Journal of Neurochemistry, 2001, 77, 16011610

Astrocytes protect neurons from nitric oxide toxicity by a glutathione-dependent mechanism

Yongmei Chen,*,1 Nina E. Vartiainen,,,1 Weihai Ying,* Pak H. Chan, Jari Koistinaho,, and Raymond A. Swanson*
*Department of Neurology, University of California and Veterans Affairs Medical Center, San Francisco, California, USA Department of Neurosurgery, Neurology, and Neurological Science, and Program in Neuroscience, Stanford University School of Medicine, Palo Alto, California, USA A. I. Virtanen Institute for Molecular Sciences and Department of Clinical Pathology, Kuopio University Hospital, University of Kuopio, Finland

Abstract Nitric oxide (NO) contributes to neuronal death in cerebral ischemia and other conditions. Astrocytes are anatomically well positioned to shield neurons from NO because astrocyte processes surround most neurons. In this study, the capacity of astrocytes to limit NO neurotoxicity was examined using a cortical co-culture system. Astrocyte-coated dialysis membranes were placed directly on top of neuronal cultures to provide a removable astrocyte layer between the neurons and the culture medium. The utility of this system was tested by comparing neuronal death produced by glutamate, which is rapidly cleared by astrocytes, and N-methyl-D-aspartate (NMDA), which is not. The presence of an astrocyte layer increased the LD50 for glutamate by approximately four-fold,

but had no effect on NMDA toxicity. Astrocyte effects on neuronal death produced by the NO donors S-nitroso-N-acetyl penicillamine and spermine NONOate were examined by placing these compounds into the medium of co-cultures containing either a control astrocyte layer or an astrocyte layer depleted of glutathione by prior exposure to buthionine sulfoximine. Neurons in culture with the glutathione-depleted astrocytes exhibited a two-fold increase in cell death over a range of NO donor concentrations. These ndings suggest that astrocytes protect neurons from NO toxicity by a glutathione-dependent mechanism. Keywords: buthionine sulfoximine, excitotoxicity, glia, glutamate, Transwell. J. Neurochem. (2001) 77, 16011610.

Nitric oxide (NO) is a signaling molecule in the CNS, acting as a neurotransmitter and a vasodilator (Garthwaite 1991; Moncada et al. 1991). However, NO can also contribute to neuronal death in cerebral ischemia (Huang et al. 1994; Samdani et al. 1997) and probably other disorders (Dawson and Dawson 1998). NO reacts with O2 to form peroxyni2 trite and its reactive intermediates, which can react indiscriminately with proteins, DNA, and other cell constituents (Beckman and Koppenol 1996; Squadrito and Pryor 1998; Szabo and Dawson 1998). NO can also damage cells by reacting with protein thiol groups and proteiniron complexes (Stamler et al. 1992; Kim et al. 1995). Glutathione (GSH) is important in limiting and repairing the deleterious actions of NO. GSH reacts directly with NO to produce nitrosoglutathione (GSNO), which acts as a sink for NO in cells (Clancy et al. 1994; Hogg et al. 1996; Singh et al. 1996). In addition, protein nitrosothiol groups formed by NO and sulfhydryl moieties undergo transnitrosation

reactions with GSH to form nitrosoglutathione (GSNO) and regenerate the native protein thiols (Singh et al. 1996;
Received February 19, 2001; revised manuscript received March 22, 2001; accepted March 23, 2001. Address correspondence and reprint requests to Raymond A. Swanson, (127) Neurology, VAMC, 4150 Clement Street, San Francisco, CA 94121, USA. E-mail: ray@itsa.ucsf.edu 1 Yongmei Chen and Nina E. Vartiainen contributed equally to this work. Abbreviations used: BSO, buthionine sulfoximine; BSS, physiological balanced salt solution; CFDA, 5(6)-carboxyuorescein diacetate; GCM, glial conditioned medium; DTNB, 5,5 H -dithio-bis(2-nitrobenzoic) acid; FBS, fetal bovine serum; GFAP, glial brillary acidic protein; GSH, glutathione; GSNO, nitrosoglutathione; LDH, lactate dehydrogenase; MAP2, microtubule-associated protein 2; MEM, minimal essential medium; NMDA, N-methyl-d-aspartate; NO, nitric oxide; SNAP, S-nitroso-N-acetyl-dl-penicillamine; Spermine NONOate, N-[4(-1-[3-aminopropyl]-2-hydroxy-2-nitrosohydrazino)butyl]-1,3-propanediamine.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 16011610

