Stephen R Robinson

Publications of Stephen R Robinson

• The metabolism and toxicity of hemin in astrocytes.

  Authors: Theresa N Dang, Glenda M Bishop, Ralf Dringen, Stephen R Robinson

  Glia. 06/2011; 59(10):1540-50.

  Hemin is cytotoxic, and contributes to the brain damage that accompanies hemorrhagic stroke. In order to better understand the basis of hemin toxicity in astrocytes, the present study quantifiedHemin is cytotoxic, and contributes to the brain damage that accompanies hemorrhagic stroke. In order to better understand the basis of hemin toxicity in astrocytes, the present study quantified hemin metabolism and compared it to the pattern of cell death. Heme oxygenase-1 (HO-1) expression was first evident after 2 h incubation with hemin, with maximal expression being observed by 24 h. Despite the induction of HO-1, it was found that the proportion of hemin metabolized by astrocytes remained fairly constant throughout the 24 h period, with 70-80% of intracellular hemin remaining intact. A period of cell loss began after 2 h exposure to hemin, which gradually increased in severity to reach a maximum by 24 h. This cell loss could not be attenuated by the iron chelator, 1,10-phenanthroline, or by several antioxidant compounds (Trolox, N-acetyl-L-cysteine and N-tert-butyl-α-phenylnitrone), indicating that the mechanism of hemin toxicity does not involve iron. While these results make it unlikely that hemin toxicity is due to interactions with endogenous H(2)O(2), hemin toxicity was increased in the presence of supraphysiological levels of H(2)O(2) and this increase was ameliorated by PHEN, indicating that the iron released from hemin can be toxic under some pathological conditions. However, when H(2)O(2) is present at physiological levels, the toxicity of hemin appears to be caused by other mechanisms that may involve bilirubin and carbon monoxide in this model system.

• Subtle cognitive impairment in elders with Mini-Mental State Examination scores within the 'normal' range.

  Authors: Timothy W Friedman, Gregory W Yelland, Stephen R Robinson

  International journal of geriatric psychiatry. 05/2011;

  OBJECTIVE: The Mini-Mental State Examination (MMSE) is commonly used as a screening test for dementia, yet MMSE scores above the cut-off for dementia (24-30) are widely thought to have limitedOBJECTIVE: The Mini-Mental State Examination (MMSE) is commonly used as a screening test for dementia, yet MMSE scores above the cut-off for dementia (24-30) are widely thought to have limited utility, particularly in older persons. The study investigates whether scores within this range can be indicative of pre-symptomatic levels of cognitive impairment. METHODS: Ninety-six community-dwelling older persons aged 62-89 years (mean = 75.2 years), who had obtained MMSE scores between 25 and 30, were tested on the computer-based Subtle Cognitive Impairment Test (SCIT). RESULTS: Compared with individuals who obtained a perfect score of 30 on the MMSE, individuals with scores of 28-29 made more errors on the SCIT, whereas those with scores of 25-27 on the MMSE made the most errors on the SCIT (F(2,94) = 9.84, p < 0.01). Individuals who made errors in the language (r(94) = -0.47, p < 0.01), attention (r(94) = 0.24, p < 0.05) and visual construction (r(94) = -0.27, p < 0.01) subtests of the MMSE were more likely to display impaired SCIT performance. CONCLUSIONS: The pattern of performance on the SCIT varied in a systematic way, depending on the MMSE subtest in which the errors were made, raising the possibility that there may be different subtypes of subtle cognitive impairment within the ostensibly normal population of older persons. Copyright © 2011 John Wiley & Sons, Ltd.

• Accumulation of non-transferrin-bound iron by neurons, astrocytes, and microglia.

  Authors: Glenda M Bishop, Theresa N Dang, Ralf Dringen, Stephen R Robinson

  Neurotoxicity research. 04/2011; 19(3):443-51.

  Neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, and hemorrhagic stroke are associated with increased levels of non-transferrin-bound iron (NTBI) in the brain, which canNeurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, and hemorrhagic stroke are associated with increased levels of non-transferrin-bound iron (NTBI) in the brain, which can promote Fenton chemistry. While all types of brain cells can take up NTBI, their efficiency of accumulation and capacity to withstand iron-mediated toxicity has not been directly compared. The present study assessed NTBI accumulation in cultures enriched in neurons, astrocytes, or microglia after exposure to ferric ammonium citrate (FAC). Microglia were found to be the most efficient in accumulating iron, followed by astrocytes, and then neurons. Exposure to 100 μM FAC for 24 h increased the specific iron content of cultured neurons, astrocytes, and microglial cells by 30-, 80-, and 100-fold, respectively. All cell types accumulated iron against the concentration gradient, resulting in intracellular iron concentrations that were several orders of magnitude higher than the extracellular iron concentrations. Accumulation of these large amounts of iron did not affect the viability of the cell cultures, indicating a high resistance to iron-mediated toxicity. These findings show that neurons, astrocytes and microglia cultured from neonatal mice all have the capacity to accumulate and safely store large quantities of iron, but that glial cells do this more efficiently than neurons. It is concluded that neurodegenerative conditions involving iron-mediated toxicity may be due to a failure of iron transport or storage mechanisms, rather than to the presence of high levels of NTBI.

• Uptake, metabolism and toxicity of hemin in cultured neurons.

  Authors: Theresa N Dang, Stephen R Robinson, Ralf Dringen, Glenda M Bishop

  Neurochemistry international. 03/2011;

  Following hemorrhagic stroke, red blood cells lyse and release neurotoxic hemin into the interstitial space. The present study investigates whether neurons can accumulate and metabolize hemin. WeFollowing hemorrhagic stroke, red blood cells lyse and release neurotoxic hemin into the interstitial space. The present study investigates whether neurons can accumulate and metabolize hemin. We demonstrate that cultured neurons express the heme carrier protein 1 (HCP1), and that this transporter appears to contribute to the time- and concentration-dependent accumulation of hemin by neurons. Although exposure of neurons to hemin stimulates the synthesis of the iron storage protein ferritin, approximately 80% of the hemin accumulated by neurons remains intact. Within 24h of incubation, substantial neurotoxicity was observed that was not attenuated by the cell permeable, selective ferrous iron chelator, 1,10-phenanthroline. These results demonstrate that while neurons efficiently accumulate hemin they slowly degrade it, and they support the conclusion that intact hemin is more neurotoxic than the iron released from the breakdown of hemin. Further investigations are required to determine the basis of this neurotoxicity.

• Impaired perceptual judgment at low blood alcohol concentrations.

  Authors: Timothy W Friedman, Stephen R Robinson, Gregory W Yelland

  Alcohol (Fayetteville, N.Y.). 12/2010; 45(7):711-8.

  Males and females show different patterns of cognitive impairment when blood alcohol concentrations (BACs) are high. To investigate whether gender differences persist at low BACs, cognitiveMales and females show different patterns of cognitive impairment when blood alcohol concentrations (BACs) are high. To investigate whether gender differences persist at low BACs, cognitive impairment was tested in 21 participants (11 female, 10 male) using a brief computerized perceptual judgment task that provides error rate and response time data. Participants consumed a measured dose of alcohol (average peak BAC: females: 0.052 g/100 mL, males: 0.055 g/100 mL), and were tested at four time points spanning both the rising and falling limbs of the BAC curve, in addition to a prealcohol time point. Comparisons were made against performance of these same participants at equivalent time points in an alcohol-free control condition. Males and females displayed a trend toward slower responses and more errors, even when mildly intoxicated. These data indicate that cognitive function can be impaired at BACs that are below the legal limit for driving in most countries.

• Two routes of iron accumulation in astrocytes: ascorbate-dependent ferrous iron uptake via the divalent metal transporter (DMT1) plus an independent route for ferric iron.

  Authors: Darius J R Lane, Stephen R Robinson, Hania Czerwinska, Glenda M Bishop, Alfons Lawen

  The Biochemical journal. 11/2010; 432(1):123-32.

