Primary Cultures Of Rat Astrocytes Research Articles

Modulation of Pyruvate Export and Extracellular Pyruvate Concentration in Primary Astrocyte Cultures

Astrocyte-derived pyruvate is considered to have neuroprotective functions. In order to investigate the processes that are involved in astrocytic pyruvate release, we used primary rat astrocyte cultures as model system. Depending on the incubation conditions and medium composition, astrocyte cultures established extracellular steady state pyruvate concentrations in the range between 150 µM and 300 µM. During incubations for up to 2 weeks in DMEM culture medium, the extracellular pyruvate concentration remained almost constant for days, while the extracellular lactate concentration increased continuously during the incubation into the millimolar concentration range as long as glucose was present. In an amino acid-free incubation buffer, glucose-fed astrocytes released pyruvate with an initial rate of around 60 nmol/(h × mg) and after around 5 h an almost constant extracellular pyruvate concentration was established that was maintained for several hours. Extracellular pyruvate accumulation was also observed, if glucose had been replaced by mannose, fructose, lactate or alanine. Glucose-fed astrocyte cultures established similar extracellular steady state concentrations of pyruvate by releasing pyruvate into pyruvate-free media or by consuming excess of extracellular pyruvate. Inhibition of the monocarboxylate transporter MCT1 by AR-C155858 lowered extracellular pyruvate accumulation, while inhibition of mitochondrial pyruvate uptake by UK5099 increased the extracellular pyruvate concentration. Finally, the presence of the uncoupler BAM15 or of the respiratory chain inhibitor antimycin A almost completely abolished extracellular pyruvate accumulation. The data presented demonstrate that cultured astrocytes establish a transient extracellular steady state concentration of pyruvate which is strongly affected by modulation of the mitochondrial pyruvate metabolism.

Modulation of Multidrug Resistance Protein 1-mediated Transport Processes by the Antiviral Drug Ritonavir in Cultured Primary Astrocytes

The Multidrug Resistance Protein 1 (Mrp1) is an ATP-dependent efflux transporter and a major facilitator of drug resistance in mammalian cells during cancer and HIV therapy. In brain, Mrp1-mediated GSH export from astrocytes is the first step in the supply of GSH precursors to neurons. To reveal potential mechanisms underlying the drug-induced modulation of Mrp1-mediated transport processes, we investigated the effects of the antiviral drug ritonavir on cultured rat primary astrocytes. Ritonavir strongly stimulated the Mrp1-mediated export of glutathione (GSH) by decreasing the Km value from 200 nmol/mg to 28 nmol/mg. In contrast, ritonavir decreased the export of the other Mrp1 substrates glutathione disulfide (GSSG) and bimane-glutathione. To give explanation for these apparently contradictory observations, we performed in silico docking analysis and molecular dynamics simulations using a homology model of rat Mrp1 to predict the binding modes of ritonavir, GSH and GSSG to Mrp1. The results suggest that ritonavir binds to the hydrophilic part of the bipartite binding site of Mrp1 and thereby differently affects the binding and transport of the Mrp1 substrates. These new insights into the modulation of Mrp1-mediated export processes by ritonavir provide a new model to better understand GSH-dependent detoxification processes in brain cells.

Steady-state intracellular pH and recovery from NH3/NH4+-induced intracellular acid loads in primary cultures of rat hippocampal neurons and astrocytes