1601

1602 Y. Chen et al.

Padgett and Whorton 1998). The formation of GSNO over other nitrosothiols is favored by the relative stability of GSNO and the normally high abundance of intracellular GSH (Singh et al. 1996). However, during exposure to high concentrations of NO, GSH is quickly consumed and cells lose this defense against NO toxicity (Clancy et al. 1994; Padgett and Whorton 1998). In addition, GSNO itself can contribute to nitrosation of protein sulfhydryl groups when the ratio of GSNO : GSH becomes elevated (Singh et al. 1996). Interaction between astrocytes and neurons has been proposed as an important factor limiting neuronal death from excitotoxins (Rosenberg and Aizenman 1989), oxidants (Wilson 1997; Tanaka et al. 1999; Dringen et al. 2000), and other stressors, but unambiguous support for specic mechanisms for these astrocyteneuron interactions has been difcult to provide. Here we introduce a new method for studying the effects of different cell populations in a co-culture system. Astrocytes are cultured on dialysis membranes for placement directly on top of neurons cultured in tissue culture wells, such that the astrocytes are interposed between the neurons and the culture medium. The method differs from standard co-culture systems in that compounds added to the culture medium can gain access to neurons only by passing through the astrocyte layer. The astrocyte-coated dialysis membrane can be removed or replaced with pharmacologically treated astrocytes in order to investigate mechanisms of astrocyteneuronal interactions. Using this approach, we conrm a role for astrocyte glutamate uptake in limiting neuronal sensitivity to glutamate excitotoxicity and show that astrocytes protect neurons from NO toxicity by a glutathione-dependent mechanism. Materials and methods
Reagents were obtained from Sigma Chemicals (St Louis, MO, USA) except where noted. Astrocyte-neuron co-cultures Primary cortical astrocyte cultures were prepared from 1-day-old mice. Brains were removed after decapitation under deep isourane anesthesia, and the meninges were stripped away. The forebrain cortices were collected and dissociated by incubation in papain/ DNAase followed by trituration. The dissociated cells were washed, suspended in a culture medium consisting of Eagle's minimal essential medium (MEM) with 10% fetal bovine serum (FBS) (Hyclone, Ogden, UT, USA) and glutamine (2 mm), and plated in 75 cm2 cell culture asks. The medium was exchanged with fresh culture medium at day 7. At conuence (day 1215), the astrocytes were trypsinized and re-plated onto 1.3-cm diameter discs of Spectra/Por 14-kDa cut-off regenerated cellulose dialysis membrane (Spectrum Laboratories, Rancho Dominguez, CA, USA) that had been sterilized by soaking in 70% ethanol and precoated with human placenta collagen (Sigma), 0.1 mg/mL in sterile distilled water. The culture medium was exchanged with fresh

Fig. 1 Schematic presentation of the co-culture model. (a) Cortical astrocytes cultures are plated onto discs cut from dialysis membranes. (b) Cortical neurons are cultured in 24-well plates. (c) The astrocyte-coated membrane discs are inverted and placed into the wells containing neurons, with the astrocyte layer facing the neuronal layer. (d) A ceramic ring is placed over the astrocyte-coated membrane. Compounds added to the culture medium can gain access to neurons only by passing through the astrocyte layer.

medium after two days. The astrocytes were used for co-cultures 7 days after replating, at which time the astrocytes formed a conuent layer across the surface of the dialysis membranes. Cortical neuron suspensions were prepared using the same procedure as described for the astrocyte cultures except that the cortices were harvested from embryonic day 15 mice, as described previously (Ying et al. 1999). The neurons were suspended in glial conditioned medium (GCM) freshly collected from conuent astrocyte asks that had been fed at least 3 days previously with MEM containing 10% FBS. The neurons were plated into 24-well culture plates coated with poly d-lysine. On the following day, astrocytes on dialysis membranes were placed on top of the neuronal layer such that the astrocyte-coated surface was resting on and facing the neurons (Fig. 1). Ceramic ring washers (1.25 cm in diameter; Small Parts Inc., Miami Lakes, FL, USA) were placed on top of the dialysis membranes to hold them down against the neuronal layers. Cytosine arabinoside (20 mm) was added to the cultures to stop cell proliferation on day 2, and on day 4 this medium was exchanged with fresh GCM. The co-cultures were used for experiments and immunocytochemical characterization between days 7 and 9 in vitro. For some studies, neurons and astrocytes were cultured in Transwell (Costar) co-culture wells, as described by Dringen et al. (1999) with minor modications. For these preparations, the cultures were prepared identically as described for the dialysis membrane co-cultures except that the astrocytes were grown to conuency on collagen-coated Transwell inserts, which are suspended approximately 2 mm above the culture well oor, instead of on dialysis membranes. The inserts with astrocytes were placed into the wells with neurons one day after the neurons were plated. Immunocytochemistry The astrocytes were removed from the co-cultures and placed in empty culture wells for xation and staining. Astrocyte and neuronal preparations were xed in 4% paraformaldehyde for 20 min. The xed cells were incubated with mouse antibodies to

