E2 and pro-inflammatory cytokine levels upon peripheral lipopolysaccharide (LPS) administration. These observations indicate that MGL-mediated degradation of 2-AG in astrocytes provides AA for prostaglandin synthesis promoting LPS-induced neuroinflammation. The beneficial effect of astrocyte-specific MGL-deficiency is not fully abrogated by the inverse cannabinoid receptor 1 agonist SR141716 (Rimonabant) suggesting that the anti-inflammatory effects are rather caused by reduced prostaglandin synthesis than by activation of cannabinoid receptors. In conclusion, our data demonstrate that MGL in astrocytes is an important regulator of 2-AG levels, AA availability, and neuroinflammation. PMID:26565024

Acidosis-Induced Dysfunction of Cortical GABAergic Neurons through Astrocyte-Related Excitotoxicity

PubMed Central

Guan, Sudong; Zhu, Yan; Wang, Jin-Hui

2015-01-01

Background Acidosis impairs cognitions and behaviors presumably by acidification-induced changes in neuronal metabolism. Cortical GABAergic neurons are vulnerable to pathological factors and their injury leads to brain dysfunction. How acidosis induces GABAergic neuron injury remains elusive. As the glia cells and neurons interact each other, we intend to examine the role of the astrocytes in acidosis-induced GABAergic neuron injury. Results Experiments were done at GABAergic cells and astrocytes in mouse cortical slices. To identify astrocytic involvement in acidosis-induced impairment, we induced the acidification in single GABAergic neuron by infusing proton intracellularly or in both neurons and astrocytes by using proton extracellularly. Compared the effects of intracellular acidification and extracellular acidification on GABAergic neurons, we found that their active intrinsic properties and synaptic outputs appeared more severely impaired in extracellular acidosis than intracellular acidosis. Meanwhile, extracellular acidosis deteriorated glutamate transporter currents on the astrocytes and upregulated excitatory synaptic transmission on the GABAergic neurons. Moreover, the antagonists of glutamate NMDA-/AMPA-receptors partially reverse extracellular acidosis-induced injury in the GABAergic neurons. Conclusion Our studies suggest that acidosis leads to the dysfunction of cortical GABAergic neurons by astrocyte-mediated excitotoxicity, in addition to their metabolic changes as indicated previously. PMID:26474076

Stochastic nanoroughness modulates neuron-astrocyte interactions and function via mechanosensing cation channels.

PubMed

Blumenthal, Nils R; Hermanson, Ola; Heimrich, Bernd; Shastri, V Prasad

2014-11-11

Extracellular soluble signals are known to play a critical role in maintaining neuronal function and homeostasis in the CNS. However, the CNS is also composed of extracellular matrix macromolecules and glia support cells, and the contribution of the physical attributes of these components in maintenance and regulation of neuronal function is not well understood. Because these components possess well-defined topography, we theorize a role for topography in neuronal development and we demonstrate that survival and function of hippocampal neurons and differentiation of telencephalic neural stem cells is modulated by nanoroughness. At roughnesses corresponding to that of healthy astrocytes, hippocampal neurons dissociated and survived independent from astrocytes and showed superior functional traits (increased polarity and calcium flux). Furthermore, telencephalic neural stem cells differentiated into neurons even under exogenous signals that favor astrocytic differentiation. The decoupling of neurons from astrocytes seemed to be triggered by changes to astrocyte apical-surface topography in response to nanoroughness. Blocking signaling through mechanosensing cation channels using GsMTx4 negated the ability of neurons to sense the nanoroughness and promoted decoupling of neurons from astrocytes, thus providing direct evidence for the role of nanotopography in neuron-astrocyte interactions. We extrapolate the role of topography to neurodegenerative conditions and show that regions of amyloid plaque buildup in brain tissue of Alzheimer's patients are accompanied by detrimental changes in tissue roughness. These findings suggest a role for astrocyte and ECM-induced topographical changes in neuronal pathologies and provide new insights for developing therapeutic targets and engineering of neural biomaterials.

Human astrocytic grid networks patterned in parylene-C inlayed SiO2 trenches.

PubMed

Jordan, M D; Raos, B J; Bunting, A S; Murray, A F; Graham, E S; Unsworth, C P

2016-10-01

Recent literature suggests that glia, and in particular astrocytes, should be studied as organised networks which communicate through gap junctions. Astrocytes, however, adhere to most surfaces and are highly mobile cells. In order to study, such organised networks effectively in vitro it is necessary to influence them to pattern to certain substrates whilst being repelled from others and to immobilise the astrocytes sufficiently such that they do not continue to migrate further whilst under study. In this article, we demonstrate for the first time how it is possible to facilitate the study of organised patterned human astrocytic networks using hNT astrocytes in a SiO2 trench grid network that is inlayed with the biocompatible material, parylene-C. We demonstrate how the immobilisation of astrocytes lies in the depth of the SiO2 trench, determining an optimum trench depth and that the optimum patterning of astrocytes is a consequence of the parylene-C inlay and the grid node spacing. We demonstrate high fidelity of the astrocytic networks and demonstrate that functionality of the hNT astrocytes through ATP evoked calcium signalling is also dependent on the grid node spacing. Finally, we demonstrate that the location of the nuclei on the grid nodes is also a function of the grid node spacing. The significance of this work, is to describe a suitable platform to facilitate the study of hNT astrocytes from the single cell level to the network level to improve knowledge and understanding of how communication links to spatial organisation at these higher order scales and trigger in vitro research further in this area with clinical applications in the area of epilepsy, stroke and focal cerebral ischemia. Copyright © 2016 Elsevier Ltd. All rights reserved.

IL-1β and IL-6 activate inflammatory responses of astrocytes against Naegleria fowleri infection via the modulation of MAPKs and AP-1.

PubMed

Kim, J-H; Song, A-R; Sohn, H-J; Lee, J; Yoo, J-K; Kwon, D; Shin, H-J

2013-01-01

Naegleria fowleri, a free-living amoeba, has been found in diverse habitats throughout the world. It causes primary amoebic meningoencephalitis in children and young adults. The amoeba attaches to nasal mucosa, migrates along olfactory nerves and enters the brain. Astrocytes are involved in the defence against infection and produce inflammatory responses. In this study, we focus on the mechanism of immune responses in astrocytes. We showed, using RNase protection assay, RT-PCR and ELISA in an in vitro culture system, that N. fowleri lysates induce interleukin-1beta (IL-1β) and IL-6 expression of astrocytes. In addition, cytokine levels of astrocytes gradually decreased due to extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 inhibitors. To determine the transcription factor, we used transcription inhibitor (AP-1 inhibitor), which downregulated IL-1β and IL-6 expression. These results show that AP-1 is related to IL-1β and IL-6 production. N. fowleri-mediated IL-1β and IL-6 expression requires ERK, JNK and p38 mitogen-activated protein kinases (MAPKs) activation in astrocytes. These findings show that N. fowleri-stimulated astrocytes in an in vitro culture system lead to AP-1 activation and the subsequent expressions of IL-1β and IL-6, which are dependent on ERK, JNK and p38 MAPKs activation. These results may imply that proinflammatory cytokines have important roles in inflammatory responses to N. fowleri infection. © 2012 Blackwell Publishing Ltd.

Might astrocytes play a role in maintaining the seizure-prone state?

PubMed

Vessal, Mani; Dugani, Chandrasagar B; Solomon, Dianand A; McIntyre Burnham, W; Ivy, Gwen O

2005-05-24

The amygdala-kindling model is used to study complex partial epilepsy with secondary generalization. The present study was designed to (A) quantify astrocytic changes in the piriform cortex of amygdala-kindled subjects over time and (B) investigate the role that astrocytes might play in maintaining the seizure-prone state. In Study A, once the experimental subjects reached five stage 5 seizures, stimulation was stopped, and both kindled and control rats were allowed to survive for the interval appropriate to their group (7, 18, 30, or 90 days). Following each interval, the kindled and control animals were given 10 intraperitoneal injections of bromodeoxyuridine (BrdU) and sacrificed 24 h following the last injection. Significantly higher numbers of dividing astrocytes (identified by co-labeling for BrdU and to one of the astrocytic intermediate filament proteins glial fibrillary acidic protein or vimentin) were found in the kindled brains. All kindled groups had significantly higher numbers of double-labeled cells on the side contralateral to the stimulation site, except for those in the 90 day survival group. In Study B, rats were implanted with chemotrodes, were kindled as in Study A, and were subsequently infused with either saline or with L alpha-AA (to lesion astrocytes) during a further 25 stimulations (1/day). L alpha-AA infused rats had significantly diminished levels of behavioral seizures, higher after discharge thresholds, lower after discharge durations, and decreased numbers of double-labeled astrocytes in piriform cortex than did saline infused rats. Together, the data indicate that astrocytes may play a role in maintaining the seizure-prone state.

