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Chronic Inflammation Alters Production and Release of Glutathione and Related Thiols in Human U373 Astroglial Cells

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Abstract

Neurons rely on glutathione (GSH) and its degradation product cysteinylglycine released by astrocytes to maintain their antioxidant defences. This is particularly important under conditions of inflammation and oxidative stress, as observed in many neurodegenerative diseases including Alzheimer’s disease (AD). The effects of inflammatory activation on intracellular GSH content and the extracellular thiol profile (including cysteinylglycine and homocysteine) of astrocytes were investigated. U373 astroglial cells exposed to IL-1β and TNF-α for up to 96 h showed a dose-dependent increase in IL-6 release, indicative of increasing pro-inflammatory cellular activation. With increasing concentrations of IL-1β and TNF-α (0.01–1 ng/ml), an increase in both intracellular and extracellular GSH levels was observed, followed by a return to control levels in response to higher concentrations of IL-1β and TNF-α. Extracellular levels of cysteinylglycine decreased in response to all concentrations of IL-1β and TNF-α. In contrast, levels of the neurotoxic thiol homocysteine increased in a dose-dependent manner to IL-1β and TNF-α-induced activation. Our results suggest that chronically activated astrocytes in the brain might fail to adequately maintain GSH substrate delivery to neurons, thus promoting neuronal vulnerability. They might also explain the elevated levels of homocysteine found in the brains and serum of patients with AD.

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Abbreviations

ABD-F:

4-Flouro-7-aminosulfonylbenzofurazan

AD:

Alzheimer’s disease

ANOVA:

Analysis of variance

ARE:

Antioxidant response element

CSF:

Cerebral spinal fluid

DMEM:

Dulbecco’s modified eagle medium

DTNB:

5,5′-Dithio-bis(2-nitrobenzoic acid)

EDTA:

Ethylenediaminetetraacetic acid

ELISA:

Enzyme-linked immunosorbent sandwich assay

FBS:

Foetal bovine serum

GCL:

Glutamate cysteine ligase

GSH:

Glutathione

GSSG:

Glutathione disulfide

HPLC:

High performance liquid chromatography

IL-1β:

Interleukin-1 beta

IL-6:

Interleukin-6

Keap1:

Kelch like-ECH-associated protein 1

MRP1:

Multidrug resistance protein 1

NADPH:

Reduced nicotinamide adenine dinucleotide phosphate

NMDA:

N-Methyl-D-aspartate

Nrf2:

Nuclear factor erythroid-2-related factor 2

PD:

Parkinson’s disease

ROS:

Reactive oxygen species

TNB:

5-Thio-2-nitrobenzoic acid

TNF-α:

Tumour necrosis factor-alpha

γ-GT:

γ-Glutamyltranspeptidase

γ-GCL-C:

Catalytic glutamate cysteine ligase subunit

γGCL-M:

Modulatory glutamate cysteine ligase subunit

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Acknowledgments

We thank UWS for support through the College Research Grant scheme and Alzheimer’s Australia for their support through the Dementia Research grants program. Megan L. Steele was supported by the Hunter Postgraduate Research Scholarship from Alzheimer’s Australia.

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Correspondence to Gerald Münch.

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Steele, M.L., Fuller, S., Maczurek, A.E. et al. Chronic Inflammation Alters Production and Release of Glutathione and Related Thiols in Human U373 Astroglial Cells. Cell Mol Neurobiol 33, 19–30 (2013). https://doi.org/10.1007/s10571-012-9867-6

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  • DOI: https://doi.org/10.1007/s10571-012-9867-6

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