Intracellular pH (pH i ) homeostasis is of vital importance because pH i fluctuations alter the net charge on intracellular weak acids and bases (e.g., amino-acid residues of proteins, metabolic intermediates, solutes like inorganic phosphate), and can result in cellular dysfunction. The rate of pH i change (dpH i /dt) is determined by the balance between acid loading (e.g., metabolic production of H + , influx of H + , efflux of HCO 3 − via transmembrane proteins of the SLC4 family) and acid extrusion (e.g., efflux of H + via Na-H exchangers in the SLC9 family, influx of HCO 3 − by members of the SLC4 family). A powerful approach for studying pH i regulation is to impose an acute intracellular acid load using the NH 4 + /NH 3 prepulse technique, and then assess dpH i /dt during the subsequent pH i recovery. Using two such prepulses and recoveries (twin pulses), one can compare 2 experimental conditions. The goal of the present study is to develop a data-analysis approach that facilitates a meaningful presentation of all twin-pulse experiments in a data set. Using primary cultures (~2 weeks) of embryonic (E18–E20) rat hippocampal neurons and astrocytes on coverslips, we loaded cells with the ratiometric pH-sensitive fluorescent dye BCECF, subjected the cells to twin NH 4 + /NH 3 pulses, and calibrated BCECF at the end of each experiment with nigericin at pH=7.0. A standard 12-point nigericin calibration curve allowed us to compute pH i vs. time. Because we studied cells in 21% O 2 but the nominal absence of CO 2 /HCO 3 − , the pH i recoveries mainly reflect Na-H exchange. We fitted the pH i -recovery time course with a double exponential (DExp) to reduce bias. Nevertheless, in both neurons and astrocytes plots of dpH i /dt vs. pH i appeared rather linear, indicating that the pH i recoveries were “quasi” single exponential (SExp). To exploit the near-linearity, for each pH i recovery, we fitted the plot of dpH i /dt vs pH i with a line, the slope of which is the quasi-single exponential rate constant (kqSExp), and the x-intercept of which is the asymptotic pH i of the recovery. We summarize the twin pH i recoveries from individual cells as miniature plots (“thumbnails”) in which we display, for each recovery, both the dpH i /dt values (from the DExp fit of pH i vs. time) vs. pH i and the line of best fit for these dpH i /dt values vs. pH i . A single figure can replicate such twin-pulse experiments from many dozens of cells, so that a reader, at a glance, can surmise the essentials of each pH i recovery. The present work provides background data for additional work in which we studied pH i recoveries at a reduced [O 2 ], with or without CO 2 /HCO 3 − . R01 NS018400, R01 HL160857, R01 DK128315 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A molecular imprint of intracellular amyloid-beta ApoE interaction correlates with its toxicity molecular imprint of intracellular amyloid beta ApoE interaction.

ApoE isoforms are major genetic risk factors related to Alzheimer's disease (AD), which is associated with the aggregation of the amyloid beta (Aβ) peptide. However, a molecular-level understanding of how ApoE is involved in AD pathology is still lacking. Here we employ a fluorescent lifetime-based assay that reports a specific ApoE-dependent modification of Aβ, both in vitro and in neuronal cells. We explore this phenomenon in several types of cells where the change of lifetime of Aβ oligomers correlates with the relative ApoE content, with the highest effect displayed by primary-cultured rat astrocytes. At the single-molecule level, ApoE-induced modification also produces distinct Aβ oligomers with increased affinity to artificial lipid bilayers. A known peptide inhibitor of Aβ-ApoE interaction, LVFFA, diminishes both the lifetime modification and toxicity of Aβ oligomers in a dose-dependent manner. The correlation between the short lifetime component and Aβ toxicity was corroborated by experiments using two nontoxic Aβ variants. Our data, therefore, establish a clear correlation between the lifetime modification of Aβ and its toxicity. Finally, neuronal stem cells derived from cells obtained from Alzheimer's patients also show the short-lifetime component, the changed oligomeric state, and the increased membrane affinity, which is likely to be relevant for AD. In summary, our assay can provide a direct readout of the toxic ApoE-Aβ interaction in vitro and in cells, which can provide a platform for AD drug discovery.