The Astrocyte: Powerhouse and Recycling Center

PubMed Central

Weber, Bruno; Barros, L. Felipe

2015-01-01

Brain metabolism is characterized by fuel monodependence, high-energy expenditure, autonomy from the rest of body, local recycling, and marked division of labor between cell types. Although neurons spend most of the brain’s energy on signaling, astrocytes bear the brunt of the metabolic load, controlling the composition of the interstitial fluid, supplying neurons with energy substrates and precursors for biosynthesis, and recycling neurotransmitters, oxidized scavengers, and other waste products. Outstanding questions in this field are the role of oligodendrocytes, the metabolic behavior of the different subtypes of astrocytes during development and disease, and the emerging notion that metabolism may participate directly in information processing. PMID:25680832

A cellular star atlas: using astrocytes from human pluripotent stem cells for disease studies

PubMed Central

Krencik, Robert; Ullian, Erik M.

2013-01-01

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. PMID:23503583

HIV-1 Nef Induces CCL5 production in astrocytes through p38-MAPK and PI3K/Akt pathway and utilizes NF-kB, CEBP and AP-1 transcription factors

NASA Astrophysics Data System (ADS)

Liu, Xun; Shah, Ankit; Gangwani, Mohitkumar R.; Silverstein, Peter S.; Fu, Mingui; Kumar, Anil

2014-03-01

The prevalence of HIV-associated neurocognitive disorders (HAND) remains high in patients infected with HIV-1. The production of pro-inflammatory cytokines by astrocytes/microglia exposed to viral proteins is thought to be one of the mechanisms leading to HIV-1- mediated neurotoxicity. In the present study we examined the effects of Nef on CCL5 induction in astrocytes. The results demonstrate that CCL5 is significantly induced in Nef-transfected SVGA astrocytes. To determine the mechanisms responsible for the increased CCL5 caused by Nef, we employed siRNA and chemical antagonists. Antagonists of NF-κB, PI3K, and p38 significantly reduced the expression levels of CCL5 induced by Nef transfection. Furthermore, specific siRNAs demonstrated that the Akt, p38MAPK, NF-κB, CEBP, and AP-1 pathways play a role in Nef-mediated CCL5 expression. The results demonstrated that the PI3K/Akt and p38 MAPK pathways, along with the transcription factors NF-κB, CEBP, and AP-1, are involved in Nef-induced CCL5 production in astrocytes.

Clinical multiplexed exome sequencing distinguishes adult oligodendroglial neoplasms from astrocytic and mixed lineage gliomas.

PubMed

Cryan, Jane B; Haidar, Sam; Ramkissoon, Lori A; Bi, Wenya Linda; Knoff, David S; Schultz, Nikolaus; Abedalthagafi, Malak; Brown, Loreal; Wen, Patrick Y; Reardon, David A; Dunn, Ian F; Folkerth, Rebecca D; Santagata, Sandro; Lindeman, Neal I; Ligon, Azra H; Beroukhim, Rameen; Hornick, Jason L; Alexander, Brian M; Ligon, Keith L; Ramkissoon, Shakti H

2014-09-30

Classifying adult gliomas remains largely a histologic diagnosis based on morphology; however astrocytic, oligodendroglial and mixed lineage tumors can display overlapping histologic features. We used multiplexed exome sequencing (OncoPanel) on 108 primary or recurrent adult gliomas, comprising 65 oligodendrogliomas, 28 astrocytomas and 15 mixed oligoastrocytomas to identify lesions that could enhance lineage classification. Mutations in TP53 (20/28, 71%) and ATRX (15/28, 54%) were enriched in astrocytic tumors compared to oligodendroglial tumors of which 4/65 (6%) had mutations in TP53 and 2/65 (3%) had ATRX mutations. We found that oligoastrocytomas harbored mutations in TP53 (80%, 12/15) and ATRX (60%, 9/15) at frequencies similar to pure astrocytic tumors, suggesting that oligoastrocytomas and astrocytomas may represent a single genetic or biological entity. p53 protein expression correlated with mutation status and showed significant increases in astrocytomas and oligoastrocytomas compared to oligodendrogliomas, a finding that also may facilitate accurate classification. Furthermore our OncoPanel analysis revealed that 15% of IDH1/2 mutant gliomas would not be detected by traditional IDH1 (p.R132H) antibody testing, supporting the use of genomic technologies in providing clinically relevant data. In all, our results demonstrate that multiplexed exome sequencing can support evaluation and classification of adult low-grade gliomas with a single clinical test.

Astrocytes influence the severity of spinal muscular atrophy

PubMed Central

Rindt, Hansjörg; Feng, Zhihua; Mazzasette, Chiara; Glascock, Jacqueline J.; Valdivia, David; Pyles, Noah; Crawford, Thomas O.; Swoboda, Kathryn J.; Patitucci, Teresa N.; Ebert, Allison D.; Sumner, Charlotte J.; Ko, Chien-Ping; Lorson, Christian L.

2015-01-01

Systemically low levels of survival motor neuron-1 (SMN1) protein cause spinal muscular atrophy (SMA). α-Motor neurons of the spinal cord are considered particularly vulnerable in this genetic disorder and their dysfunction and loss cause progressive muscle weakness, paralysis and eventually premature death of afflicted individuals. Historically, SMA was therefore considered a motor neuron-autonomous disease. However, depletion of SMN in motor neurons of normal mice elicited only a very mild phenotype. Conversely, restoration of SMN to motor neurons in an SMA mouse model had only modest effects on the SMA phenotype and survival. Collectively, these results suggested that additional cell types contribute to the pathogenesis of SMA, and understanding the non-autonomous requirements is crucial for developing effective therapies. Astrocytes are critical for regulating synapse formation and function as well as metabolic support for neurons. We hypothesized that astrocyte functions are disrupted in SMA, exacerbating disease progression. Using viral-based restoration of SMN specifically to astrocytes, survival in severe and intermediate SMA mice was observed. In addition, neuromuscular circuitry was improved. Astrogliosis was prominent in end-stage SMA mice and in post-mortem patient spinal cords. Increased expression of proinflammatory cytokines was partially normalized in treated mice, suggesting that astrocytes contribute to the pathogenesis of SMA. PMID:25911676

Glucose regulates hypothalamic long-chain fatty acid metabolism via AMP-activated kinase (AMPK) in neurons and astrocytes.

PubMed

Taïb, Bouchra; Bouyakdan, Khalil; Hryhorczuk, Cécile; Rodaros, Demetra; Fulton, Stephanie; Alquier, Thierry

2013-12-27

Hypothalamic controls of energy balance rely on the detection of circulating nutrients such as glucose and long-chain fatty acids (LCFA) by the mediobasal hypothalamus (MBH). LCFA metabolism in the MBH plays a key role in the control of food intake and glucose homeostasis, yet it is not known if glucose regulates LCFA oxidation and esterification in the MBH and, if so, which hypothalamic cell type(s) and intracellular signaling mechanisms are involved. The aim of this study was to determine the impact of glucose on LCFA metabolism, assess the role of AMP-activated Kinase (AMPK), and to establish if changes in LCFA metabolism and its regulation by glucose vary as a function of the kind of LCFA, cell type, and brain region. We show that glucose inhibits palmitate oxidation via AMPK in hypothalamic neuronal cell lines, primary hypothalamic astrocyte cultures, and MBH slices ex vivo but not in cortical astrocytes and slice preparations. In contrast, oleate oxidation was not affected by glucose or AMPK inhibition in MBH slices. In addition, our results show that glucose increases palmitate, but not oleate, esterification into neutral lipids in neurons and MBH slices but not in hypothalamic astrocytes. These findings reveal for the first time the metabolic fate of different LCFA in the MBH, demonstrate AMPK-dependent glucose regulation of LCFA oxidation in both astrocytes and neurons, and establish metabolic coupling of glucose and LCFA as a distinguishing feature of hypothalamic nuclei critical for the control of energy balance.

Endogenous IL-6 of mesenchymal stem cell improves behavioral outcome of hypoxic-ischemic brain damage neonatal rats by supressing apoptosis in astrocyte

PubMed Central

Gu, Yan; He, Mulan; Zhou, Xiaoqin; Liu, Jinngjing; Hou, Nali; Bin, Tan; Zhang, Yun; Li, Tingyu; Chen, Jie

2016-01-01

Mesenchymal stem cell (MSC) transplantation reduces the neurological impairment caused by hypoxic-ischemic brain damage (HIBD) via immunomodulation. In the current study, we found that MSC transplantation improved learning and memory function and enhanced long-term potentiation in neonatal rats subjected to HIBD and the amount of IL-6 released from MSCs was far greater than that of other cytokines. However, the neuroprotective effect of MSCs infected with siIL-6-transduced recombinant lentivirus (siIL-6 MSCs) was significantly weakened in the behavioural tests and electrophysiological analysis. Meanwhile, the hippocampal IL-6 levels were decreased following siIL-6 MSC transplantation. In vitro, the levels of IL-6 release and the levels of IL-6R and STAT3 expression were increased in both primary neurons and astrocytes subjected to oxygen and glucose deprivation (OGD) following MSCs co-culture. The anti-apoptotic protein Bcl-2 was upregulated and the pro-apoptotic protein Bax was downregulated in OGD-injured astrocytes co-cultured with MSCs. However, the siIL-6 MSCs suppressed ratio of Bcl-2/Bax in the injured astrocytes and induced apoptosis number of the injured astrocytes. Taken together, these data suggest that the neuroprotective effect of MSC transplantation in neonatal HIBD rats is partly mediated by IL-6 to enhance anti-apoptosis of injured astrocytes via the IL-6/STAT3 signaling pathway. PMID:26766745

Endogenous IL-6 of mesenchymal stem cell improves behavioral outcome of hypoxic-ischemic brain damage neonatal rats by supressing apoptosis in astrocyte.