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 16011610

Astrocytes block NO-mediated neurotoxicity

1603

microtubule-associated protein 2 (MAP2) (Boeringer-Mannheim, Indianapolis, IN, USA) diluted 1 : 800 to identify neurons, or with glial brillary acidic protein (GFAP; ICN, Costa Mesa, CA, USA) diluted 1 : 5000, or vimentin (DAKO, Carpenteria, CA, USA) diluted 1 : 2000 to identify astrocytes. After 18 h at 48C, excess antibody was removed and the cultures were incubated with biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA) diluted 1 : 200 for 1 h at room temperature. After removal of excess antibody, cultures were treated with the ABC streptavidin detection system (Vector Laboratories, Burlingame, CA, USA) for 1 h, and the resulting horseradish peroxidase signal was detected using 3,3 H -diaminobenzidene.
Fig. 2 The CDFA and LDH methods of determining cell death yield comparable results. Neuronal cultures were incubated 20 min with NMDA at the designated concentrations. Percent cell death was determined both by the LDH method and the CDFA method in the same culture wells. CDFA, 5(6)-carboxyuorescein diacetate; LDH, lactate dehydrogenase; NMDA, N-methyl-D-aspartate.

Exposure to NO donors and excitotoxins The test reagents were prepared as concentrated stocks in physiological balanced salt solution (BSS) buffered at pH 7.2 with CO2/HCO32 during equilibrium with a 5% CO2 atmosphere. The BSS consisted of 3.1 mm KCl, 134 mm NaCl, 1.2 mm CaCl2, 1.2 mm MgSO4, 0.25 mm KH2PO4, 15.7 mm NaHCO3, and 2 mm glucose. Osmolarity was adjusted when necessary with small amounts of H2O or NaCl to attain a nal value of 290310 mOsm. Drugs were added from 20 stocks, prepared and pH adjusted to pH 7.2 in BSS. Stocks of the NO donors S-nitroso-N-acetyl-dlpenicillamine (SNAP) and N-[4-(-1-[3-aminopropyl]-2-hydroxy-2nitrosohydrazino)butyl]-1,3-propanediamine (spermine NONOate) (both from Alexis, San Diego, CA, USA) were prepared immediately before use and kept on ice until added to the cultures. Experiments using the dialysis membrane co-cultures were preceded by removing the astrocyte-coated dialysis membranes from the co-culture wells and replacing them with either (i) astrocyte-coated dialysis membranes that had been treated for 24 h with 0.25 mm buthionine sulfoximine (BSO) in culture medium (to deplete astrocyte glutathione); (ii) control astrocyte-coated dialysis membranes (not treated with BSO); or (iii) with collagen-coated dialysis membranes that had not been plated with astrocytes. (All cultures underwent the replacement of the dialysis membranes to control for potential neuronal death caused by trauma from this exchange). A 1.25-cm outer diameter ceramic ring (Small Parts Inc., Miami Lakes, FL, USA) was placed over each membrane disc to ensure that substances placed in the culture media could gain access to the neurons only by rst passing through the astrocyte layer. Experiments were begun by replacing the culture medium in each well with 0.5 mL CO2-equilibrated BSS. NMDA, glutamate, or NO donors were added from stock solutions and incubations were performed at 378C in a 5% CO2 atmosphere. For studies of NO toxicity, the NO generating compounds remained in the BSS throughout the 24 h interval until assessment of neuron survival. In some studies the NO donors were hydrolyzed prior to use by incubating the stocks at 378C for 5 h (spermine NONOate) or 24 h (SNAP). These hydrolyzed stocks were used to control for potential toxicity of products other than NO, namely NO2, NO2, and the 2 3 parent donor adducts. Experiments using Transwell co-cultures were begun by replacing the culture medium with 700 mL BSS. This total volume included 100 mL above the astrocytes on the Transwell insert. Glutamate, NMDA, or NO donors were added to the volume above the inserts at concentrations calculated to achieve the designated nal concentrations in the wells assuming complete diffusion and

equilibration through the Transwell inserts (i.e. at seven times the designated nal concentrations). Assessment of neuronal survival Neuronal viability after excitotoxic and NO insults were assessed 24 h after addition of the test compounds by counting 5(6)carboxyuorescein diacetate (CFDA)-labeled cells. CFDA, like uorescein diacetate, is a non-uorescent hydrophobic dye that penetrates cell membranes (Bruning et al. 1980). Once inside the cell, the dye is cleaved by cytosolic esterases into the uorescent product, 5(6)-carboxyuorescein. The carboxyl group improves retention of the dye in viable neurons (Bruning et al. 1980; Petroski and Geller 1994). CDFA was incubated with the neurons for 30 min at a nal concentration of 50 mm. Overlying dialysis membranes were not moved in order to prevent damage or displacement of the neurons. After the dye loading, cells were washed twice with BSS and uorescing cells were counted using Nikon inverted uorescence microscope equipped with appropriate lter set. Counts of 35 random 400 mm diameter elds in each culture well were averaged and normalized to counts obtained from four control wells. Cell survival was assessed using CFDA staining of live cells rather than the more standard propidium iodide staining of dead cell nuclei (Ying et al. 1999) because typically 2030% of the neurons initially plated subsequently died and fragmented during the 79 day culture period preceding the experiments. Since phagocytic cells were not present in the neuronal layers, fragmented nuclei from these cells were present at the onset of the experiments and these were often difcult to distinguish from nuclei of neurons killed by the experimental conditions. In some experiments, cell death determinations by the CFDA method were corroborated by measuring lactate dehydrogenase (LDH) activity in the cell culture medium (Swanson and Choi 1993). LDH activity was determined by the method of Koh and Choi (1987) using a kinetic plate reader (Molecular Devices, Menlo Park, CA, USA). In brief, 200 mL medium was mixed with 100 mL 500 mm potassium phosphate buffer (pH 7.5) containing 1.5 mm NADH and 7.5 mm sodium pyruvate. The A340 nm change was linear over 90 s. The CDFA and

