Characterization of mouse cell line IMA 2.1 as a potential model system to study astrocyte functions

Schildknecht, Stefan; Kirner, Susanne; Henn, Anja; Gasparic, Karlo; Pape, Regina; Efremova, Liudmila V.; Maier, Olaf; Fischer, Roman; Leist, Marcel

doi:10.14573/altex.2012.3.261

Cited by 19 publications

(23 citation statements)

References 35 publications

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“…For differentiation, the cells were seeded at a density of 1.5*10 5 cells per cm 2 in differentiation medium containing 1 mM dibutyryl cAMP (Sigma-Aldrich), 10 μg/ml tetracycline (Sigma-Aldrich), and 2 ng/ml human GDNF (R&D Systems). The immortalized murine astrocytes (IMA2.1) were passaged in DMEM supplemented with 10 % FCS (fetal calf serum, PAA) and 1 % of penicillin/streptomycin (Schildknecht et al 2012). Normal human astrocytes (NHA) were purchased from LONZA (catalog number CC-2565, Cologne, Germany) and grown in medium consisting of astrocyte basal medium (ABM™, catalog number CC-3187, LONZA) supplemented with AGM SingleQuot Kit Supplement & Growth Factors (catalog number CC-4123, LONZA).…”

Section: Cell Culturementioning

confidence: 99%

“…Nitrite (NO 2 − ), the stable autoxidation product of · NO, was measured by the Griess assay exactly as described earlier (Schildknecht et al 2012). Nitrite concentrations were calculated from a NaNO 2 standard curve.…”

Section: No Releasementioning

confidence: 99%

“…To model an inflammatory situation, as it occurs during brain damage and neurodegenerative disease, we used a cytokine mix (TNF-α and IL-1β) known to be produced in neuroinflammatory situations (mainly by microglia) and known to activate astrocytic cells, including IMA (Falsig et al 2006;Henn et al 2011;Kuegler et al 2012;Schildknecht et al 2012). This CM had no effect on the viability and neurite structure of LUHMES monocultures (Fig.…”

Section: Switching From Neuroprotective To Neurotoxic Properties Of Amentioning

confidence: 99%

“…The pathological and toxicological role of astrocytes may be highly model-specific, as they contribute to the metabolism of some toxicants (Schildknecht et al 2012) and they show a large plasticity, e.g., by taking the role of stem cells (Robel et al 2011). Moreover, astrocytes and microglia produce a large variety of different mediators that may affect neurons, e.g., direct excitotoxicants, like glutamate, or indirect excitotoxic mediators, such as NO (Bal-Price and Brown 2001;Bal-Price et al 2002;Gandelman et al 2010;Gegg and Clark 1036;Mander et al 2005), lipid mediators/small molecules (Mayo et al 2014;Simon et al 2002;Wang et al 2012), reactive oxygen species (Ma et al 2013), proteases (cathepsin B), cytokines (Lee et al 2013a;Mattson et al 1997), complement factors (Pekny et al 2007;Walker et al 1998), but their respective contribution to human pathology needs further clarification (Lioy et al 2011;Williams et al 2014).…”

Section: Introductionmentioning

confidence: 99%

“…These cells are conditionally immortalized, and they assume neuronal morphological, electrophysiological, and neurochemical properties upon the switching off of the v-myc transgene by tetracycline. The cells can be cultured on a layer of astrocytes (Efremova et al 2015;Kuegler et al 2012), and they form highly reproducible co-cultures when combined with the immortalized murine astrocytic cell line IMA (Schildknecht et al 2012). The latter cells can metabolize drugs, such as 1-methyl-4-phenyl-tetrahydropyridine (MPTP), and are well characterized in their inflammatory response.…”

Section: Introductionmentioning

confidence: 99%

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Switching from astrocytic neuroprotection to neurodegeneration by cytokine stimulation

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were involved in this neurodegeneration. The neurotoxicity-mediating effect of IMA was faithfully reproduced by human astrocytes. Moreover, glia-dependent toxicity was also observed, when IMA cultures were stimulated with CM, and the culture medium was transferred to neurons. Such neurotoxicity was prevented when astrocytes were treated by p38 kinase inhibitors or dexamethasone, whereas such compounds had no effect when added to neurons. Conversely, treatment of neurons with five different drugs, including resveratrol and CEP1347, prevented toxicity of astrocyte supernatants. Thus, the sequential IMA-LUHMES neuroinflammation model is suitable for separate profiling of both glial-directed and directly neuroprotective strategies. Moreover, direct evaluation in co-cultures of the same cells allows for testing of therapeutic effectiveness in more complex settings, in which astrocytes affect pharmacological properties of neurons.Keywords LUHMES · Astrocyte · p38 kinase · Neuroinflammation · Neuropharmacology Abstract Astrocytes, the largest cell population in the human brain, are powerful inflammatory effectors. Several studies have examined the interaction of activated astrocytes with neurons, but little is known yet about human neurotoxicity under such situations and about strategies of neuronal rescue. To address this question, immortalized murine astrocytes (IMA) were combined with human LUHMES neurons and stimulated with an inflammatory (TNF, IL-1) cytokine mix (CM). Neurotoxicity was studied both in co-cultures and in monocultures after transfer of conditioned medium from activated IMA. Interventions with >20 drugs were used to profile the model system. Control IMA supported neurons and protected them from neurotoxicants. Inflammatory activation reduced this protection, and prolonged exposure of co-cultures to CM triggered neurotoxicity. Neither the added cytokines nor the release of NO from astrocytes Liudmila Efremova and Petra Chovancova have contributed equally to this study.