TB-3 A GINGER EXTRACT COMPOUND A REVEALED ANTITUMOR ACTIVITY AGAINST GLIOMA

Abstract Purpose Malignant neoplasms arising in the brain and central nervous system have a poor prognosis and present with symptoms such as headache, epileptic seizures, and paralysis of the arms and legs. Treatment of glioma, the most frequent primary brain tumor, is based on surgical removal of the tumor, radiation therapy, and chemotherapy. However, since gliomas grow invasively, surgical removal of the entire tumor is considered difficult. In addition, the standard treatment, temozolomide, possesses the problem of resistance. Therefore, the risk of recurrence is high, and the development of noble therapeutic agents for Glioma is required. We have screened for compounds with anti-tumor activity against Glioma from our natural compound library and focused on ginger extract, Compound A. In this study, we investigated the effect of Compound A on Glioma cell lines. Methods and Results To evaluate the antitumor activity of Compound A on Glioma in vitro , MTT assay was performed. Human glioma cell lines U87-MG and U251 cells were treated with Compound A. After 96 hours, cell viability was evaluated using CCK-8. The IC50 value of Compound A in U87-MG cells was calculated to be 16.3 μM. The IC50 of Compound A in U251 cells was 10.8 μM. Next, to examine the effect of Compound A on normal cells, we performed the same MTT assay using primary cultured rat astrocytes. The MTT assay showed no decrease in cell viability in primary cultured rat astrocytes at 15 μM, whereas an antitumor effect was observed in a human glioma cell line. Discussion In this study, Compound A showed antitumor activity against Glioma without affecting normal cells. In the future, we will examine the effects of Compound A in an immunocompromised mouse brain in which U87-MG-RFP cells are implanted, which may lead to the development of therapeutic agents against Glioma.

CBMS-3 SEARCH FOR COMPOUNDS WITH ANTI-TUMOUR EFFECTS AGAINST GLIOBLASTOMA

Abstract Purpose Glioblastoma (GBM) has a high risk of recurrence and a poor prognosis due to the difficulty of surgical resection and the resistance to temozolomide, the standard treatment for GBM. Therefore, the development of new therapeutic agents for GBM is desired. We searched our compound library for compounds with anti-tumor activity against GBM and identified Curcumin (Cur) derivatives, Compound A and B. The purpose of this study was to investigate the antitumor activity of Compound A and B against GBM. Methods and Results To evaluate the antitumor activity of Compound A and B against GBM, we performed the MTT assay. Human glioblastoma cell lines, U87-MG and U251 cells, were treated with Compound A and B. After 96 hours, cell viability was evaluated using CCK-8, and the IC50 values of Compound A and B were calculated. In U87-MG, the IC50 of Cur was 9.78 μM, whereas the that of Compound A was 2.42 μM and that of Compound B was 1.28 μM. In U251 cells, the IC50 of Cur was 9.50 μM, whereas that of Compound A was 2.27 μM and that of Compound B was 0.64 μM. Next, to examine the effects of Compound A and B on normal cells, we performed the same MTT assay using primary cultured rat astrocytes. At the concentrations at which antitumor effects were observed (Compound A; 3 μM, B; 1.5 μM), there was no reduction in cell viability in primary rat cultured astrocytes. Discussion The present study shows that Compound A and B exhibit antitumor activity against human glioblastoma cells at lower concentrations than Cur without affecting normal cells. We will examine the effects of Compound A and B in a mouse model of brain tumor transplanted with U87-RFP cells in the future, which may lead to the development of noble brain tumor therapeutics.

Poly(I:C) promotes neurotoxic amyloid β accumulation through reduced degradation by decreasing neprilysin protein levels in astrocytes.

Inflammation associated with viral infection of the nervous system has been involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease (AD) and multiple sclerosis. Polyinosinic:polycytidylic acid (poly[I:C]) is a Toll-like receptor 3 (TLR3) agonist that mimics the inflammatory response to systemic viral infections. Despite growing recognition of the role of glial cells in AD pathology, their involvement in the accumulation and clearance of amyloid β (Aβ) in the brain of patients with AD is poorly understood. Neprilysin (NEP) and insulin-degrading enzyme (IDE) are the main Aβ-degrading enzymes in the brain. This study investigated whether poly(I:C) regulated Aβ degradation and neurotoxicity by modulating NEP and IDE protein levels through TLR3 in astrocytes. To this aim, primary rat primary astrocyte cultures were treated with poly(I:C) and inhibitors of the TLR3 signaling. Protein levels were assessed by Western blot. Aβ toxicity to primary neurons was measured by lactate dehydrogenase release. Poly(I:C) induced a significant decrease in NEP levels on the membrane of astrocytes as well as in the culture medium. The degradation of exogenous Aβ was markedly delayed in poly(I:C)-treated astrocytes. This delay significantly increased the neurotoxicity of exogenous Aβ1-42. Altogether, these results suggest that viral infections induce Aβ neurotoxicity by decreasing NEP levels in astrocytes and consequently preventing Aβ degradation.