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 16011610

1604 Y. Chen et al.

Fig. 4 Effects of astrocytes on excitotoxic neuronal death. (a) Neuronal survival of glutamate exposure is increased in the presence of astrocytes. (b) Neuronal survival after incubation with NMDA, which is not taken up by astrocytes, is unaffected by the presence of astrocytes. n 8, means ^ SEM. **p , 0.01. vs. the `with astrocyte' condition; NMDA, N-methyl-D-aspartate; B, with astrocytes; W, without astrocytes. Fig. 3 Immunocytochemical characterization of the cell cultures. (a) Astrocytes on dialysis membranes express glial brillary acidic protein (GFAP) and form a conuent monolayer. (b) Neurons on the culture plate surface beneath the astrocytes are identied by expression of microtubule-associated protein 2. (c) GFAP immunostaining of the neuronal culture layer shows rare astrocytes (most elds contained no astrocytes). Photograph (c) is at one-half magnication of (a) and (b).

LDH methods were compared on the same set of culture wells after exposure to NMDA, using sham wash and 10 mm NMDA for the control and 100% neuronal death conditions, respectively. As shown in Fig. 2, the two methods provide similar doseresponse curves for NMDA neurotoxicity. The LDH method was not used throughout the studies because of the possibility that the higher concentrations of NO donors could produce LDH release from astrocytes as well as from neurons. Measurements of astrocyte glutathione content Astrocytes cultured on dialysis membranes were placed in culture wells without neurons, then exposed to buthionine sulfoximine

(BSO) or NO donors as described for the co-cultures. Total glutathione content (reduced glutathione content plus twice glutathione disulde content) was measured by the cycling method of Baker et al. (1990). Astrocytes were lysed on ice in 1% sulfosalicylic acid, centrifuged to remove denatured protein, and brought to pH 7.0 with NaOH. A reaction mixture was added containing NADPH, glutathione reductase, and 5,5 H -dithio-bis(2nitrobenzoic) acid (DTNB). The rate of DTNB reduction was determined by following the rate of absorbance increase at 405 nm over 90 s on a kinetic plate reader. Results were calibrated against known glutathione standards that were treated in parallel with the cell extracts and normalized to protein content measured in sister culture wells by the bicinchonic acid (Pierce, Rockford, IL, USA) method (Smith et al. 1985), using bovine serum albumin standards. To conrm that low levels of glutathione were not simply due to cell death and lysis, medium from the culture wells was sampled and measured for LDH activity at the time the cells were harvested for glutathione measurements. Statistics Comparisons between two treatment groups were performed with Student's t-test. Comparisons between multiple groups were

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 16011610

Astrocytes block NO-mediated neurotoxicity

1605

performed with anova followed by Tukey's test for multiple comparisons between groups or Dunnett's test for multiple comparisons against the control group.