PubMed

Gu, Yan; He, Mulan; Zhou, Xiaoqin; Liu, Jinngjing; Hou, Nali; Bin, Tan; Zhang, Yun; Li, Tingyu; Chen, Jie

2016-01-14

Mesenchymal stem cell (MSC) transplantation reduces the neurological impairment caused by hypoxic-ischemic brain damage (HIBD) via immunomodulation. In the current study, we found that MSC transplantation improved learning and memory function and enhanced long-term potentiation in neonatal rats subjected to HIBD and the amount of IL-6 released from MSCs was far greater than that of other cytokines. However, the neuroprotective effect of MSCs infected with siIL-6-transduced recombinant lentivirus (siIL-6 MSCs) was significantly weakened in the behavioural tests and electrophysiological analysis. Meanwhile, the hippocampal IL-6 levels were decreased following siIL-6 MSC transplantation. In vitro, the levels of IL-6 release and the levels of IL-6R and STAT3 expression were increased in both primary neurons and astrocytes subjected to oxygen and glucose deprivation (OGD) following MSCs co-culture. The anti-apoptotic protein Bcl-2 was upregulated and the pro-apoptotic protein Bax was downregulated in OGD-injured astrocytes co-cultured with MSCs. However, the siIL-6 MSCs suppressed ratio of Bcl-2/Bax in the injured astrocytes and induced apoptosis number of the injured astrocytes. Taken together, these data suggest that the neuroprotective effect of MSC transplantation in neonatal HIBD rats is partly mediated by IL-6 to enhance anti-apoptosis of injured astrocytes via the IL-6/STAT3 signaling pathway.

Astrocyte-neuron lactate transport is required for long-term memory formation.

PubMed

Suzuki, Akinobu; Stern, Sarah A; Bozdagi, Ozlem; Huntley, George W; Walker, Ruth H; Magistretti, Pierre J; Alberini, Cristina M

2011-03-04

We report that, in the rat hippocampus, learning leads to a significant increase in extracellular lactate levels that derive from glycogen, an energy reserve selectively localized in astrocytes. Astrocytic glycogen breakdown and lactate release are essential for long-term but not short-term memory formation, and for the maintenance of long-term potentiation (LTP) of synaptic strength elicited in vivo. Disrupting the expression of the astrocytic lactate transporters monocarboxylate transporter 4 (MCT4) or MCT1 causes amnesia, which, like LTP impairment, is rescued by L-lactate but not equicaloric glucose. Disrupting the expression of the neuronal lactate transporter MCT2 also leads to amnesia that is unaffected by either L-lactate or glucose, suggesting that lactate import into neurons is necessary for long-term memory. Glycogenolysis and astrocytic lactate transporters are also critical for the induction of molecular changes required for memory formation, including the induction of phospho-CREB, Arc, and phospho-cofilin. We conclude that astrocyte-neuron lactate transport is required for long-term memory formation. Copyright © 2011 Elsevier Inc. All rights reserved.

Astrocyte-neuron lactate transport is required for long-term memory formation

PubMed Central

Suzuki, Akinobu; Stern, Sarah A.; Bozdagi, Ozlem; Huntley, George W.; Walker, Ruth H.; Magistretti, Pierre J.; Alberini, Cristina M.

2011-01-01

SUMMARY We report that in the rat hippocampus learning leads to a significant increase in extracellular lactate levels, which derive from glycogen, an energy reserve selectively localized in astrocytes. Astrocytic glycogen breakdown and lactate release are essential for long-term but not short-term memory formation, and for the maintenance of long-term potentiation (LTP) of synaptic strength elicited in-vivo. Disrupting the expression of the astrocytic lactate transporters monocarboxylate transporter 4 (MCT4) or MCT1 causes amnesia, which, like LTP impairment, is rescued by lactate but not equicaloric glucose. Disrupting the expression of the neuronal lactate transporter MCT2 also leads to amnesia that is unaffected by either L-lactate or glucose, suggesting that lactate import into neurons is necessary for long-term memory. Glycogenolysis and astrocytic lactate transporters are also critical for the induction of molecular changes required for memory formation, including the induction of phospho-CREB, Arc and phospho-cofilin. We conclude that astrocyte-neuron lactate transport is required for long-term memory formation. PMID:21376239

Glycine Receptor Activation Impairs ATP-Induced Calcium Transients in Cultured Cortical Astrocytes

PubMed Central

Morais, Tatiana P.; Coelho, David; Vaz, Sandra H.; Sebastião, Ana M.; Valente, Cláudia A.

2018-01-01

In central nervous system, glycine receptor (GlyR) is mostly expressed in the spinal cord and brainstem, but glycinergic transmission related elements have also been identified in the brain. Astrocytes are active elements at the tripartite synapse, being responsible for the maintenance of brain homeostasis and for the fine-tuning of synaptic activity. These cells communicate, spontaneously or in response to a stimulus, by elevations in their cytosolic calcium (calcium transients, Ca2+T) that can be propagated to other cells. How these Ca2+T are negatively modulated is yet poorly understood. In this work, we evaluated GlyR expression and its role on calcium signaling modulation in rat brain astrocytes. We first proved that GlyR, predominantly subunits α2 and β, was expressed in brain astrocytes and its localization was confirmed in the cytoplasm and astrocytic processes by immunohistochemistry assays. Calcium imaging experiments in cultured astrocytes showed that glycine (500 μM), a GlyR agonist, caused a concentration-dependent reduction in ATP-induced Ca2+T, an effect abolished by the GlyR antagonist, strychnine (0.8 μM), as well as by nocodazole (1 μM), known to impair GlyR anchorage to the plasma membrane. This effect was mimicked by activation of GABAAR, another Cl--permeable channel. In summary, we demonstrated that GlyR activation in astrocytes mediates an inhibitory effect upon ATP induced Ca2+T, which most probably involves changes in membrane permeability to Cl- and requires GlyR anchorage at the plasma membrane. GlyR in astrocytes may thus be part of a mechanism to modulate astrocyte-to-neuron communication. PMID:29386993

An Excitatory Loop with Astrocytes Contributes to Drive Neurons to Seizure Threshold

PubMed Central

Chiavegato, Angela; Zonta, Micaela; Cammarota, Mario; Brondi, Marco; Vetri, Francesco; Uva, Laura; Pozzan, Tullio; de Curtis, Marco; Ratto, Gian Michele; Carmignoto, Giorgio

2010-01-01

Seizures in focal epilepsies are sustained by a highly synchronous neuronal discharge that arises at restricted brain sites and subsequently spreads to large portions of the brain. Despite intense experimental research in this field, the earlier cellular events that initiate and sustain a focal seizure are still not well defined. Their identification is central to understand the pathophysiology of focal epilepsies and to develop new pharmacological therapies for drug-resistant forms of epilepsy. The prominent involvement of astrocytes in ictogenesis was recently proposed. We test here whether a cooperation between astrocytes and neurons is a prerequisite to support ictal (seizure-like) and interictal epileptiform events. Simultaneous patch-clamp recording and Ca2+ imaging techniques were performed in a new in vitro model of focal seizures induced by local applications of N-methyl-D-aspartic acid (NMDA) in rat entorhinal cortex slices. We found that a Ca2+ elevation in astrocytes correlates with both the initial development and the maintenance of a focal, seizure-like discharge. A delayed astrocyte activation during ictal discharges was also observed in other models (including the whole in vitro isolated guinea pig brain) in which the site of generation of seizure activity cannot be precisely monitored. In contrast, interictal discharges were not associated with Ca2+ changes in astrocytes. Selective inhibition or stimulation of astrocyte Ca2+ signalling blocked or enhanced, respectively, ictal discharges, but did not affect interictal discharge generation. Our data reveal that neurons engage astrocytes in a recurrent excitatory loop (possibly involving gliotransmission) that promotes seizure ignition and sustains the ictal discharge. This neuron–astrocyte interaction may represent a novel target to develop effective therapeutic strategies to control seizures. PMID:20405049

Channel-mediated lactate release by K⁺-stimulated astrocytes.