Aβ1-42 peptides blunt the adenosine A2A receptor-mediated control of the interplay between P2X7 and P2Y1 receptors mediated calcium responses in astrocytes.

The contribution of astrocytes to Alzheimer's disease (AD) is still ill defined. AD involves an abnormal accumulation of amyloid-β peptides (Aβ) and increased production of danger signals such as ATP. ATP can direct or indirectly, through its metabolism into adenosine, trigger adaptive astrocytic responses resulting from intracellular Ca2+ oscillations. AD also triggers an upregulation of astrocytic adenosine A2A receptors (A2AR), which blockade prevents memory dysfunction in AD. We now investigated how Aβ peptides affect ATP-mediated Ca2+ responses in astrocytes measured by fluorescence live-cell imaging and whether A2AR control astrocytic Ca2+ responses mediated by ATP receptors, mainly P2X7R and P2Y1R. In primary cultures of rat astrocytes exposed to Aβ1-42, ATP-evoked Ca2+ responses had a lower amplitude but a longer duration than in control astrocytes and involved P2X7R and P2Y1R, the former potentiating the later. Moreover, Aβ1-42 exposure increased protein levels of P2Y1R in astrocytes. A2AR antagonism with SCH58261 controlled in a protein kinase A-dependent manner both P2X7R- and P2Y1R-mediated Ca2+ responses in astrocytes. The interplay between these purinoceptors in astrocytes was blunted upon exposure to Aβ1-42. These findings uncover the ability of A2AR to regulate the inter-twinned P2X7R- and P2Y1R-mediated Ca2+ dynamics in astrocytes, which is disrupted in conditions of early AD.

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis.

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells. The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

Brivaracetam and Levetiracetam Suppress Astroglial L-Glutamate Release through Hemichannel via Inhibition of Synaptic Vesicle Protein.

To explore the pathophysiological mechanisms of antiseizure and adverse behavioural/psychiatric effects of brivaracetam and levetiracetam, in the present study, we determined the effects of brivaracetam and levetiracetam on astroglial L-glutamate release induced by artificial high-frequency oscillation (HFO) bursts using ultra-high-performance liquid chromatography. Additionally, the effects of brivaracetam and levetiracetam on protein expressions of connexin43 (Cx43) and synaptic vesicle protein 2A (SV2A) in the plasma membrane of primary cultured rat astrocytes were determined using a capillary immunoblotting system. Acutely artificial fast-ripple HFO (500 Hz) burst stimulation use-dependently increased L-glutamate release through Cx43-containing hemichannels without affecting the expression of Cx43 or SV2A in the plasma membrane, whereas acute physiological ripple HFO (200 Hz) stimulation did not affect astroglial L-glutamate release or expression of Cx43 or SV2A. Contrarily, subchronic ripple HFO and acute pathological fast-ripple HFO (500 Hz) stimulations use-dependently increased L-glutamate release through Cx43-containing hemichannels and Cx43 expression in the plasma membrane. Subchronic fast-ripple HFO-evoked stimulation produced ectopic expression of SV2A in the plasma membrane, but subchronic ripple HFO stimulation did not generate ectopic SV2A. Subchronic administration of brivaracetam and levetiracetam concentration-dependently suppressed fast-ripple HFO-induced astroglial L-glutamate release and expression of Cx43 and SV2A in the plasma membrane. In contrast, subchronic ripple HFO-evoked stimulation induced astroglial L-glutamate release, and Cx43 expression in the plasma membrane was inhibited by subchronic levetiracetam administration, but was not affected by brivaracetam. These results suggest that brivaracetam and levetiracetam inhibit epileptogenic fast-ripple HFO-induced activated astroglial transmission associated with hemichannels. In contrast, the inhibitory effect of therapeutic-relevant concentrations of levetiracetam on physiological ripple HFO-induced astroglial responses probably contributes to the adverse behavioural/psychiatric effects of levetiracetam.