Results GFAP immunostaining of the astrocyte-coated dialysis membranes showed a conuent layer of astrocytes (Fig. 3a). Immunostaining of the neuronal cultures with antibody to MAP2 at 9 days in vitro showed dispersed neurons with rare clumping (Fig. 3b). The neuronal cultures had rare (, 1%) contaminating astrocytes as evidenced by staining for GFAP and vimentin: while most microscopic elds showed no astrocytes, some elds showed isolated astrocytes (Fig. 3c). The neurons could be maintained with at least 50% survival for up to 18 days as long as they remained under the astrocyte layer. After culture day 5, however, removal of the astrocyte layer for more than 34 h led to death of most of the neurons in the wells. The mechanism of this neuronal death was not further evaluated. Glutamate, but not NMDA, is rapidly taken up by astrocytes (Skerritt and Johnston 1981; Rosenberg et al. 1992; Tanaka 1993; Robinson and Dowd 1997). As shown in Fig. 4(a), neurons overlaid with astrocyte-coated dialysis membranes were more resistant to glutamate toxicity than were neurons overlaid with the dialysis membranes alone (without astrocytes) during the 20 minute glutamate exposures. As expected, the presence or absence of astrocytes in the co-cultures had no discernible effect on NMDA toxicity (Fig. 4b), consistent with rapid uptake as the mechanism by which astrocytes provide neuroprotection from glutamate. These results conrm that the astrocyte layer can have a signicant effect on the composition of the medium reaching the neurons. Glutamate and NMDA neurotoxicity were also compared using the commercially available Transwell system. Transwell co-cultures, like the dialysis membrane co-cultures, allow cells to be plated on separate surfaces and share a common medium. The Transwell cultures differ from the dialysis membrane co-cultures in that the upper layer of cells (astrocytes in the present study) is plated on a rigid support that is suspended above the neuronal layer rather than directly on top of it. Diffusion through this support is slower than through a dialysis membrane, and unlike the dialysis membrane co-cultures, the cells on the upper layer are not interposed between the lower layer and the bulk of the culture medium. Glutamate and NMDA placed in the small uid compartment above the Transwell insert at concentrations to achieve 100 mm with uniform diffusion through the culture medium produced no detectable neuronal death with incubations of up to 30 min (data not shown). The lack of neurotoxicity was, presumably, due to slow diffusion through the inserts, as 20 mm of either

Fig. 5 Effects of BSO and NO donors on astrocyte glutathione content and astrocyte viability. (a) Incubation with the NO donors produced dose- and time-dependent reductions in glutathione content. Near-total depletion was produced by extended incubations at the higher NO donor concentrations. BSO pre-incubation also produced near total glutathione depletion. (b) Measurements of extracellular lactate dehydrogenase (LDH) activity performed at the same time as the glutathione determinations showed that neither the BSO pretreatment nor the NO donors produced signicant astrocyte death at the concentrations employed. BSO, buthionine sulfoximine; SNAP, S-nitroso-N-acetyl-DL-penicillamine; spermine NO, N-[4-(-1-[3-aminopropyl]-2-hydroxy-2-nitrosohydrazino)butyl]-1,3-propanediamine. n 6, means ^ SEM. **p , 0.01 vs. the control group at the same incubation interval.

NMDA or glutamate added to the medium below the inserts caused death of more than 50% of the neurons. Since the NO donors employed have half-lives in the range of minutes to hours, the subsequent studies used only the dialysis membrane co-cultures.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 16011610

1606 Y. Chen et al.

Fig. 6 Photomicrographs showing the effect of astrocyte BSO treatment on neuronal survival of NO exposure. Neuronal viability is established by 5(6)-carboxyuorescein diacetate (CDFA) uorescence, assessed 24 h after treatment with SNAP. Neurons were placed in co-culture with astrocytes that were sham treated (a and c) or pre-incubated with BSO to deplete glutathione (b and d). The co-cultures were then treated with 0 mM SNAP (a and b) or 200 mM SNAP (c and d), and photographed 24 h later. BSO, buthionine sulfoximine; SNAP, S-nitroso-N-acetyl-DL-penicillamine.

Astrocyte glutathione levels were manipulated without affecting neuronal glutathione by placing astrocyte-coated dialysis membranes in fresh MEM containing 5% FBS, with or without 0.25 mm buthionine sulfoximine (BSO) to inhibit glutathione synthesis (Grifth and Meister 1979). As shown in Fig. 5, the BSO-treated astrocytes had glutathione levels of less than 5% of the non-treated, control astrocytes. The BSO-treated astrocytes were then compared to control astrocytes with respect to their effects on NO-mediated neurotoxicity. Astrocyte glutathione content was found to have a large effect on neuronal survival when the NO generating compounds were added to the co-cultures. Figure 6 shows representative photomicrographs of CDFA stained (live) neurons under various treatment conditions. In the absence of NO donors, neuronal survival was not signicantly different in cultures with BSO-treated or control astrocytes. Treatment with 200 mm SNAP, however, produced much greater neuronal death in neurons overlaid with the BSO-treated (glutathione-depleted) astrocytes. Figure 7 shows dose-response curves for the effects of SNAP and spermine NONOate on neuronal survival. Spermine NONOate and SNAP are both hydrolyzed in solution to form NO, but the rates of these hydrolyses are different: the half-life at pH 7.4, 378C is approximately 0.6 h for spermine NONOate and greater than 4 h for SNAP (Bauer and Fung 1991; Maragos et al. 1991; Kroncke et al. 1993; Morley et al. 1993). Both compounds produced a