PubMed

Sotelo-Hitschfeld, Tamara; Niemeyer, María I; Mächler, Philipp; Ruminot, Iván; Lerchundi, Rodrigo; Wyss, Matthias T; Stobart, Jillian; Fernández-Moncada, Ignacio; Valdebenito, Rocío; Garrido-Gerter, Pamela; Contreras-Baeza, Yasna; Schneider, Bernard L; Aebischer, Patrick; Lengacher, Sylvain; San Martín, Alejandro; Le Douce, Juliette; Bonvento, Gilles; Magistretti, Pierre J; Sepúlveda, Francisco V; Weber, Bruno; Barros, L Felipe

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. Copyright © 2015 the authors 0270-6474/15/354168-11$15.00/0.

Astrocytic modulation of blood brain barrier: perspectives on Parkinson’s disease

PubMed Central

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

2014-01-01

The blood–brain barrier (BBB) is a tightly regulated interface in the Central Nervous System (CNS) that regulates the exchange of molecules in and out from the brain thus maintaining the CNS homeostasis. It is mainly composed of endothelial cells (ECs), pericytes and astrocytes that create a neurovascular unit (NVU) with the adjacent neurons. Astrocytes are essential for the formation and maintenance of the BBB by providing secreted factors that lead to the adequate association between the cells of the BBB and the formation of strong tight junctions. Under neurological disorders, such as chronic cerebral ischemia, brain trauma, Epilepsy, Alzheimer and Parkinson’s Diseases, a disruption of the BBB takes place, involving a lost in the permeability of the barrier and phenotypical changes in both the ECs and astrocytes. In this aspect, it has been established that the process of reactive gliosis is a common feature of astrocytes during BBB disruption, which has a detrimental effect on the barrier function and a subsequent damage in neuronal survival. In this review we discuss the implications of astrocyte functions in the protection of the BBB, and in the development of Parkinson’s disease (PD) and related disorders. Additionally, we highlight the current and future strategies in astrocyte protection aimed at the development of restorative therapies for the BBB in pathological conditions. PMID:25136294

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

PubMed Central

Jiang, Ruotian; Haustein, Martin D.; Sofroniew, Michael V.; Khakh, Baljit S.

2014-01-01

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry. PMID:25490346

Synergistic induction of astrocytic differentiation by factors secreted from meninges in the mouse developing brain.

PubMed

Kawamura, Yoichiro; Katada, Sayako; Noguchi, Hirofumi; Yamamoto, Hiroyuki; Sanosaka, Tsukasa; Iihara, Koji; Nakashima, Kinichi

2017-11-01

Astrocytes, which support diverse neuronal functions, are generated from multipotent neural stem/precursor cells (NS/PCs) during brain development. Although many astrocyte-inducing factors have been identified and studied in vitro, the regions and/or cells that produce these factors in the developing brain remain elusive. Here, we show that meninges-produced factors induce astrocytic differentiation of NS/PCs. Consistent with the timing when astrocytic differentiation of NS/PCs increases, expression of astrocyte-inducing factors is upregulated. Meningeal secretion-mimicking combinatorial treatment of NS/PCs with bone morphogenetic protein 4, retinoic acid and leukemia inhibitory factor synergistically activate the promoter of a typical astrocytic marker, glial fibrillary acidic protein. Taken together, our data suggest that meninges play an important role in astrocytic differentiation of NS/PCs in the developing brain. © 2017 Federation of European Biochemical Societies.

Imbalance between Glutamate and GABA in Fmr1 Knockout Astrocytes Influences Neuronal Development

PubMed Central

Wang, Lu; Wang, Yan; Zhou, Shimeng; Yang, Liukun; Shi, Qixin; Li, Yujiao; Zhang, Kun; Yang, Le; Zhao, Minggao; Yang, Qi

2016-01-01

Fragile X syndrome (FXS) is a form of inherited mental retardation that results from the absence of the fragile X mental retardation protein (FMRP), the product of the Fmr1 gene. Numerous studies have shown that FMRP expression in astrocytes is important in the development of FXS. Although astrocytes affect neuronal dendrite development in Fmr1 knockout (KO) mice, the factors released by astrocytes are still unclear. We cultured wild type (WT) cortical neurons in astrocyte-conditioned medium (ACM) from WT or Fmr1 KO mice. Immunocytochemistry and Western blotting were performed to detect the dendritic growth of both WT and KO neurons. We determined glutamate and γ-aminobutyric acid (GABA) levels using high-performance liquid chromatography (HPLC). The total neuronal dendritic length was reduced when cultured in the Fmr1 KO ACM. This neurotoxicity was triggered by an imbalanced release of glutamate and GABA from Fmr1 KO astrocytes. We found increased glutaminase and GABA transaminase (GABA-T) expression and decreased monoamine oxidase B expression in Fmr1 KO astrocytes. The elevated levels of glutamate contributed to oxidative stress in the cultured neurons. Vigabatrin (VGB), a GABA-T inhibitor, reversed the changes caused by glutamate and GABA release in Fmr1 KO astrocytes and the abnormal behaviors in Fmr1 KO mice. Our results indicate that the imbalance in the astrocytic glutamate and GABA release may be involved in the neuropathology and the underlying symptoms of FXS, and provides a therapeutic target for treatment. PMID:27517961

Features of morfological changes in primary thyroid gland CTLL cultures of rats descendants prenatally exposed by radioisotopes of iodine-131.

PubMed

Boiko, O A; Lavrenchuk, H Yo; Lypska, A I; Talko, V V; Asmolkov, V S

2017-12-01

to investigate morphological changes in the primary thyroid cell culture of rat infants whose parents were prenatally exposed by radioisotope iodine 131. obtaining and culturing of thyroid tissue primary cell cultures of newborn rats, cytological (receipt and analysis of cell cultures agents for optical microscopy), biophysical (flow cytometry), statistics. It was shown that cells in thyroid primary culture of offspring rats prenatally exposed by radioisotopes of iodine 131 signs of destructive degenerative changes were observed mostly when animals of both sexes were irra diated. Increased number of two and three nuclear cells and induction of ring like cells is an evidence of signifi cant genotoxic violation and points to the genome instability in offspring of animals exposed by radioisotope iodine 131. Analysis and quantitative morphological parameters of cells in thyroid primary culture of newborn rats whose parents were exposed prenatally by radioisotopes of iodine 131 showed that upon exposure to radiation thy roid undergoes destructive changes at the cellular level and, even in the second generation of offspring, leads to disruption of its functions. O. A. Boiko, H. Yo. Lavrenchuk, A. I. Lypska, V. V. Talko, V. S. Asmolkov.

A computational study of astrocytic glutamate influence on post-synaptic neuronal excitability.

PubMed

Flanagan, Bronac; McDaid, Liam; Wade, John; Wong-Lin, KongFatt; Harkin, Jim

2018-04-01

The ability of astrocytes to rapidly clear synaptic glutamate and purposefully release the excitatory transmitter is critical in the functioning of synapses and neuronal circuits. Dysfunctions of these homeostatic functions have been implicated in the pathology of brain disorders such as mesial temporal lobe epilepsy. However, the reasons for these dysfunctions are not clear from experimental data and computational models have been developed to provide further understanding of the implications of glutamate clearance from the extracellular space, as a result of EAAT2 downregulation: although they only partially account for the glutamate clearance process. In this work, we develop an explicit model of the astrocytic glutamate transporters, providing a more complete description of the glutamate chemical potential across the astrocytic membrane and its contribution to glutamate transporter driving force based on thermodynamic principles and experimental data. Analysis of our model demonstrates that increased astrocytic glutamate content due to glutamine synthetase downregulation also results in increased postsynaptic quantal size due to gliotransmission. Moreover, the proposed model demonstrates that increased astrocytic glutamate could prolong the time course of glutamate in the synaptic cleft and enhances astrocyte-induced slow inward currents, causing a disruption to the clarity of synaptic signalling and the occurrence of intervals of higher frequency postsynaptic firing. Overall, our work distilled the necessity of a low astrocytic glutamate concentration for reliable synaptic transmission of information and the possible implications of enhanced glutamate levels as in epilepsy.

Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways

NASA Astrophysics Data System (ADS)

Chung, Won-Suk; Clarke, Laura E.; Wang, Gordon X.; Stafford, Benjamin K.; Sher, Alexander; Chakraborty, Chandrani; Joung, Julia; Foo, Lynette C.; Thompson, Andrew; Chen, Chinfei; Smith, Stephen J.; Barres, Ben A.

2013-12-01

To achieve its precise neural connectivity, the developing mammalian nervous system undergoes extensive activity-dependent synapse remodelling. Recently, microglial cells have been shown to be responsible for a portion of synaptic pruning, but the remaining mechanisms remain unknown. Here we report a new role for astrocytes in actively engulfing central nervous system synapses. This process helps to mediate synapse elimination, requires the MEGF10 and MERTK phagocytic pathways, and is strongly dependent on neuronal activity. Developing mice deficient in both astrocyte pathways fail to refine their retinogeniculate connections normally and retain excess functional synapses. Finally, we show that in the adult mouse brain, astrocytes continuously engulf both excitatory and inhibitory synapses. These studies reveal a novel role for astrocytes in mediating synapse elimination in the developing and adult brain, identify MEGF10 and MERTK as critical proteins in the synapse remodelling underlying neural circuit refinement, and have important implications for understanding learning and memory as well as neurological disease processes.