Curcumin Facilitates Aryl Hydrocarbon Receptor Activation to Ameliorate Inflammatory Astrogliosis.

Curcumin is an anti-inflammatory and neuroprotective compound in turmeric. It is a potential ligand of the aryl hydrocarbon receptor (AhR) that mediates anti-inflammatory signaling. However, the AhR-mediated anti-inflammatory effect of curcumin within the brain remains unclear. We investigated the role of AhR on the curcumin effect in inflammatory astrogliosis. Curcumin attenuated lipopolysaccharide (LPS)-induced proinflammatory IL-6 and TNF-α gene expression in primary cultured rat astrocytes. When AhR was knocked down, LPS-induced IL-6 and TNF-α were increased and curcumin-decreased activation of the inflammation mediator NF-κB p65 by LPS was abolished. Although LPS increased AhR and its target gene CYP1B1, curcumin further enhanced LPS-induced CYP1B1 and indoleamine 2,3-dioxygenase (IDO), which metabolizes tryptophan to AhR ligands kynurenine (KYN) and kynurenic acid (KYNA). Potential interactions between curcumin and human AhR analyzed by molecular modeling of ligand–receptor docking. We identified a new ligand binding site on AhR different from the classical 2,3,7,8-tetrachlorodibenzo-p-dioxin site. Curcumin docked onto the classical binding site, whereas KYN and KYNA occupied the novel one. Moreover, curcumin and KYNA collaboratively bound onto AhR during molecular docking, potentially resulting in synergistic effects influencing AhR activation. Curcumin may enhance the inflammation-induced IDO/KYN axis and allosterically regulate endogenous ligand binding to AhR, facilitating AhR activation to regulate inflammatory astrogliosis.

Activated glia cells cause bioenergetic impairment of neurons that can be rescued by knock-down of the mitochondrial calcium uniporter

Neuroinflammation is a hallmark of various neurological disorders including autoimmune-, neurodegenerative and neuropsychiatric diseases. In neuroinflammation, activated microglia and astrocytes release soluble mediators such as cytokines, glutamate, and reactive oxygen species that negatively affect neuronal function and viability, and thus contribute to neurodegeneration during disease progression. Therefore, the development of neuroprotective strategies might be important in addition to treating inflammation in these diseases. Mitochondria are promising cellular targets for neuroprotective interventions: They are among the first structures affected in many neuroinflammatory diseases, with mitochondrial impairment ranging from impaired respiratory activity and reduced mitochondrial membrane potential to mitochondrial oxidation and fragmentation. Therefore, we developed a cell culture model that resembles an early state of inflammation-induced neuronal mitochondrial dysfunction preceding neuronal cell death, and can be used to test mito- and neuroprotective strategies. Rat primary cortical neurons were challenged with conditioned medium from mixed primary cultures of rat microglia and astrocytes that had been activated with lipopolysaccharide and ATP. When sublethal amounts of glia-conditioned medium were added to neurons for 24 h, mitochondrial membrane potential and ATP levels were decreased, whereas mitochondrial redox state remained unaffected. Effects on mitochondrial membrane potential and ATP levels were ameliorated by knock-down of the mitochondrial calcium uniporter in neurons. This study suggests that neuronal bioenergetic failure is an early event during neuroinflammation and it identifies the mitochondrial calcium uniporter as a candidate target for neuroprotection in this context.

Effect of Thiazolidin-4-one Against Lipopolysaccharide-Induced Oxidative Damage, and Alterations in Adenine Nucleotide Hydrolysis and Acetylcholinesterase Activity in Cultured Astrocytes.