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Section: Cell Culturementioning

confidence: 99%

Section: No Releasementioning

confidence: 99%

Section: Switching From Neuroprotective To Neurotoxic Properties Of Amentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Switching from astrocytic neuroprotection to neurodegeneration by cytokine stimulation

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In vitro acute and developmental neurotoxicity screening: an overview of cellular platforms and high-throughput technical possibilities

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Neurotoxicity and developmental neurotoxicity are important issues of chemical hazard assessment. Since the interpretation of animal data and their extrapolation to man is challenging, and the amount of substances with information gaps exceeds present animal testing capacities, there is a big demand for in vitro tests to provide initial information and to prioritize for further evaluation. During the last decade, many in vitro tests emerged. These are based on animal cells, human tumour cell lines, primary cells, immortalized cell lines, embryonic stem cells, or induced pluripotent stem cells. They differ in their read-outs and range from simple viability assays to complex functional endpoints such as neural crest cell migration. Monitoring of toxicological effects on differentiation often requires multiomics approaches, while the acute disturbance of neuronal functions may be analysed by assessing electrophysiological features. Extrapolation from in vitro data to humans requires a deep understanding of the test system biology, of the endpoints used, and of the applicability domains of the tests. Moreover, it is important that these be combined in the right way to assess toxicity. Therefore, knowledge on the advantages and disadvantages of all cellular platforms, endpoints, and analytical methods is essential when establishing in vitro test systems for different aspects of neurotoxicity. The elements of a test, and their evaluation, are discussed here in the context of comprehensive prediction of potential hazardous effects of a compound. We summarize the main cellular characteristics underlying neurotoxicity, present an overview of cellular platforms and read-out combinations assessing distinct parts of acute and developmental neurotoxicology, and highlight especially the use of stem cell-based test systems to close gaps in the available battery of tests.

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Epithelial growth factor receptor expression influences 5-ALA induced glioblastoma fluorescence

et al. 2017

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The extent of 5-aminolevulinic acid (5-ALA) guided tumor resection has a determining impact in high-grade glioma and glioblastoma surgery. Yet the intensity of the 5-ALA induced fluorescence may vary within the tumor. We aimed to correlate 5-ALA induced fluorescence with the expression of epithelial growth factor receptor (EGFR) and its constitutively active version EGFRvIII in different glioblastoma (GBM) cell lines. To elucidate the role of EGFR in the metabolism of 5-ALA in GBM cell lines with variable EGFR expression status, we analyzed the activation of EGFR by its primary ligand EGF, and its downstream effect on Heme oxygenase-1 (HO-1), a key enzyme regulating the metabolism of Protoporphyrin IX (PpIX), the fluorescent metabolite of 5-ALA. Effects of direct pharmacological inhibition by Tin(IV)-Protoporphyrin (SnPP) or gene knockdown by small interfering RNA (siRNA) on HO-1 enzyme were analyzed in respect to 5-ALA induced fluorescence. Furthermore, inhibition of EGFR by Gefitinib was tested. A significant difference in 5-ALA induced fluorescence was obtained in U87MG (low EGFR expression) and LN229EGFR cells (EGFR overexpression) compared to BS153 (EGFR overexpression/EGFRvIII+). Treatment of U87MG and LN229EGFR cells with EGF significantly reduced cellular fluorescence, by promoting HO-1 transcription and expression in a concentration-dependent manner. This effect could be reversed by EGFR-specific siRNA treatment, which reduced protein expression of about 80% in U87MG. Remarkably, inhibition of HO-1 activity by SnPP or reduction of HO-1 protein levels by siHO-1 treatment restored fluorescence in all cell lines, independently of EGFR quantitative and qualitative expression. Gefitinib treatment was able to restore fluorescence after EGF stimulation in U87MG cells but not in BS153 cells, overexpressing EGFR/EGFRvIII. In GBM cell lines, 5-ALA induced fluorescence is variable and influenced by EGF-induced downstream activation of HO-1. HO-1 protein expression was identified as a negative regulator of 5-ALA induced fluorescence in GBM cells. We further propose that co-expression of EGFRvIII but not quantitative EGFR expression influence HO-1 activity and therefore cellular fluorescence.Electronic supplementary materialThe online version of this article (doi:10.1007/s11060-017-2474-0) contains supplementary material, which is available to authorized users.

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Characterization of mouse cell line IMA 2.1 as a potential model system to study astrocyte functions

Abstract: Summary Astrocytes are activated in most chronic neurodegenerative diseases associated with inflammatory

Cited by 19 publications

References 35 publications

Switching from astrocytic neuroprotection to neurodegeneration by cytokine stimulation

Switching from astrocytic neuroprotection to neurodegeneration by cytokine stimulation

In vitro acute and developmental neurotoxicity screening: an overview of cellular platforms and high-throughput technical possibilities

Epithelial growth factor receptor expression influences 5-ALA induced glioblastoma fluorescence

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