Astrocyte Transforming Growth Factor Beta 1 Protects Synapses against Aβ Oligomers in Alzheimer's Disease Model.

PubMed

Diniz, Luan Pereira; Tortelli, Vanessa; Matias, Isadora; Morgado, Juliana; Bérgamo Araujo, Ana Paula; Melo, Helen M; Seixas da Silva, Gisele S; Alves-Leon, Soniza V; de Souza, Jorge M; Ferreira, Sergio T; De Felice, Fernanda G; Gomes, Flávia Carvalho Alcantara

2017-07-12

Alzheimer's disease (AD) is characterized by progressive cognitive decline, increasingly attributed to neuronal dysfunction induced by amyloid-β oligomers (AβOs). Although the impact of AβOs on neurons has been extensively studied, only recently have the possible effects of AβOs on astrocytes begun to be investigated. Given the key roles of astrocytes in synapse formation, plasticity, and function, we sought to investigate the impact of AβOs on astrocytes, and to determine whether this impact is related to the deleterious actions of AβOs on synapses. We found that AβOs interact with astrocytes, cause astrocyte activation and trigger abnormal generation of reactive oxygen species, which is accompanied by impairment of astrocyte neuroprotective potential in vitro We further show that both murine and human astrocyte conditioned media (CM) increase synapse density, reduce AβOs binding, and prevent AβO-induced synapse loss in cultured hippocampal neurons. Both a neutralizing anti-transforming growth factor-β1 (TGF-β1) antibody and siRNA-mediated knockdown of TGF-β1, previously identified as an important synaptogenic factor secreted by astrocytes, abrogated the protective action of astrocyte CM against AβO-induced synapse loss. Notably, TGF-β1 prevented hippocampal dendritic spine loss and memory impairment in mice that received an intracerebroventricular infusion of AβOs. Results suggest that astrocyte-derived TGF-β1 is part of an endogenous mechanism that protects synapses against AβOs. By demonstrating that AβOs decrease astrocyte ability to protect synapses, our results unravel a new mechanism underlying the synaptotoxic action of AβOs in AD. SIGNIFICANCE STATEMENT Alzheimer's disease is characterized by progressive cognitive decline, mainly attributed to synaptotoxicity of the amyloid-β oligomers (AβOs). Here, we investigated the impact of AβOs in astrocytes, a less known subject. We show that astrocytes prevent synapse loss induced by A�

Ulinastatin suppresses lipopolysaccharide induced neuro-inflammation through the downregulation of nuclear factor-κB in SD rat hippocampal astrocyte

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Yuting; Zhao, Lei; Fu, Huiqun

Astrocyte activation plays a pivotal role in neuroinflammation, which contributes to neuronal damage, so the inhibition of astrocyte activation may alleviate the progression of neurodegeneration. Recent studies have proved that urinary trypsin inhibitor ulinastatin could inhibit NF-kB activation. In our study, the inhibitory effects of ulinastatin on the production of pro-inflammatory mediators were investigated in lipopolysaccharide (LPS)-reduced primary astrocyte. Our results showed that ulinastatin significantly inhibited LPS-induced astrogliosis, which is measured by MTT and BrdU. Ulinastatin decreased the production of pro-inflammatory cytokines, such as TNF-α, IL-6, IL-1β, it significantly decreased both the mRNA and the protein levels of these pro-inflammatorymore » cytokines and also increased the protein levels of IκB-α binded to NF-κB, which blocked NF-κB translocation to the nucleus and prevented its activity. Our results suggest that ulinastatin is able to inhibit neuroinflammation by interfering with NF-κB signaling. The study provides direct evidence of potential therapy methods of ulinastatin for the treatment of neuroinflammatory diseases. - Highlights: • The anti-inflammatory effect of UTI on hippocampal astrocyte. • UTI showed protective effect on neuroinflammation by the downregulation of NF-κB. • UTI led to expression of cytokines decreased in concentration and time dependence.« less

GABA from reactive astrocytes impairs memory in mouse models of Alzheimer's disease.

PubMed

Jo, Seonmi; Yarishkin, Oleg; Hwang, Yu Jin; Chun, Ye Eun; Park, Mijeong; Woo, Dong Ho; Bae, Jin Young; Kim, Taekeun; Lee, Jaekwang; Chun, Heejung; Park, Hyun Jung; Lee, Da Yong; Hong, Jinpyo; Kim, Hye Yun; Oh, Soo-Jin; Park, Seung Ju; Lee, Hyo; Yoon, Bo-Eun; Kim, YoungSoo; Jeong, Yong; Shim, Insop; Bae, Yong Chul; Cho, Jeiwon; Kowall, Neil W; Ryu, Hoon; Hwang, Eunmi; Kim, Daesoo; Lee, C Justin

2014-08-01

In Alzheimer's disease (AD), memory impairment is the most prominent feature that afflicts patients and their families. Although reactive astrocytes have been observed around amyloid plaques since the disease was first described, their role in memory impairment has been poorly understood. Here, we show that reactive astrocytes aberrantly and abundantly produce the inhibitory gliotransmitter GABA by monoamine oxidase-B (Maob) and abnormally release GABA through the bestrophin 1 channel. In the dentate gyrus of mouse models of AD, the released GABA reduces spike probability of granule cells by acting on presynaptic GABA receptors. Suppressing GABA production or release from reactive astrocytes fully restores the impaired spike probability, synaptic plasticity, and learning and memory in the mice. In the postmortem brain of individuals with AD, astrocytic GABA and MAOB are significantly upregulated. We propose that selective inhibition of astrocytic GABA synthesis or release may serve as an effective therapeutic strategy for treating memory impairment in AD.

Astrocytic and neuronal oxidative metabolism are coupled to the rate of glutamate-glutamine cycle in the tree shrew visual cortex.

PubMed

Sonnay, Sarah; Poirot, Jordan; Just, Nathalie; Clerc, Anne-Catherine; Gruetter, Rolf; Rainer, Gregor; Duarte, João M N

2018-03-01

Astrocytes play an important role in glutamatergic neurotransmission, namely by clearing synaptic glutamate and converting it into glutamine that is transferred back to neurons. The rate of this glutamate-glutamine cycle (V NT ) has been proposed to couple to that of glucose utilization and of neuronal tricarboxylic acid (TCA) cycle. In this study, we tested the hypothesis that glutamatergic neurotransmission is also coupled to the TCA cycle rate in astrocytes. For that we investigated energy metabolism by means of magnetic resonance spectroscopy (MRS) in the primary visual cortex of tree shrews (Tupaia belangeri) under light isoflurane anesthesia at rest and during continuous visual stimulation. After identifying the activated cortical volume by blood oxygenation level-dependent functional magnetic resonance imaging, 1 H MRS was performed to measure stimulation-induced variations in metabolite concentrations. Relative to baseline, stimulation of cortical activity for 20 min caused a reduction of glucose concentration by -0.34 ± 0.09 µmol/g (p < 0.001), as well as a -9% ± 1% decrease of the ratio of phosphocreatine-to-creatine (p < 0.05). Then 13 C MRS during [1,6- 13 C]glucose infusion was employed to measure fluxes of energy metabolism. Stimulation of glutamatergic activity, as indicated by a 20% increase of V NT , resulted in increased TCA cycle rates in neurons by 12% ( VTCAn, p < 0.001) and in astrocytes by 24% ( VTCAg, p = 0.007). We further observed linear relationships between V NT and both VTCAn and VTCAg. Altogether, these results suggest that in the tree shrew primary visual cortex glutamatergic neurotransmission is linked to overall glucose oxidation and to mitochondrial metabolism in both neurons and astrocytes. © 2017 Wiley Periodicals, Inc.

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

PubMed

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

2017-06-01

Astrocytes respond to various stimuli resulting in intracellular Ca 2+ signals that can propagate through organized functional networks. Recent literature calls for the development of techniques that can stimulate astrocytes in a fast and highly localized manner to emulate more closely the characteristics of astrocytic Ca 2+ signals in vivo. In this article we demonstrate, for the first time, how nanosecond UV lasers are capable of reproducibly stimulating Ca 2+ transients in human hNT astrocytes. We report that laser pulses with a beam energy of 4-29 µJ generate transient increases in cytosolic Ca 2+ . These Ca 2+ transients then propagate to adjacent astrocytes as intercellular Ca 2+ waves. We propose that nanosecond laser stimulation provides a valuable tool for enabling the study of Ca 2+ dynamics in human astrocytes at both a single cell and network level. Compared to previously developed techniques nanosecond laser stimulation has the advantage of not requiring loading of photo-caged or -sensitising agents, is non-contact, enables stimulation with a high spatiotemporal resolution and is comparatively cost effective.

Effect of Huperzine A on Aβ-induced p65 of astrocyte in vitro.