Astrocytes play multiple important roles in brain physiology. However, depending on the stimuli, astrocytes may exacerbate inflammatory reactions, contributing to the development and progression of neurological diseases. Therefore, therapies targeting astrocytes represent a promising area for the development of new brain drugs. Thiazolidinones are heterocyclic compounds that have a sulfur and nitrogen atom and a carbonyl group in the ring and represent a class of compounds of great scientific interest due to their pharmacological properties. The aim of this study was to investigate the effect of 3-(3-(diethylamino)propyl)-2-(4-(methylthio)phenyl)thiazolidin-4-one (DS27) on cell proliferation and morphology, oxidative stress parameters, activity of the enzymes ectonucleotidases and acetylcholinesterase (AChE) and interleukin 6 (IL-6) levels in primary astrocyte cultures treated with lipopolysaccharide (LPS), to model neuroinflammation. The astrocyte culture was exposed to LPS (10μg/ml) for 3h and subsequently treated with compound DS27 for 24 and 48h (concentrations ranging to 10-100μM). LPS induced an increase in astrocyte proliferation, AChE activity, IL-6 levels, oxidative damage, ATP and ADP and a reduction in AMP hydrolysis in rat primary astrocyte cultures. DS27 treatment was effective in reversing these alterations induced by LPS. Our findings demonstrated that DS27 is able to modulate cholinergic and purinergic signaling, redox status, and the levels of pro-inflammatory cytokines in LPS-induced astrocyte damage. These glioprotective effects of DS27 may be very important for improving neuroinflammation, which is associated with many brain diseases.

Thrombospondin type 1 repeat-derived C-mannosylated peptide attenuates synaptogenesis of cortical neurons induced by primary astrocytes via TGF-β.

C-Mannosylation is a rare type of protein glycosylation and is reportedly critical for the proper folding and secretion of parental proteins. Still, the effects of C-mannosylation on the biological functions of these modified proteins remain to be elucidated. The Trp-x-x-Trp (WxxW) sequences, whose first tryptophan (Trp) can be C-mannosylated, constitute the consensus motifs for this glycosylation modification and are commonly found in thrombospondin type 1 repeats that regulate molecular functions of thrombospondin 1 in binding and activation of transforming growth factor β (TGF-β). TGF-β plays critical roles in the control of the central nervous system including synaptogenesis. Here, we investigated whether C-mannosylation of the synthetic Trp-Ser-Pro-Trp (WSPW) peptide may confer certain functions to this peptide in TGF-β-mediated synaptogenesis. By using primary cultured rat astrocytes and cortical neurons, we found that the C-mannosylated WSPW (C-Man-WSPW) peptide, but not non-mannosylated WSPW peptide, suppressed astrocyte-conditioned medium (ACM)-stimulated synaptogenesis. C-Man-WSPW peptide inhibited both ACM- and recombinant mature TGF-β1-induced activations of Smad 2, an important mediator in TGF-β signaling. Interactions of recombinant mature TGF-β with the C-Man-WSPW peptide were similar to those with non-C-mannosylated WSPW peptide. Taken together, our results reveal a novel function of C-mannosylation of the WxxW motif in signaling and synaptogenesis mediated by TGF-β. Molecular details of how C-mannosylation affects the biological functions of WxxW motifs deserve future study for clarification.

Reactive astrocytes induced by 2-chloroethanol modulate microglia polarization through IL-1β, TNF-α, and iNOS upregulation