  Astrocytes are central to iron and ascorbate homoeostasis within the brain. Although NTBI (non-transferrin-bound iron) may be a major form of iron imported by astrocytes in vivo, the mechanismsAstrocytes are central to iron and ascorbate homoeostasis within the brain. Although NTBI (non-transferrin-bound iron) may be a major form of iron imported by astrocytes in vivo, the mechanisms responsible remain unclear. The present study examines NTBI uptake by cultured astrocytes and the involvement of ascorbate and DMT1 (divalent metal transporter 1). We demonstrate that iron accumulation by ascorbate-deficient astrocytes is insensitive to both membrane-impermeant Fe(II) chelators and to the addition of the ferroxidase caeruloplasmin. However, when astrocytes are ascorbate-replete, as occurs in vivo, their rate of iron accumulation is doubled. The acquisition of this additional iron depends on effluxed ascorbate and can be blocked by the DMT1 inhibitor ferristatin/NSC306711. Furthermore, the calcein-accessible component of intracellular labile iron, which appears during iron uptake, appears to consist of only Fe(III) in ascorbate-deficient astrocytes, whereas that of ascorbate-replete astrocytes comprises both valencies. Our data suggest that an Fe(III)-uptake pathway predominates when astrocytes are ascorbate-deficient, but that in ascorbate-replete astrocytes, at least half of the accumulated iron is initially reduced by effluxed ascorbate and then imported by DMT1. These results suggest that ascorbate is intimately involved in iron accumulation by astrocytes, and is thus an important contributor to iron homoeostasis in the mammalian brain.

  Download

• Neurones express glutamine synthetase when deprived of glutamine or interaction with astrocytes.

  Authors: Samantha P Fernandes, Ralf Dringen, Alfons Lawen, Stephen R Robinson

  Journal of neurochemistry. 09/2010; 114(5):1527-36.

  Glutamine synthetase (GS) forms glutamine by catalyzing the ATP-dependent amidation of glutamate. In healthy brains, GS is restricted to astrocytes but in Alzheimer's disease and cell culture, GS hasGlutamine synthetase (GS) forms glutamine by catalyzing the ATP-dependent amidation of glutamate. In healthy brains, GS is restricted to astrocytes but in Alzheimer's disease and cell culture, GS has been detected in neurones. The present study demonstrates the expression of functional GS in cultured cerebellar granule cells and investigates conditions required to reduce this expression. Cerebellar granule cells from neonatal rats were grown in the absence of glutamine. Immunostaining revealed that the majority of neurones contained GS in their somata and dendrites. Treatment of neuronal cultures with glutamine greatly reduced the enzymatic activity of GS and also reduced the intensity of GS immunolabelling in dendrites. GS activity was reduced by 32% in neurones that had been transiently co-cultured with astrocytes, whereas GS immunoreactivity was largely abolished from neurones that had been directly seeded onto astrocytic monolayers. These results imply that GS expression in neurones occurs in response to a reduced availability of glutamine from astrocytes, and that neuronal GS expression represents a default phenotype which is normally suppressed via direct contacts with astrocytes. The aberrant expression of GS in sporadic neurones in Alzheimer's disease may indicate an impairment of such interactions.

  Download

• Astrocytes retain their antioxidant capacity into advanced old age.

  Authors: Jeff R Liddell, Stephen R Robinson, Ralf Dringen, Glenda M Bishop

  Glia. 09/2010; 58(12):1500-9.

  Oxidative stress has been implicated in the progression of ageing and in many age-related neurodegenerative conditions. Astrocytes play a major role in the antioxidant protection of the brain, yetOxidative stress has been implicated in the progression of ageing and in many age-related neurodegenerative conditions. Astrocytes play a major role in the antioxidant protection of the brain, yet little is known about how the antioxidant defenses of astrocytes change across the lifespan. This study assessed the antioxidant capacity and glutathione metabolism of astrocytes cultured from the brains of neonatal (<24 h old), mature (12-month-old), old (25-month-old), and senescent (31-month-old) C57BL/6J mice. When exposed to 100 microM hydrogen peroxide, mature, old, and senescent astrocytes cleared the peroxide approximately 30% more slowly than neonatal astrocytes. This difference persisted when catalase was inhibited by 3-aminotriazole, but was abolished when glutathione was depleted by application of buthionine sulfoximine, suggesting a deficit in the glutathione system. Correspondingly, the specific glutathione reductase activity of mature, old, and senescent astrocytes was approximately 30% lower than that of neonatal cultures, whereas no age-related change was observed in the specific activities of glutathione peroxidase, catalase, or in total antioxidant capacity. In addition, the specific rate of glutathione export was almost identical in mature, old, and senescent astrocytes, but was more than double that of neonatal astrocytes. These results indicate that the antioxidant capacity and glutathione metabolism of astrocytes are preserved from mature adulthood into senescence. It is concluded that the oxidative stress seen in ageing brains is likely due to factors extrinsic to astrocytes, rather than to an impairment of the antioxidative functions of astrocytes.