PubMed

Xie, Lushuang; Jiang, Cen; Wang, Zhang; Yi, Xiaohong; Gong, Yuanyuan; Chen, Yunhui; Fu, Yan

2016-12-01

Alzheimer's disease (AD) is the most common cause of dementia. Its pathology often accompanies inflammatory action, and astrocytes play important roles in such procedure. Rela(p65) is one of significant message factors in NF-κB pathway which has been reported high expression in astrocyte treated by Aβ. HupA, an alkaloid isolated from Chinese herb Huperzia serrata, has been widely used to treat AD and observations reflected that it improves memory and cognitive capacity of AD patients. To reveal its molecular mechanisms on p65, we cultured astrocytes, built Aβ-induced AD model, treated astrocytes with HupA at different concentrations, assayed cell viability with MTT, and detected p65 expression by immunohistochemistry and PCR. Our results revealed that treatment with 10 μM Aβ1-42 for 24 h induced a significant increase of NF-κB in astrocytes; HupA significantly down-regulated p65 expression induced by Aβ in astrocytes. This study infers that HupA can regulate NF-κB pathway to treat AD.

Cultured astrocytes do not release adenosine during hypoxic conditions

PubMed Central

Fujita, Takumi; Williams, Erika K; Jensen, Tina K; Smith, Nathan A; Takano, Takahiro; Tieu, Kim; Nedergaard, Maiken

2012-01-01

Recent reports based on a chemiluminescent enzymatic assay for detection of adenosine conclude that cultured astrocytes release adenosine during mildly hypoxic conditions. If so, astrocytes may suppress neural activity in early stages of hypoxia. The aim of this study was to reevaluate the observation using high-performance liquid chromatography (HPLC). The HPLC analysis showed that exposure to 20 or 120 minutes of mild hypoxia failed to increase release of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine from cultured astrocytes. Similar results were obtained using a chemiluminescent enzymatic assay. Moreover, since the chemiluminescent enzymatic assay relies on hydrogen peroxide generation, release of free-radical scavengers from hypoxic cells can interfere with the assay. Accordingly, adenosine added to samples collected from hypoxic cultures could not be detected using the chemiluminescent enzymatic assay. Furthermore, addition of free-radical scavengers sharply reduced the sensitivity of adenosine detection. Conversely, use of a single-step assay inflated measured values due to the inability of the assay to distinguish adenosine and its metabolite inosine. These results show that cultured astrocytes do not release adenosine during mild hypoxia, an observation consistent with their high resistance to hypoxia. PMID:21989480

Impairment of astrocytic glutaminolysis in glutaric aciduria type I.

PubMed

Komatsuzaki, Shoko; Ediga, Raga Deepthi; Okun, Jürgen G; Kölker, Stefan; Sauer, Sven W

2018-01-01

Glutaric aciduria type I is a rare, autosomal recessive, inherited defect of glutaryl-CoA dehydrogenase. Deficiency of this protein in L-lysine degradation leads to the characteristic accumulation of nontoxic glutarylcarnitine and neurotoxic glutaric acid (GA), glutaryl-CoA, and 3-hydroxyglutaric acid. Untreated patients develop bilateral lesions of basal ganglia resulting in a complex movement disorder with predominant dystonia in infancy and early childhood. The current pathomechanistic concept strongly focuses on imbalanced neuronal energy metabolism due to accumulating metabolites, whereas little is known about the pathomechanistic role of astrocytes, which are thought to be in constant metabolic crosstalk with neurons. We found that glutaric acid (GA) causes astrocytic cell death under starvation cell culture conditions, i.e. low glucose, without glutamine and fetal calf serum. Glutamine completely abolished GA-induced toxicity, suggesting involvement of glutaminolysis. Increasing dependence on glutaminolysis by chemical induction of hypoxia signaling-potentiated GA-induced toxicity. We further show that GA disturbs glutamine degradation by specifically inhibiting glutamate dehydrogenase. Summarizing our study shows that pathologically relevant concentrations of GA block an important step in the metabolic crosstalk between neurons and astrocytes, ultimately leading to astrocytic cell death.

Astrocyte-neuron interaction in diphenyl ditelluride toxicity directed to the cytoskeleton.

PubMed

Heimfarth, Luana; da Silva Ferreira, Fernanda; Pierozan, Paula; Mingori, Moara Rodrigues; Moreira, José Cláudio Fonseca; da Rocha, João Batista Teixeira; Pessoa-Pureur, Regina

2017-03-15

Diphenylditelluride (PhTe) 2 is a neurotoxin that disrupts cytoskeletal homeostasis. We are showing that different concentrations of (PhTe) 2 caused hypophosphorylation of glial fibrillary acidic protein (GFAP), vimentin and neurofilament subunits (NFL, NFM and NFH) and altered actin organization in co-cultured astrocytes and neurons from cerebral cortex of rats. These mechanisms were mediated by N-methyl-d-aspartate (NMDA) receptors without participation of either L-type voltage-dependent calcium channels (L-VDCC) or metabotropic glutamate receptors. Upregulated Ca 2+ influx downstream of NMDA receptors activated Ca 2+ -dependent protein phosphatase 2B (PP2B) causing hypophosphorylation of astrocyte and neuron IFs. Immunocytochemistry showed that hypophosphorylated intermediate filaments (IF) failed to disrupt their organization into the cytoskeleton. However, phalloidin-actin-FITC stained cytoskeleton evidenced misregulation of actin distribution, cell spreading and increased stress fibers in astrocytes. βIII tubulin staining showed that neurite meshworks are not altered by (PhTe) 2 , suggesting greater susceptibility of astrocytes than neurons to (PheTe) 2 toxicity. These findings indicate that signals leading to IF hypophosphorylation fail to disrupt the cytoskeletal IF meshwork of interacting astrocytes and neurons in vitro however astrocyte actin network seems more susceptible. Our findings support that intracellular Ca 2+ is one of the crucial signals that modulate the action of (PhTe) 2 in co-cultured astrocytes and neurons and highlights the cytoskeleton as an end-point of the neurotoxicity of this compound. Cytoskeletal misregulation is associated with cell dysfunction, therefore, the understanding of the molecular mechanisms mediating the neurotoxicity of this compound is a matter of increasing interest since tellurium compounds are increasingly released in the environment. Copyright © 2017 Elsevier B.V. All rights reserved.

Regulation of neuron-astrocyte metabolic coupling across the sleep-wake cycle.

PubMed

Petit, J-M; Magistretti, P J

2016-05-26

Over the last thirty years, a growing number of studies showed that astrocytes play a pivotal role in the energy support to synapses. More precisely, astrocytes adjust energy production to neuronal energy needs through different mechanisms grouped under the term "neurometabolic coupling" (NMC). In this review we describe these mechanisms of coupling and how they involve astrocytes. From a physiological point of view, these mechanisms of coupling are particularly important to ensure normal synaptic functioning when neurons undergo rapid and repetitive changes in the firing rate such as during the sleep/wake transitions. Investigations into brain energy metabolism during the sleep/wake cycle have been mainly focused on glucose (Gluc) consumption and on glycogen metabolism. However, the recent development of substrate-specific biosensors allowed measurements of the variation in extracellular levels of glutamate, Gluc and lactate (Lac) with a time resolution compatible with sleep stage duration. Together with gene expression data these experiments allowed to better define the variations of energy metabolite regulation across the sleep/wake cycle. The aim of this review is to bring into perspective the role of astrocytes and NMC in the regulation of the sleep/wake cycle. The data reviewed also suggest an important role of the astrocytic network. In addition, the role of astrocytes in NMC mechanisms is consistent with the "local and use dependent" sleep hypothesis. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Astrocytes Increase ATP Exocytosis Mediated Calcium Signaling in Response to Microgroove Structures

PubMed Central

Singh, Ajay V.; Raymond, Michael; Pace, Fabiano; Certo, Anthony; Zuidema, Jonathan M.; McKay, Christopher A.; Gilbert, Ryan J.; Lu, X. Lucas; Wan, Leo Q.