The synthetic organic chemical, 1,2-dichloroethane (1,2-DCE), can cause brain edemas under subacute poisoning. Our previous studies indicated that neuroinflammation could be induced due to astrocytes and microglia activation during brain edemas in 1,2-DCE-intoxicated mice. However, the crosstalk between these two glial cells in 1,2-DCE-induced neuroinflammation remained unclear. In this study, primary cultured rat astrocytes and microglia, as well as an immortalized microglia cell line were employed to study the effects of 2-chloroethanol (2-CE, a 1,2-DCE intermediate metabolite in vivo) treated astrocytes on microglia polarization. Our current results revealed that 2-CE treated rat astrocytes were activated through p38 mitogen-activated protein kinase (p38 MAPK)/nuclear factor-κB (NF-κB), and activator protein-1 (AP-1) signaling pathways. Theses pathways were triggered by reactive oxygen species (ROS) produced during 2-CE metabolism. Also, astrocytes were more sensitive to 2-CE effects than microglia. Interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase (iNOS) expressions were upregulated in 2-CE-induced reactive astrocytes, enhancing IL-1β, TNF-α, and nitric oxide (NO) excretions, which stimulated microglia polarization. Therefore, the neuroinflammation induced by 1,2-DCE in mice's brains is probably triggered by reactive astrocytes.

Differential production of interleukin‐6 and tumor necrosis factor‐α in primary rat astrocyte cultures using two distinct methods of microglia elimination

AbstractHepatic encephalopathy (HE) is a neurocognitive disorder due to acute or chronic liver failure as well as portosystemic bypass. Both neuroinflammation and oxidative stress have been strongly implicated in the pathogenesis of HE. An increasing body of evidence suggests that in the development of these mechanisms astrocytes may be involved. With regard of HE, this aspect of astrocytes is, however, neglected. While several studies focus on astrocytes related to the above‐mentioned pathological processes, the applied in vitro models are generated by diverse methods, which make the results difficult to compare with each other. In addition, these astrocyte cultures frequently contain microglia as well, complicating the interpretation of results. Therefore, we applied enriched primary rat astrocyte cultures generated by microglia elimination via shaking or the combination of cytosine β‐d‐arabinofuranoside hydrochloride (AraC) and l‐leucine methyl ester (LME). We examine whether some HE‐relevant pathological factors, such as ammonia, manganese (Mn), lipopolysaccharide (LPS), and H2O2 could elicit oxidative stress and cytokine production in astrocytes. Mn exposure increased the oxidative stress in astrocytes in a time‐dependent manner; furthermore, we could detect tumor necrosis factor (TNF)‐α production in both cultures at resting state. In addition, it was around 10‐fold higher in the cultures after shaking. LPS increased TNF‐α secretion in cultures after shaking, but not in cultures after AraC + LME treatment. In contrast, interleukin (IL)‐6 was detectable only in the AraC + LME‐treated cultures. In conclusion, we have demonstrated that HE‐relevant factors evoke oxidative stress and cytokine production, albeit depending on the elimination method used.

Regulation and role of Acyl-CoA synthetase 4 in glial cells

Acyl-CoA synthetase 4 (Acsl4), an enzyme involved in arachidonic acid (AA) metabolism, participates in physiological and pathological processes such as steroidogenesis and cancer. The role of Acsl4 in neurons and in nervous system development has also been documented but little is known regarding its functionality in glial cells. In turn, several processes in glial cells, including neurosteroidogenesis, stellation and AA uptake, are regulated by cyclic adenosine monophosphate (cAMP) signal. In this context, the aim of this work was to analyze the expression and functional role of Acsl4 in primary rat astrocyte cultures and in the C6 glioma cell line by chemical inhibition and stable silencing, respectively. Results show that Acsl4 expression was regulated by cAMP in both models and that cAMP stimulation of steroidogenic acute regulatory protein mRNA levels was reduced by Acsl4 inhibition or silencing. Also, Acsl4 inhibition reduced progesterone synthesis stimulated by cAMP and also affected cAMP-induced astrocyte stellation, decreasing process elongation and increasing branching complexity. Similar effects were observed for Acsl4 silencing on cAMP-induced C6 cell morphological shift. Moreover, Acsl4 inhibition and silencing reduced proliferation and migration of both cell types. Acsl4 silencing in C6 cells reduced the capacity for colony proliferation and neurosphere formation, the latter ability also being abolished by Acsl4 inhibition. In sum, this work presents novel evidence of Acsl4 involvement in neurosteroidogenesis and the morphological changes of glial cells promoted by cAMP. Furthermore, Acsl4 participates in migration and proliferation, also affecting cell self-renewal. Altogether, these findings provide insights into Acsl4 functions in glial cells.