• Histidine, cystine, glutamine, and threonine collectively protect astrocytes from the toxicity of zinc.

  Authors: David M Ralph, Stephen R Robinson, Melissa S Campbell, Glenda M Bishop

  Free radical biology & medicine. 08/2010; 49(4):649-57.

  In epilepsy, traumatic brain injury, and ischemic stroke, toxic levels of zinc released from neurons contribute to the brain damage associated with these disorders. Zinc causes oxidative stress byIn epilepsy, traumatic brain injury, and ischemic stroke, toxic levels of zinc released from neurons contribute to the brain damage associated with these disorders. Zinc causes oxidative stress by increasing the generation of reactive oxygen species and by inhibiting glutathione reductase (GR). This study investigated whether naturally occurring amino acids can protect cultured mouse astrocytes from zinc. Astrocytes incubated for 24h with 33 microM zinc acetate in a minimal medium displayed a loss of GR activity, a depletion of total glutathione, and a loss of total antioxidant capacity. These changes were accompanied by extensive cell death. Four amino acids (200 microM L-histidine, 201 microM L-cystine, 4mM L-glutamine, 798 microM L-threonine), which are a subset of the 15 present in Dulbecco's modified Eagle medium, were found to collectively prevent zinc toxicity. Histidine was the most protective, followed by cystine, glutamine, and threonine. It is proposed that each of these amino acids protects against different aspects of zinc toxicity by chelating zinc, by serving as precursors for glutathione, or by converting to tricarboxylic acid cycle intermediates. It is possible that these 4 amino acids contribute in vivo to the protection of brain cells from the toxic effects of zinc.

• Synergistic accumulation of iron and zinc by cultured astrocytes.

  Authors: Glenda M Bishop, Ivo F Scheiber, Ralf Dringen, Stephen R Robinson

  Journal of neural transmission (Vienna, Austria : 1996). 07/2010; 117(7):809-17.

  Iron and zinc are essential for normal brain function, yet the mechanisms used by astrocytes to scavenge non-transferrin-bound iron (NTBI) and zinc are not well understood. Ischaemic stroke,Iron and zinc are essential for normal brain function, yet the mechanisms used by astrocytes to scavenge non-transferrin-bound iron (NTBI) and zinc are not well understood. Ischaemic stroke, traumatic brain injury and Alzheimer's disease are associated with perturbations in the metabolism of NTBI and zinc, suggesting that these two metals may collectively contribute to pathology. The present study has investigated the accumulation of NTBI and zinc by rat primary astrocyte cultures. It was found that astrocytes express mRNA for both divalent metal transporter 1 (DMT1) and Zip14, indicating the potential for these transporters to contribute to the accumulation of NTBI and zinc by these cells. Astrocytes were found to accumulate iron from ferric chloride in a time- and dose-dependent manner, and the rate of accumulation was strongly stimulated by co-incubation with zinc acetate. In addition, cultured astrocytes rapidly accumulated zinc from zinc acetate, and this accumulation was stimulated by co-incubation with ferric chloride. Because a synergistic stimulation of iron and zinc accumulation is inconsistent with the known properties of DMT1 and Zip14, the present results suggest that additional mechanisms assist astrocytes to scavenge iron and zinc when they are present together in the extracellular compartment. These mechanisms may be involved in disorders that involve elevations in the extracellular concentrations of these metal ions.

• A role for Na+/H+ exchangers and intracellular pH in regulating vitamin C-driven electron transport across the plasma membrane.

  Authors: Darius J R Lane, Stephen R Robinson, Hania Czerwinska, Alfons Lawen

  The Biochemical journal. 03/2010; 428(2):191-200.

