Neuroscience Letters 490 (2011) 27–30 Contents lists available at ScienceDirect Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet Inactivation of astrocytic glutamine synthetase by hydrogen peroxide requires iron S.P. Fernandes a , R. Dringen a,b , A. Lawen c , S.R. Robinson a,∗ a Blood-Brain Interactions Group, School of Psychology and Psychiatry, Monash University, Victoria 3800, Australia b Center for Biomolecular Interactions Bremen, University of Bremen, Bremen, Germany c Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Victoria 3800, Australia article info Article history: Received 1 October 2010 Received in revised form 3 December 2010 Accepted 8 December 2010 Keywords: Glutamate–ammonia ligase Fenton reaction Oxidative stress Iron chelator Primary astrocyte culture abstract The specific activity of brain glutamine synthetase (GS) is lowered in several neurodegenerative diseases that involve iron-mediated oxidative stress. The present study has investigated whether H 2 O 2 directly inactivates GS or whether GS is primarily inactivated by hydroxyl radicals that are produced by the Fenton reaction when H 2 O 2 reacts with ferrous iron. Exposure of purified sheep brain GS to supraphysiological concentrations of H 2 O 2 (1 mM for 30 min) reduced its specific activity by only 41%, indicating that the enzyme is fairly resistant to oxidation by peroxide. However, the enzyme was completely inactivated when co-incubated with H 2 O 2 , iron and ascorbate, indicating a vulnerability to oxidation by conditions that favour the production of hydroxyl radicals. Similarly, specific GS activity in cultured mouse astrocytes was resistant to supraphysiological concentrations of H 2 O 2 , with approximately 37% of activity remaining 3 h after incubation with 1 mM H 2 O 2 . This inactivation was prevented by the iron chelators 2,2 ′ -dipyridyl or 1,10-phenanthroline, but not by their non-chelating analogues. These data suggest that inactivation of astrocytic GS is caused by H 2 O 2 indirectly via the Fenton reaction as it required the presence of chelatable intracellular iron. © 2010 Elsevier Ireland Ltd. All rights reserved. Iron-mediated oxidative stress is thought to contribute to the neu- rodegeneration that occurs in disorders such as stroke, Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis [23]. Exposure to reactive oxygen species can cause lipid peroxidation, protein carbonyl formation, DNA damage and ultimately, cell death [26]. Many enzymes are vulnerable to inactivation and degrada- tion by reactive oxygen species, including glutamate–ammonia ligase, more commonly known as glutamine synthetase (GS; EC 6.3.1.2) which keeps glutamate and ammonia at low levels by combining them to form glutamine [16]. In healthy human brains, GS is concentrated in the astrocytic endfeet that abut blood vessels, in the astrocytic processes that surround synapses, and in their perineuronal cytoplasm; whereas in Alzheimer’s disease, GS is depleted from synapses and astrocytic endfeet [33]. In disease states where high levels of iron-mediated oxida- tive stress are observed, the expression and activity levels of GS are often lower than normal [8,27]. Post-mortem studies of brains from patients with Alzheimer’s disease reveal a loss of GS activity and expression throughout the brain, and a redistri- ∗ Corresponding author at: Blood-Brain Interactions Group, School of Psychology and Psychiatry, Building 17, Monash University, Wellington Rd., Clayton, Victoria 3800, Australia. Tel.: +61 3 9905 3950; fax: +61 3 9905 3948. E-mail address: steve.robinson@monash.edu (S.R. Robinson). bution of GS expression to neurones and the cerebrospinal fluid [15,33,38]. GS is reputed to be highly sensitive to oxidation, and the specific activity of GS in brain homogenates has often been used as a proxy for oxidative stress [6,30]. In vitro [19] and in vivo studies [6,28] have shown that treatment with H 2 O 2 , a normal by-product of oxidative metabolism [29,39], can result in the inactivation of GS. However, it is unclear whether H 2 O 2 directly inactivates GS or whether GS is primarily inactivated by hydroxyl radicals that are produced by the Fenton reaction when H 2 O 2 reacts with ferrous iron or other metals. Several studies have shown that the inactivation of bacte- rial GS by H 2 O 2 proceeds efficiently in the presence of iron [12,21]. Furthermore, Tiffany-Castiglioni et al. [37] have shown that when primary rat astrocytes are exposed to 100 M ferrous chloride they exhibit a reduction in their GS activity. Thus, iron-mediated inac- tivation of GS in mammalian brain could account for the fact that the specific activity of GS is often lower in those neurodegenerative disorders that are characterized by abnormally high levels of iron, which can reach 1 mM in the -amyloid plaques of Alzheimer’s disease [2,4,34]. Oxidative stress or exogenous H 2 O 2 can inactivate GS in astrocytes [19], yet as far as we are aware, no studies have investigated whether this inactivation depends on the avail- ability of redox-active metals like iron. Although cultured astrocytes contain relatively low levels of free iron [32], the 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.12.019