Neuroprotective potential of isothiocyanates in an in vitro model of neuroinflammation

Isothiocyanates (ITCs), present as glucosinolate precursors in cruciferous vegetables, have shown anti-inflammatory, antioxidant and anticarcinogenic activities. Here, we compared the effects of three different ITCs on ROS production and on the expression of matrix metalloproteinase (MMP)-2 and -9, which represent important pathogenetic factors of various neurological diseases. Primary cultures of rat astrocytes were activated by LPS and simultaneously treated with different doses of Allyl isothiocyanate (AITC), 2-Phenethyl isothiocyanate (PEITC) and 2-Sulforaphane (SFN). Results showed that SFN and PEITC were able to counteract ROS production induced by H2O2. The zymographic analysis of cell culture supernatants evidenced that PEITC and SFN were the most effective inhibitors of MMP-9, whereas, only SFN significantly inhibited MMP-2 activity. PCR analysis showed that all the ITCs used significantly inhibited both MMP-2 and MMP-9 expression. The investigation on the mitogen-activated protein kinase (MAPK) signaling pathway demonstrated that ITCs modulate MMP transcription by inhibition of extracellular-regulated protein kinase (ERK) activity. Results of this study suggest that ITCs could be promising nutraceutical agents for the prevention and complementary treatment of neurological diseases associated with MMP involvement.

Astrocytes from cortex and striatum show differential responses to mitochondrial toxin and BDNF: implications for protection of striatal neurons expressing mutant huntingtin

BackgroundEvidence shows significant heterogeneity in astrocyte gene expression and function. We previously demonstrated that brain-derived neurotrophic factor (BDNF) exerts protective effects on whole brain primary cultured rat astrocytes treated with 3-nitropropionic acid (3NP), a mitochondrial toxin widely used as an in vitro model of Huntington’s disease (HD). Therefore, we now investigated 3NP and BDNF effects on astrocytes from two areas involved in HD: the striatum and the entire cortex, and their involvement in neuron survival.MethodsWe prepared primary cultured rat cortical or striatal astrocytes and treated them with BDNF and/or 3NP for 24 h. In these cells, we assessed expression of astrocyte markers, BDNF receptor, and glutamate transporters, and cytokine release. We prepared astrocyte-conditioned medium (ACM) from cortical and striatal astrocytes and tested its effect on a cellular model of HD.ResultsBDNF protected astrocytes from 3NP-induced death, increased expression of its own receptor, and activation of ERK in both cortical and striatal astrocytes. However, BDNF modulated glutamate transporter expression differently by increasing GLT1 and GLAST expression in cortical astrocytes but only GLT1 expression in striatal astrocytes. Striatal astrocytes released higher amounts of tumor necrosis factor-α than cortical astrocytes in response to 3NP but BDNF decreased this effect in both populations. 3NP decreased transforming growth factor-β release only in cortical astrocytes, whereas BDNF treatment increased its release only in striatal astrocytes. Finally, we evaluated ACM effect on a cellular model of HD: the rat striatal neuron cell line ST14A expressing mutant human huntingtin (Q120) or in ST14A cells expressing normal human huntingtin (Q15). Neither striatal nor cortical ACM modified the viability of Q15 cells. Only ACM from striatal astrocytes treated with BDNF and ACM from 3NP + BDNF-treated striatal astrocytes protected Q120 cells, whereas ACM from cortical astrocytes did not.ConclusionsData suggest that cortical and striatal astrocytes respond differently to mitochondrial toxin 3NP and BDNF. Moreover, striatal astrocytes secrete soluble neuroprotective factors in response to BDNF that selectively protect neurons expressing mutant huntingtin implicating that BDNF modulation of striatal astrocyte function has therapeutic potential against neurodegeneration.Graphical abstract