  Ascorbate (vitamin C) is the major electron donor to a tPMET (transplasma membrane electron transport) system that was originally identified in human erythrocytes. This plasma membrane redox systemAscorbate (vitamin C) is the major electron donor to a tPMET (transplasma membrane electron transport) system that was originally identified in human erythrocytes. This plasma membrane redox system appears to transfer electrons from intracellular ascorbate to extracellular oxidants (e.g. non-transferrin-bound iron). Although this phenomenon has been observed in nucleated cells, its mechanism and regulation are not well understood. In the present study we have examined both facets of this phenomenon in K562 cells and primary astrocyte cultures. Using ferricyanide as the analytical oxidant we demonstrate that tPMET is enhanced by dehydroascorbate uptake via facilitative glucose transporters, and subsequent accumulation of intracellular ascorbate. Additionally, we demonstrate that this stimulation is not due to ascorbate that is released from the cells, but is dependent only on a restricted intracellular pool of the vitamin. Substrate-saturation kinetics suggest an enzyme-catalysed reaction across the plasma membrane by an as-yet-unidentified reductase that relies on extensive recycling of intracellular ascorbate. Inhibition of ascorbate-stimulated tPMET by the NHE (Na(+)/H(+)-exchanger) inhibitors amiloride and 5-(N-ethyl-N-isopropyl)amiloride, which is diminished by bicarbonate, suggests that tPMET activity may be regulated by intracellular pH. In support of this hypothesis, tPMET in astrocytes was significantly inhibited by ammonium chloride-pulse-induced intracellular acidification, whereas it was significantly stimulated by bicarbonate-induced intracellular alkalinization. These results suggest that ascorbate-dependent tPMET is enzyme-catalysed and is modulated by NHE activity and intracellular pH.

  Download

• Effects of carboxylic acids on the uptake of non-transferrin-bound iron by astrocytes.

  Authors: Belinda M Keenan, Stephen R Robinson, Glenda M Bishop

  Neurochemistry international. 03/2010; 56(6-7):843-9.

  The concentrations of non-transferrin-bound iron are elevated in the brain during pathological conditions such as stroke and Alzheimer's disease. Astrocytes are specialised for sequestering thisThe concentrations of non-transferrin-bound iron are elevated in the brain during pathological conditions such as stroke and Alzheimer's disease. Astrocytes are specialised for sequestering this iron, however little is known about the mechanisms involved. Carboxylates, such as citrate, have been reported to facilitate iron uptake by intestinal cells. Citrate binds iron and limits its redox activity. The presence of high citrate concentrations in the interstitial fluid of the brain suggests that citrate may be an important ligand for iron transport by astrocytes. This study investigates whether iron accumulation by cultured rat astrocytes is facilitated by citrate or other carboxylates. Contrary to expectations, citrate, tartrate and malate were found to block iron accumulation in a concentration-dependent manner; alpha-ketoglutarate had limited effects, while fumarate, succinate and glutarate had no effect. This blockade was not due to an inhibition of ferric reductase activity. Instead, it appeared to be related to the capacity of these carboxylates to bind iron, since phosphate, which also binds iron, diminished the capacity of citrate, tartrate and malate to block the cellular accumulation of iron. These findings raise the possibility that citrate may have therapeutic potential in the management of neurodegenerative conditions that involve cellular iron overload.

• Uptake of ferrous iron by cultured rat astrocytes.

  Authors: Ketki Tulpule, Stephen R Robinson, Glenda M Bishop, Ralf Dringen

  Journal of neuroscience research. 09/2009;

  Astrocytes are considered to play an important role in iron homeostasis of the brain, yet the mechanisms involved in the uptake of iron into astrocytes remain elusive. To investigate the uptake ofAstrocytes are considered to play an important role in iron homeostasis of the brain, yet the mechanisms involved in the uptake of iron into astrocytes remain elusive. To investigate the uptake of iron into astrocytes, we have applied ferric ammonium citrate (FAC) to rat astrocyte-rich primary cultures. These cultures express the mRNAs of two membrane-bound ferric reductases, Dcytb and SDR2, and reduce extracellular ferric iron (100 muM) with a rate of 3.2 +/- 0.4 nmol/(hr x mg). This reduction rate is substantially lower than the rate of cellular iron accumulation from 100 muM FAC [24.7 +/- 8.9 nmol/(hr x mg)], which suggests that iron accumulation from FAC does at best partially depend on extracellular ferric reduction. Nonetheless, when the iron in FAC was almost completely reduced by an excess of exogenous ascorbate, astrocytes accumulated iron in a time- and concentration-dependent manner with specific iron accumulation rates that increased linearly for concentrations of up to 100 muM ferrous iron. This accumulation was attenuated by lowering the incubation temperature, by the presence of ferrous iron chelators, or by lowering the pH from 7.4 to 6.8. These data indicate that, in addition to the DMT1-mediated uptake of ferrous iron, astrocytes can accumulate ferric and ferrous iron by mechanisms that are independent of DMT1 or transferrin. (c) 2009 Wiley-Liss, Inc.