2015-01-01

Following central nervous system (CNS) injury, activated astrocytes form glial scars, which inhibit axonal regeneration, leading to long-term functional deficits. Engineered nanoscale scaffolds guide cell growth and enhance regeneration within models of spinal cord injury. However, the effects of micro-/nanosize scaffolds on astrocyte function are not well characterized. In this study, a high throughput (HTP) microscale platform was developed to study astrocyte cell behavior on micropatterned surfaces containing 1 μm spacing grooves with a depth of 250 or 500 nm. Significant changes in cell and nuclear elongation and alignment on patterned surfaces were observed, compared to on flat surfaces. The cytoskeleton components (particularly actin filaments and focal adhesions) and nucleus-centrosome axis were aligned along the grooved direction as well. More interestingly, astrocytes on micropatterned surfaces showed enhanced mitochondrial activity with lysosomes localized at the lamellipodia of the cells, accompanied by enhanced adenosine triphosphate (ATP) release and calcium activities. These data indicate that the lysosome-mediated ATP exocytosis and calcium signaling may play an important role in astrocytic responses to substrate topology. These new findings have furthered our understanding of the biomechanical regulation of astrocyte cell–substrate interactions, and may benefit the optimization of scaffold design for CNS healing. PMID:25597401

Photolysis of Caged Ca2+ But Not Receptor-Mediated Ca2+ Signaling Triggers Astrocytic Glutamate Release

PubMed Central

Smith, Nathan A.; Xu, Qiwu; Goldman, Siri; Peng, Weiguo; Huang, Jason H.; Takano, Takahiro; Nedergaard, Maiken

2013-01-01

Astrocytes in hippocampal slices can dynamically regulate synaptic transmission in a process mediated by increases in intracellular Ca2+. However, it is debated whether astrocytic Ca2+ signals result in release of glutamate. We here compared astrocytic Ca2+ signaling triggered by agonist exposure versus photolysis side by side. Using transgenic mice in which astrocytes selectively express the MrgA1 receptor, we found that receptor-mediated astrocytic Ca2+ signaling consistently triggered neuronal hyperpolarization and decreased the frequency of miniature excitatory postsynaptic currents (EPSCs). In contrast, photolysis of caged Ca2+ (o-nitrophenyl–EGTA) in astrocytes led to neuronal depolarization and increased the frequency of mEPSCs through a metabotropic glutamate receptor-mediated pathway. Analysis of transgenic mice in which astrocytic vesicular release is suppressed (dominant-negative SNARE mice) and pharmacological manipulations suggested that glutamate is primarily released by opening of anion channels rather than exocytosis. Combined, these studies show that photolysis but not by agonists induced astrocytic Ca2+ signaling triggers glutamate release. PMID:24174673

Astrocyte activation and wound healing in intact-skull mouse after focal brain injury.

PubMed

Suzuki, Takayuki; Sakata, Honami; Kato, Chiaki; Connor, John A; Morita, Mitsuhiro

2012-12-01

Localised brain tissue damage activates surrounding astrocytes, which significantly influences subsequent long-term pathological processes. Most existing focal brain injury models in rodents employ craniotomy to localise mechanical insults. However, the craniotomy procedure itself induces gliosis. To investigate perilesional astrocyte activation under conditions in which the skull is intact, we created focal brain injuries using light exposure through a cranial window made by thinning the skull without inducing gliosis. The lesion size was maximal at ~ 12 h and showed substantial recovery over the subsequent 30 days. Two distinct types of perilesional reactive astrocyte, identified by GFAP upregulation and hypertrophy, were found. In proximal regions the reactive astrocytes proliferated and expressed nestin, whereas in regions distal to the injury core the astrocytes showed increased GFAP expression but did not proliferate, lacked nestin expression, and displayed different morphology. Simply making the window did not induce any of these changes. There were also significant numbers of neurons in the recovering cortical tissue. In the recovery region, reactive astrocytes radially extended processes which appeared to influence the shapes of neuronal nuclei. The proximal reactive astrocytes also formed a cell layer which appeared to serve as a protective barrier, blocking the spread of IgG deposition and migration of microglia from the lesion core to surrounding tissue. The recovery was preceded by perilesional accumulation of leukocytes expressing vascular endothelial growth factor. These results suggest that, under intact skull conditions, focal brain injury is followed by perilesional reactive astrocyte activities that foster cortical tissue protection and recovery. © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

CDK5-induced p-PPARγ(Ser 112) downregulates GFAP via PPREs in developing rat brain: effect of metal mixture and troglitazone in astrocytes.

PubMed

Rai, A; Tripathi, S; Kushwaha, R; Singh, P; Srivastava, P; Sanyal, S; Bandyopadhyay, S

2014-01-30

The peroxisome proliferator-activated receptor gamma (PPARγ), a group of ligand-activated transcriptional factors, is expressed in glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes. Here, we investigated the role of PPARγ in regulating GFAP using a mixture of As, Cd and Pb (metal mixture, MM) that induces apoptosis and aberrant morphology in rat brain astrocytes. We observed a phospho PPARγ (serine 112 (S112)) (p-PPARγ (S112))-mediated downregulation of GFAP in the MM-exposed astrocytes. We validated this using pure PPARγ agonist, troglitazone (TZ). As reported with MM, TZ induced astrocyte damage owing to reduced GFAP. In silico analysis in the non-coding region of GFAP gene revealed two PPARγ response elements (PPREs); inverted repeat 10 and direct repeat 1 sequences. Gel shift and chromatin immunoprecipitation assays demonstrated enhancement in binding of p-PPARγ (S112) to the sequences, and luciferase reporter assay revealed strong repression of GFAP via PPREs, in response to both MM and TZ. This indicated that suppression in GFAP indeed occurs through direct regulation of these elements by p-PPARγ (S112). Signaling studies proved that MM, as well as TZ, activated the cyclin-dependent kinase 5 (CDK5) and enhanced its interaction with PPARγ resulting into increased p-PPARγ (S112). The p-CDK5 levels were dependent on proximal activation of extracellular signal-regulated protein kinase 1/2 and downstream Jun N-terminal kinase. Taken together, these results are the first to delineate downregulation of GFAP through genomic and non-genomic signaling of PPARγ. It also brings forth a resemblance of TZ with MM in terms of astrocyte disarray in developing brain.

Astrocyte deletion of Bmal1 alters daily locomotor activity and cognitive functions via GABA signalling

PubMed Central

Barca-Mayo, Olga; Pons-Espinal, Meritxell; Follert, Philipp; Armirotti, Andrea; Berdondini, Luca; De Pietri Tonelli, Davide

2017-01-01

Circadian rhythms are controlled by a network of clock neurons in the central pacemaker, the suprachiasmatic nucleus (SCN). Core clock genes, such as Bmal1, are expressed in SCN neurons and in other brain cells, such as astrocytes. However, the role of astrocytic clock genes in controlling rhythmic behaviour is unknown. Here we show that ablation of Bmal1 in GLAST-positive astrocytes alters circadian locomotor behaviour and cognition in mice. Specifically, deletion of astrocytic Bmal1 has an impact on the neuronal clock through GABA signalling. Importantly, pharmacological modulation of GABAA-receptor signalling completely rescues the behavioural phenotypes. Our results reveal a crucial role of astrocytic Bmal1 for the coordination of neuronal clocks and propose a new cellular target, astrocytes, for neuropharmacology of transient or chronic perturbation of circadian rhythms, where alteration of astrocytic clock genes might contribute to the impairment of the neurobehavioural outputs such as cognition. PMID:28186121

Effect of dibutyryl cyclic AMP on the kinetics of myo-inositol transport in cultured astrocytes.

PubMed

Isaacks, R E; Bender, A S; Reuben, J S; Kim, C Y; Shi, Y F; Norenberg, M D

1999-07-01

Dibutyryl cyclic AMP (dBcAMP) is known to induce maturation and differentiation in astrocytes. As myo-inositol is an important osmoregulator in astrocytes, we examined the effects of maturation and biochemical differentiation on the kinetic properties of myo-inositol transport. Treatment of astrocytes with dBcAMP significantly decreased the Vmax of myo-inositol uptake, but the effect on Km was not significant. The myo-inositol content of astrocytes was significantly decreased in cells treated for 5 days with dBcAMP as compared with untreated controls. Maximum suppression of myo-inositol uptake occurred 7 days after exposure of astrocytes to dBcAMP; this was gradually reversible when dBcAMP was removed from the medium. After exposure to hypertonic medium for 6 h, mRNA expression of the myo-inositol co-transporter was diminished by approximately 36% in astrocytes treated with dBcAMP as compared with untreated cells. It appears that myo-inositol transporters in astrocytes treated with dBcAMP are either decreased in number or inactivated during maturation and differentiation, suggesting that the stage of differentiation and biochemical maturation of astrocytes is an important factor in osmoregulation.

Differential involvement of ipsilateral and contralateral spinal cord astrocyte D-serine in carrageenan-induced mirror-image pain: role of σ1 receptors and astrocyte gap junctions.

PubMed

Choi, Hoon-Seong; Roh, Dae-Hyun; Yoon, Seo-Yeon; Choi, Sheu-Ran; Kwon, Soon-Gu; Kang, Suk-Yun; Moon, Ji-Young; Han, Ho-Jae; Beitz, Alvin J; Lee, Jang-Hern

2018-02-01

Although we have recently demonstrated that spinal astrocyte gap junctions mediate the development of mirror-image pain (MIP), it is still unclear which astrocyte-derived factor is responsible for the development of MIP and how its production is controlled. In the present study, we focused on the role of ipsilateral versus contralateral D-serine in the development of MIP and investigated the possible involvement of σ1 receptors and gap junctions in astrocyte D-serine production. Following carrageenan injection, mechanical allodynia was tested at various time points to examine the effect of individual drugs. Immunohistochemistry and Western blot analyses were performed to clarify the expression levels of spinal D-serine, serine racemase, σ1 receptors and connexin 43. The expression of ipsilateral D-serine was up-regulated during the early phase of inflammation, while contralateral D-serine increased during the later phase of inflammation. The pharmacological inhibition of D-serine during the early phase blocked the development of both ipsilateral and contralateral mechanical allodynia. However, the inhibition of D-serine during the later phase of inflammation blocked contralateral, but not ipsilateral mechanical allodynia. Furthermore, the inhibition of σ1 receptors during the earlier phase of inflammation inhibited the increase in ipsilateral D-serine. Conversely, the blockade of astrocyte gap junctions suppressed the up-regulation of contralateral D-serine during the later phase of inflammation. Spinal astrocyte D-serine plays an important role in the development of mirror-image pain. Furthermore, σ1 receptors and astrocyte gap junction signalling mediate ipsilateral and contralateral D-serine production respectively. © 2017 The British Pharmacological Society.