dose-dependent decrease in neuronal viability, with spermine NONOate showing more potent effects than SNAP. Cultures incubated with hydrolyzed SNAP (1 mm) or hydrolyzed spermine NONOate (250 mm) did not increase neuronal death over control values (not shown). The primary nding shown here is that the neuronal death produced by the NO donors was markedly inuenced by the astrocytes present in co-culture. Neurons in co-culture with astrocytes depleted of glutathione by BSO pretreatment exhibited signicantly greater vulnerability to the NO donors than did neurons in co-culture with control astrocytes not pretreated with BSO. If the astrocyte glutathione content is important in limiting NO-mediated neuronal death, astrocyte glutathione levels would be expected to decrease during NO exposure. Figure 5(a) shows that glutathione levels are depleted by the higher concentrations of NO donors employed, and also shows that BSO pretreatment causes nearly complete GSH depletion. Astrocyte survival was not signicantly affected by the NO generators at these concentrations, as shown by the preserved intracellular LDH activity (Fig. 5b). Discussion These ndings suggest that astrocytes can protect neurons from NO toxicity by a mechanism dependent on astrocyte glutathione content. In most regions of the CNS, astrocyte

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 16011610

Astrocytes block NO-mediated neurotoxicity

1607

Fig. 7 Effects of astrocyte BSO treatment on neuronal survival after 24 hour exposures to NO generating compounds. (a) Co-cultures treated with SNAP exhibited decreased neuronal survival when the astrocytes were pretreated with BSO to deplete glutathione. (b) Similar results were observed in co-cultures treated with spermineNONOate, which like SNAP produces NO in aqueous solution. n 610, means ^ SEM. *p , 0.05, **p , 0.01 vs. the control astrocyte condition. BSO, buthionine sulfoximine; SNAP, S-nitroso-N-acetyl-DLpenicillamine; spermine NONOate, N-[4-(-1-[3-aminopropyl]-2hydroxy-2-nitrosohydrazino)butyl]-1,3-propanediamine; B, control astrocytes; X, BSO-treated astrocytes.

processes form a nearly continuous membrane around neuronal cell bodies and processes (Peters et al. 1976). Standard astrocyteneuron co-cultures, in which the two cell types are intermingled on a common surface, do not fully replicate this anatomical relationship, since electron microscopy has shown that neuronal cell somas are exposed to the medium in these cultures (Harris and Rosenberg 1993). An important feature of the co-culture system presented here is that the astrocyte-coated dialysis membranes are interposed between the bulk of the culture medium and the neuronal layer. The aim of this arrangement was to allow the astrocytes to modify the composition of the medium reaching the neurons. This effect was conrmed by the studies comparing the effects of the layer on glutamate and NMDA toxicity. The astrocyte layer reduced the toxicity of glutamate, which is taken up by astrocytes, but

not the toxicity of NMDA, which is cleared very slowly by astrocytes (Skerritt and Johnston 1981; Rosenberg et al. 1992; Tanaka 1993; Robinson and Dowd 1997). Of note, the astrocyte layer does not provide a impermeable barrier to NMDA. This resembles conditions in situ, where NMDA is a potent neurotoxin despite the presence of astrocyte processes around most neurons. The glutamate sensitivity of cortical neurons during removal of the astrocyte layer in the present studies is comparable to the glutamate sensitivity previously observed in cortical neurons cultured under astrocyte-poor conditions, and much greater than the glutamate sensitivity of cortical neurons in astrocyte-rich cultures (Rosenberg and Aizenman 1989). Comparisons between astrocyte-poor and astrocyterich neuronal cultures have been used by several groups to investigate astrocyteneuron interactions. These studies have provided support for the idea that astrocytes protect neurons from many excitotoxins and oxidants (Vibulsreth et al. 1987; Rosenberg and Aizenman 1989; Swanson and Choi 1993; Desagher et al. 1996; Lucius and Sievers 1996; Drukarch et al. 1997; Drukarch et al. 1998; Ye and Sontheimer 1998; Brown 1999; Tanaka et al. 1999; Xu et al. 1999). In studies comparing the two culture conditions, however, it is difcult to exclude the possibility that differences in neuronal survival might be due to intrinsic differences in the neurons cultured with and without abundant astrocytes, rather than to specic astrocyte functions during the toxin exposures. With studies of reactive oxygen species it is additionally difcult to exclude the possibility that the astrocytes in the astrocyte-rich cultures provide a non-specic protective effect by simply providing other targets for the oxidants. These problems were eliminated in the present study by (i) culturing all the neurons identically up to the start of experiments, and (ii) maintaining the presence of an astrocyte layer in all wells during the experiments. NO diffuses freely across cell membranes (Garthwaite 1991; Moncada et al. 1991). Since astrocyte processes surround neurons in most brain regions (Peters et al. 1976), glutathione localized within astrocytes can trap NO and thereby reduce neuronal NO exposure. Glutathione has previously been shown to limit cell sensitivity to NO-mediated mitochondrial injury (Bolanos et al. 1996), and it is likely that glutathione can likewise limit other NO-mediated cytotoxic processes. The major nding presented here is that the ability to astrocytes to protect neurons from NO toxicity is dependent at least in part on astrocyte glutathione content. Astrocyte glutathione content was manipulated by incubation with BSO, an irreversible inhibitor of g-glutamylcysteine synthetase (Grifth and Meister 1979). The plating of astrocytes on removable membranes allowed the astrocytes to be incubated with BSO without exposing the neurons to BSO, as performed previously by McNaught and Jenner (1999). Astrocytes