• The putative heme transporter HCP1 is expressed in cultured astrocytes and contributes to the uptake of hemin.

  Authors: Theresa N Dang, Glenda M Bishop, Ralf Dringen, Stephen R Robinson

  Glia. 07/2009;

  Hemin, which is toxic to brain cells, has been reported to be taken up by cultured astrocytes; however, the mechanism of uptake is currently unknown. The present study investigated the mechanism ofHemin, which is toxic to brain cells, has been reported to be taken up by cultured astrocytes; however, the mechanism of uptake is currently unknown. The present study investigated the mechanism of hemin uptake by rat primary astrocyte cultures. In medium containing 10% fetal calf serum, cultured astrocytes failed to accumulate significant amounts of heme-iron, while in serum-free medium the accumulation of heme-iron was found to be time- and concentration-dependent. After 6 h of incubation with 24 muM hemin, cells contained 36.2 +/- 2.4 nmol heme-iron/mg protein, which was 21% of the applied hemin. These results suggest that the accumulation of hemin in astrocytes does not require serum proteins such as hemopexin. A potential mechanism of hemin uptake in astrocytes involves the heme carrier protein 1 (HCP1), which is reported to mediate hemin uptake into intestinal cells. RT-PCR analysis revealed that astrocyte cultures contained HCP1 mRNA, and immunocytochemical staining and Western blot analysis confirmed the expression of HCP1 protein in cultured astrocytes. The functionality of HCP1 in astrocytes was demonstrated by incubating cells with zinc protoporphyrin IX (ZnPPIX), which is known to be transported into cells via HCP1, and ZnPPIX autofluorescence was detected in HCP1-positive astrocytes. In addition, ZnPPIX was found to attenuate the accumulation of heme-iron by astrocytes. These results are the first to demonstrate that cultured astrocytes contain functional HCP1 and that this transporter contributes to hemin uptake by astrocytes. HCP1 may therefore provide a new target for reducing hemin-related toxicity in brain cells. (c) 2009 Wiley-Liss, Inc.

• Sustained hydrogen peroxide stress decreases lactate production by cultured astrocytes.

  Authors: Jeff R Liddell, Claudia Zwingmann, Maike M Schmidt, Anette Thiessen, Dieter Leibfritz, Stephen R Robinson, Ralf Dringen

  Journal of neuroscience research. 05/2009;

  Oxidative stress and disrupted energy metabolism are common to many pathological conditions of the brain. Because astrocytes play an important role in the glucose metabolism of the brain, we haveOxidative stress and disrupted energy metabolism are common to many pathological conditions of the brain. Because astrocytes play an important role in the glucose metabolism of the brain, we have investigated whether sustained oxidative stress affects astroglial glucose metabolism with cultured primary rat astrocytes as a model system. Cultured astrocytes were exposed to a sustained concentration of approximately 50 muM H(2)O(2) in the presence of [U-(13)C]glucose, and cellular and extracellular contents of lactate and glucose were analysed by enzymatic assays and NMR spectroscopy. Exposure of the cells to sustained H(2)O(2) stress for up to 120 min significantly lowered the rate of lactate accumulation in the media to 61% +/- 14% of that in cultures incubated without peroxide. In addition, the ratio of lactate release to glucose consumption was lowered in peroxide-treated astrocytes to 77% +/- 13% of that in control cells, and the specific activity of glyceraldehyde-3-phosphate dehydrogenase had declined to about 10% of control cells within 90 min. In addition, the (13)C enrichment of intracellular and extracellular [(13)C]lactate was about 30% and 95%, respectively, and was not affected by the presence of peroxide, demonstrating that two metabolic pools of lactate are present in cultured astrocytes. The decreased rate of lactate production by astrocytes that have been exposed to peroxide stress is a new example of an alteration by oxidative stress of an important metabolic pathway in astrocytes. Such alterations could contribute to the pathological conditions that have been connected with oxidative stress and disrupted energy metabolism in the brain. (c) 2009 Wiley-Liss, Inc.