Diffusion of D-glucose measured in the cytosol of a single astrocyte.

PubMed

Kreft, Marko; Lukšič, Miha; Zorec, Tomaž M; Prebil, Mateja; Zorec, Robert

2013-04-01

Astrocytes interact with neurons and endothelial cells and may mediate exchange of metabolites between capillaries and nerve terminals. In the present study, we investigated intracellular glucose diffusion in purified astrocytes after local glucose uptake. We used a fluorescence resonance energy transfer (FRET)-based nano sensor to monitor the time dependence of the intracellular glucose concentration at specific positions within the cell. We observed a delay in onset and kinetics in regions away from the glucose uptake compared with the region where we locally super-fused astrocytes with the D-glucose-rich solution. We propose a mathematical model of glucose diffusion in astrocytes. The analysis showed that after gradual uptake of glucose, the locally increased intracellular glucose concentration is rapidly spread throughout the cytosol with an apparent diffusion coefficient (D app) of (2.38 ± 0.41) × 10(-10) m(2) s(-1) (at 22-24 °C). Considering that the diffusion coefficient of D-glucose in water is D = 6.7 × 10(-10) m(2) s(-1) (at 24 °C), D app determined in astrocytes indicates that the cytosolic tortuosity, which hinders glucose molecules, is approximately three times higher than in aqueous solution. We conclude that the value of D app for glucose measured in purified rat astrocytes is consistent with the view that cytosolic diffusion may allow glucose and glucose metabolites to traverse from the endothelial cells at the blood-brain barrier to neurons and neighboring astrocytes.

General anesthesia selectively disrupts astrocyte calcium signaling in the awake mouse cortex

PubMed Central

Thrane, Alexander Stanley; Zeppenfeld, Douglas; Lou, Nanhong; Xu, Qiwu; Nagelhus, Erlend Arnulf; Nedergaard, Maiken

2012-01-01

Calcium signaling represents the principle pathway by which astrocytes respond to neuronal activity. General anesthetics are routinely used in clinical practice to induce a sleep-like state, allowing otherwise painful procedures to be performed. Anesthetic drugs are thought to mainly target neurons in the brain and act by suppressing synaptic activity. However, the direct effect of general anesthesia on astrocyte signaling in awake animals has not previously been addressed. This is a critical issue, because calcium signaling may represent an essential mechanism through which astrocytes can modulate synaptic activity. In our study, we performed calcium imaging in awake head-restrained mice and found that three commonly used anesthetic combinations (ketamine/xylazine, isoflurane, and urethane) markedly suppressed calcium transients in neocortical astrocytes. Additionally, all three anesthetics masked potentially important features of the astrocyte calcium signals, such as synchronized widespread transients that appeared to be associated with arousal in awake animals. Notably, anesthesia affected calcium transients in both processes and soma and depressed spontaneous signals, as well as calcium responses, evoked by whisker stimulation or agonist application. We show that these calcium transients are inositol 1,4,5-triphosphate type 2 receptor (IP3R2)-dependent but resistant to a local blockade of glutamatergic or purinergic signaling. Finally, we found that doses of anesthesia insufficient to affect neuronal responses to whisker stimulation selectively suppressed astrocyte calcium signals. Taken together, these data suggest that general anesthesia may suppress astrocyte calcium signals independently of neuronal activity. We propose that these glial effects may constitute a nonneuronal mechanism for sedative action of anesthetic drugs. PMID:23112168

Synchronized Astrocytic Ca2+ Responses in Neurovascular Coupling during Somatosensory Stimulation and for the Resting State.

PubMed

Gu, Xiaochun; Chen, Wei; Volkow, Nora D; Koretsky, Alan P; Du, Congwu; Pan, Yingtian

2018-06-26

The role of astrocytes in neurovascular coupling (NVC) is unclear. Here, we applied a multimodality imaging approach to concomitantly measure synchronized neuronal or astrocytic Ca 2+ and hemodynamic changes in the mouse somatosensory cortex at rest and during sensory electrical stimulation. Strikingly, we found that low-frequency stimulation (0.3-1 Hz), which consistently evokes fast neuronal Ca 2+ transients (6.0 ± 2.7 ms latency) that always precede vascular responses, does not always elicit astrocytic Ca 2+ transients (313 ± 65 ms latency). However, the magnitude of the hemodynamic response is increased when astrocytic transients occur, suggesting a facilitatory role of astrocytes in NVC. High-frequency stimulation (5-10 Hz) consistently evokes a large, delayed astrocytic Ca 2+ accumulation (3.48 ± 0.09 s latency) that is temporarily associated with vasoconstriction, suggesting a role for astrocytes in resetting NVC. At rest, neuronal, but not astrocytic, Ca 2+ fluctuations correlate with hemodynamic low-frequency oscillations. Taken together, these results support a role for astrocytes in modulating, but not triggering, NVC. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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

PubMed

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.

Activity-dependent ATP-waves in the mouse neocortex are independent from astrocytic calcium waves.

PubMed

Haas, Brigitte; Schipke, Carola G; Peters, Oliver; Söhl, Goran; Willecke, Klaus; Kettenmann, Helmut

2006-02-01

In the corpus callosum, astrocytic calcium waves propagate via a mechanism involving ATP-release but not gap junctional coupling. In the present study, we report for the neocortex that calcium wave propagation depends on functional astrocytic gap junctions but is still accompanied by ATP-release. In acute slices obtained from the neocortex of mice deficient for astrocytic expression of connexin43, the calcium wave did not propagate. In contrast, in the corpus callosum and hippocampus of these mice, the wave propagated as in control animals. In addition to calcium wave propagation in astrocytes, ATP-release was recorded as a calcium signal from 'sniffer cells', a cell line expressing high-affinity purinergic receptors placed on the surface of the slice. The astrocyte calcium wave in the neocortex was accompanied by calcium signals in the 'sniffer cell' population. In the connexin43-deficient mice we recorded calcium signals from sniffer cells also in the absence of an astrocytic calcium wave. Our findings indicate that astrocytes propagate calcium signals by two separate mechanisms depending on the brain region and that ATP release can propagate within the neocortex independent from calcium waves.

Sodium Benzoate, a Metabolite of Cinnamon and a Food Additive, Upregulates Ciliary Neurotrophic Factor in Astrocytes and Oligodendrocytes.

PubMed

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

2015-11-01

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

Glucose Regulates Hypothalamic Long-chain Fatty Acid Metabolism via AMP-activated Kinase (AMPK) in Neurons and Astrocytes*

PubMed Central

Taïb, Bouchra; Bouyakdan, Khalil; Hryhorczuk, Cécile; Rodaros, Demetra; Fulton, Stephanie; Alquier, Thierry

2013-01-01

Hypothalamic controls of energy balance rely on the detection of circulating nutrients such as glucose and long-chain fatty acids (LCFA) by the mediobasal hypothalamus (MBH). LCFA metabolism in the MBH plays a key role in the control of food intake and glucose homeostasis, yet it is not known if glucose regulates LCFA oxidation and esterification in the MBH and, if so, which hypothalamic cell type(s) and intracellular signaling mechanisms are involved. The aim of this study was to determine the impact of glucose on LCFA metabolism, assess the role of AMP-activated Kinase (AMPK), and to establish if changes in LCFA metabolism and its regulation by glucose vary as a function of the kind of LCFA, cell type, and brain region. We show that glucose inhibits palmitate oxidation via AMPK in hypothalamic neuronal cell lines, primary hypothalamic astrocyte cultures, and MBH slices ex vivo but not in cortical astrocytes and slice preparations. In contrast, oleate oxidation was not affected by glucose or AMPK inhibition in MBH slices. In addition, our results show that glucose increases palmitate, but not oleate, esterification into neutral lipids in neurons and MBH slices but not in hypothalamic astrocytes. These findings reveal for the first time the metabolic fate of different LCFA in the MBH, demonstrate AMPK-dependent glucose regulation of LCFA oxidation in both astrocytes and neurons, and establish metabolic coupling of glucose and LCFA as a distinguishing feature of hypothalamic nuclei critical for the control of energy balance. PMID:24240094

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