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 16011610

1608 Y. Chen et al.

depleted of GSH by prior incubation with BSO were less effective than control astrocytes in preventing NO-induced neuronal death. Moreover, death of neurons in co-culture with control astrocytes (not treated with BSO) occurred only at NO donor concentrations sufcient to signicantly deplete astrocyte glutathione content. The most straightforward explanation for these observations is that astrocytes reduce the amount of NO reaching the neurons by trapping NO as GSNO within their cytoplasm. GSNO may also have intrinsic antioxidant properties (Konorev et al. 1995; Rauhala et al. 1998). Alternatively, it is possible that astrocyte glutathione fuels an exchange of reducing equivalents between astrocytes and neurons. For example, astrocytes can take up oxidized vitamin C (dehydroascorbic acid), reduce it to ascorbate at the expense of glutathione, and then release ascorbate to the extracellular uid (Wilson 1997). Astrocytes can also release glutathione itself, but there is little evidence to suggest that neurons can directly import glutathione from the extracellular space (Yudkoff et al. 1990; Sagara et al. 1996; Dringen et al. 1999). Last, depletion of glutathione may facilitate production some reactive oxygen species by astrocytes (McNaught and Jenner 1999), and this could contribute to neuronal death during NO exposure. The present ndings do not exclude the presence of other, glutathione-independent mechanisms by which astrocytes limit NO neurotoxicity. In particular, Tanaka et al. (1999) have shown that neurons grown on an astrocyte-derived extracellular matrix have increased resistance to NO, and Dringen et al. (1999) have shown that astrocyte-derived precursors are taken up by neurons and used for neuronal glutathione synthesis. It is also possible that protein sulfhydryls and other reactive moieties within astrocytes can function, like glutathione, to bind or trap NO. NMDA receptor activation stimulates NO production by neurons, and in many systems NMDA neurotoxicity is substantially reduced by blockade of NO production or scavenging of the NO produced (Dawson and Dawson 1998). However, astrocytes had no effect on neuronal vulnerability to NMDA in the present study despite a large effect of astrocyte glutathione content on neuronal vulnerability to exogenous NO donors. It is possible that NO is only minor aspect of NMDA toxicity in this culture system. As reviewed by Aizenman et al. (1998), the degree to which NMDA neurotoxicity is mediated by NO depends on several factors and varies considerably among different systems and conditions. It should also be noted that astrocytes in this system are well positioned to shield neurons from NO donors in the medium, but not to shield neurons from one another, and consequently the astrocyte layer may have little inuence neuronal exposure to NO generated by other neurons. Astrocyte processes do separate neurons in situ, however, and this together with the present ndings suggests that astrocyte glutathione content could inuence the

vulnerability of neurons to NMDA receptor-mediated death in situ. Several reports suggest that concentrations of glutathione and glutathione synthesizing enzymes are generally higher in astrocytes than in neurons (Slivka et al. 1987); reviewed in (Makar et al. 1994). This arrangement is consistent with a signicant role for astrocyte glutathione in brain oxidant scavenging, and reductions in astrocyte glutathione may therefore endanger neurons under oxidative stress. Glutathione depletion has previously been shown to exacerbate cerebral ischemic injury in rats (Mizui et al. 1992). In light of the current results, this exacerbation could be attributable in part to reduced capacity of astrocytes to limit NO neurotoxicity during ischemia. These results demonstrate a glutathione-dependent interaction between neurons and astrocytes and further support the idea that free radical scavenging is an important astrocyte function in the CNS. Acknowledgements We thank Sophie Parmentier-Batteur for comments on the manuscript and Jill Guenza for expert technical support. This study was supported by grants from Saastamoinen Foundation, the Finnish Cultural Foundation of Northern Savo (NV), and the US National Institutes of Health (grant P50 NS 14543). References
Aizenman E., Brimecombe J. C., Potthoff W. K. and Rosenberg P. A. (1998) Why is the role of nitric oxide in NMDA receptor function and dysfunction so controversial? Prog. Brain Res. 118, 5371. Baker M. A., Cerniglia G. J. and Zaman A. (1990) Microtiter plate assay for the measurement of glutathione and glutathione disulde in large numbers of biological samples. Anal Biochem. 190, 360365. Bauer J. A. and Fung H. L. (1991) Chemical stabilization of a vasoactive S-nitrosothiol with cyclodextrins without loss of pharmacologic activity. Pharm. Res. 8, 13291334. Beckman J. S. and Koppenol W. H. (1996) Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am. J. Physiol. 271, C1424C1437. Bolanos J. P., Heales S. J. R., Peuchen S., Barker J. E., Land J. M. and Clark J. B. (1996) Nitric oxide-mediated mitochondrial damage: a potential neuroprotective role for glutathione. Free Radic. Biol. Med. 21, 9951001. Brown D. R. (1999) Neurons depend on astrocytes in a coculture system for protection from glutamate toxicity. Mol. Cell. Neurosci. 13, 379389. Bruning J. W., Kardol M. J. and Arentzen R. (1980) Carboxyuorescein uorochromasia assays. I. Non-radioactively labeled cell mediated lympholysis. J. Immunol. Methods 33, 3344. Clancy R. M., Levartovsky D., Leszczynska-Piziak J., Yegudin J. and Abramson S. B. (1994) Nitric oxide reacts with intracellular glutathione and activates the hexose monophosphate shunt in human neutrophils: evidence for S-nitrosoglutathione as a bioactive intermediary. Proc. Natl Acad. Sci. USA 91, 36803684. Dawson V. L. and Dawson T. M. (1998) Nitric oxide in neurodegeneration. Prog. Brain Res. 118, 215229.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 16011610