• Hemin toxicity: a preventable source of brain damage following hemorrhagic stroke.

  Authors: Stephen R Robinson, Theresa N Dang, Ralf Dringen, Glenda M Bishop

  Redox report : communications in free radical research. 01/2009; 14(6):228-35.

  Hemorrhagic stroke is a common cause of permanent brain damage, with a significant amount of the damage occurring in the weeks following a stroke. This secondary damage is partly due to the toxicHemorrhagic stroke is a common cause of permanent brain damage, with a significant amount of the damage occurring in the weeks following a stroke. This secondary damage is partly due to the toxic effects of hemin, a breakdown product of hemoglobin. The serum proteins hemopexin and albumin can bind hemin, but these natural defenses are insufficient to cope with the extremely high amounts of hemin (10 mM) that can potentially be liberated from hemoglobin in a hematoma. The present review discusses how hemin gets into brain cells, and examines the multiple routes through which hemin can be toxic. These include the release of redox-active iron, the depletion of cellular stores of NADPH and glutathione, the production of superoxide and hydroxyl radicals, and the peroxidation of membrane lipids. Important gaps are revealed in contemporary knowledge about the metabolism of hemin by brain cells, particularly regarding how hemin interacts with hydrogen peroxide. Strategies currently being developed for the reduction of hemin toxicity after hemorrhagic stroke include chelation therapy, antioxidant therapy and the modulation of heme oxygenase activity. Future strategies may be directed at preventing the uptake of hemin into brain cells to limit the opportunity for toxic interactions.

• The pivotal role of astrocytes in the metabolism of iron in the brain.

  Authors: Ralf Dringen, Glenda M Bishop, Maico Koeppe, Theresa N Dang, Stephen R Robinson

  Neurochemical research. 12/2007; 32(11):1884-90.

  Iron is essential for the normal functioning of cells but since it is also capable of generating toxic reactive oxygen species, the metabolism of iron is tightly regulated. The present articleIron is essential for the normal functioning of cells but since it is also capable of generating toxic reactive oxygen species, the metabolism of iron is tightly regulated. The present article advances the view that astrocytes are largely responsible for distributing iron in the brain. Capillary endothelial cells are separated from the neuropil by the endfeet of astrocytes, so astrocytes are ideally positioned to regulate the transport of iron to other brain cells and to protect them if iron breaches the blood-brain barrier. Astrocytes do not appear to have a high metabolic requirement for iron yet they possess transporters for transferrin, haemin and non-transferrin-bound iron. They store iron efficiently in ferritin and can export iron by a mechanism that involves ferroportin and ceruloplasmin. Since astrocytes are a common site of abnormal iron accumulation in ageing and neurodegenerative disorders, they may represent a new therapeutic target for the treatment of iron-mediated oxidative stress.

• Zinc stimulates the production of toxic reactive oxygen species (ROS) and inhibits glutathione reductase in astrocytes.

  Authors: Glenda M Bishop, Ralf Dringen, Stephen R Robinson

  Free radical biology & medicine. 05/2007; 42(8):1222-30.

  The release of zinc (Zn) from glutamatergic synapses contributes to the neuropathology of ischemia, traumatic brain injury, and stroke. Astrocytes surround glutamatergic synapses and are vulnerableThe release of zinc (Zn) from glutamatergic synapses contributes to the neuropathology of ischemia, traumatic brain injury, and stroke. Astrocytes surround glutamatergic synapses and are vulnerable to the toxicity of Zn, which impairs their antioxidative glutathione (GSH) system and elevates the production of reactive oxygen species (ROS). It is not known whether one or both of these actions are the primary cause of Zn-induced cell death in astrocytes. Using primary rat astrocyte cultures we have examined whether Zn-mediated impairment of GSH redox cycling is the main source of its toxicity. Zn acetate at concentrations of 100 microM or greater were found to inactivate glutathione reductase (GR) via an NADPH-dependent mechanism, while concentrations of 150 microM and above caused substantial cell death. Furthermore, Zn increased the ratio of GSSG:GSH in astrocytes, increased their export of GSSG, slowed their clearance of exogenous H2O2, and promoted the intracellular production of ROS. In contrast, the GR inhibitor, carmustine, did not induce cell death, cause the production of ROS, or alter the GSH thiol redox balance. Taken together these results indicate that Zn toxicity in astrocytes is primarily associated with the generation of intracellular ROS, rather than the inhibition of GR.

• Glutathione peroxidase 1 and a high cellular glutathione concentration are essential for effective organic hydroperoxide detoxification in astrocytes.

  Authors: Jeff R Liddell, Ralf Dringen, Peter J Crack, Stephen R Robinson

  Glia. 01/2007; 54(8):873-9.

  Organic hydroperoxides are produced in the eicosanoid metabolism and by lipid peroxidation. To examine the contribution of glutathione peroxidase-1 (GPx1) and glutathione (GSH) in the disposal ofOrganic hydroperoxides are produced in the eicosanoid metabolism and by lipid peroxidation. To examine the contribution of glutathione peroxidase-1 (GPx1) and glutathione (GSH) in the disposal of organic hydroperoxides in brain astrocytes, primary astrocyte cultures from wild type or GPx1-deficient (GPx1(-/-)) mice were exposed to cumene hydroperoxide (CHP). After application of 100 microM CHP, the peroxide disappeared quickly from the incubation medium of wild type cells with a half-life of 9 min, whereas CHP clearance was strongly retarded in GPx1(-/-) astrocytes. Depletion of GSH by pre-incubation with buthionine sulfoximine (BSO) significantly slowed CHP clearance by wild type astrocytes, while almost completely preventing peroxide disposal by GPx1(-/-) cells. In contrast, the catalase inhibitor 3-aminotriazole (3AT) had no effect on CHP clearance. Application of CHP to wild type astrocytes was followed by a rapid and transient accumulation of GSSG, whereas in GPx1(-/-) cells no increase in the GSSG content was detected. Astrocytes from both mouse lines remained viable for up to 24 h following CHP exposure, however depletion of cellular GSH by pre-treatment with BSO compromised the viability of astrocytes, an effect that was stronger in GPx1(-/-) than in wild type cells. This cell death was almost completely prevented by iron chelators, whereas pre-incubation with iron increased CHP toxicity. These novel data demonstrate that the toxicity of organic hydroperoxides in astrocytes is iron-mediated, and that an intact GSH system is required for the effective removal of organic hydroperoxides and for protection from these peroxides.

• Glutathione peroxidase 1 and glutathione are required to protect mouse astrocytes from iron-mediated hydrogen peroxide toxicity.

  Authors: Jeff R Liddell, Hans H Hoepken, Peter J Crack, Stephen R Robinson, Ralf Dringen

  Journal of neuroscience research. 09/2006; 84(3):578-86.

  The enzyme glutathione peroxidase 1 (GPx1) is involved in the cellular detoxification of peroxides. To test for the consequences of GPx deficiency in astrocytes, astrocyte-rich primary cultures fromThe enzyme glutathione peroxidase 1 (GPx1) is involved in the cellular detoxification of peroxides. To test for the consequences of GPx deficiency in astrocytes, astrocyte-rich primary cultures from wild-type and GPx1-deficient [GPx1(-/-)] mice were exposed to H(2)O(2). In GPx1(-/-) astrocytes, the clearance rate of H(2)O(2) was slower than in wild-type cells. In contrast to GPx1-deficient astrocytes, wild-type cells exhibited, within 2 min of H(2)O(2) application, a rapid and transient accumulation of cellular glutathione disulfide that amounted to 60% of total glutathione. The peroxide treatment did not affect the viability of wild-type astrocytes, whereas 45% of the GPx1(-/-) cells died within 8 hr. However, the viability of both types of astrocytes was strongly compromised by lowering cellular glutathione content before peroxide application. In contrast, inactivation of catalase caused substantial cell death only in GPx1(-/-) cells but not in wild-type astrocytes. The cell death observed was prevented by the iron chelators deferoxamine, 1,10-phenathroline, or 2,2'-dipyridyl, whereas preincubation with ferric ammonium citrate increased the toxicity of peroxide treatments. These results demonstrate that GPx1 contributes to the rapid clearance of H(2)O(2) by mouse astrocytes and that both GPx1 and a high concentration of glutathione are required to protect these cells from iron-dependent peroxide damage.