Astrocytes block NO-mediated neurotoxicity

1609

Desagher S., Glowinski J. and Premont J. (1996) Astrocytes protect neurons from hydrogen peroxide toxicity. J. Neurosci 16, 25532562. Dringen R., Pfeiffer B. and Hamprecht B. (1999) Synthesis of the antioxidant glutathione in neurons: supply by astrocytes of CysGly as precursor for neuronal glutathione. J. Neurosci. 19, 562569. Dringen R., Gutterer J. M. and Hirrlinger J. (2000) Glutathione metabolism in brain metabolic interaction between astrocytes and neurons in the defense against reactive oxygen species. Eur. J. Biochem. 267, 49124916. Drukarch B., Schepens E., Jongenelen C. A., Stoof J. C. and Langeveld C. H. (1997) Astrocyte-mediated enhancement of neuronal survival is abolished by glutathione deciency. Brain Res. 770, 123130. Drukarch B., Schepens E., Stoof J. C., Langeveld C. H. and Van Muiswinkel F. L. (1998) Astrocyte-enhanced neuronal survival is mediated by scavenging of extracellular reactive oxygen species. Free Radic. Biol. Med. 25, 217220. Garthwaite J. (1991) Glutamate, nitric oxide and cellcell signalling in the nervous system. Trends Neurosci. 14, 6067. Grifth O. W. and Meister A. (1979) Potent and specic inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J. Biol. Chem. 254, 75587560. Harris K. M. and Rosenberg P. A. (1993) Localization of synapses in rat cortical cultures. Neuroscience 53, 495508. Hogg N., Singh R. J. and Kalyanaraman B. (1996) The role of glutathione in the transport and catabolism of nitric oxide. FEBS Lett. 382, 223228. Huang Z., Huang P. L., Panahian N., Dalkara T., Fishman M. C. and Moskowitz M. A. (1994) Effects of cerebral ischemia in mice decient in neuronal nitric oxide synthase. Science 265, 18831885. Kim Y. M., Bergonia H. A., Muller C., Pitt B. R., Watkins W. D. and Lancaster J. R. Jr (1995) Loss and degradation of enzyme-bound heme induced by cellular nitric oxide synthesis. J. Biol. Chem. 270, 57105713. Koh J. Y. and Choi D. W. (1987) Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efux assay. J. Neurosci. Methods 20, 8390. Konorev E. A., Tarpey M. M., Joseph J., Baker J. E. and Kalyanaraman B. (1995) S-Nitrosoglutathione improves functional recovery in the isolated rat heart after cardioplegic ischemic arrest-evidence for a cardioprotective effect of nitric oxide. J. Pharmacol. Exp. Ther. 274, 200206. Kroncke K. D., Brenner H. H., Rodriguez M. L., Etzkorn K., Noack E. A., Kolb H. and Kolb-Bachofen V. (1993) Pancreatic islet cells are highly susceptible towards the cytotoxic effects of chemically generated nitric oxide. Biochim. Biophys. Acta 1182, 221229. Lucius R. and Sievers J. (1996) Postnatal retinal ganglion cells in vitro: protection against reactive oxygen species (ROS)-induced axonal degeneration by cocultured astrocytes. Brain Res. 743, 5662. McNaught K. S. and Jenner P. (1999) Altered glial function causes neuronal death and increases neuronal susceptibility to 1-methyl4-phenylpyridinium- and 6-hydroxydopamine-induced toxicity in astrocytic/ventral mesencephalic co-cultures. J. Neurochem. 73, 24692476. Makar T. K., Nedergaard M., Preuss A., Gelbard A. S., Perumal A. S. and Cooper A. J. (1994) Vitamin E, ascorbate, glutathione, glutathione disulde, and enzymes of glutathione metabolism in cultures of chick astrocytes and neurons: evidence that astrocytes

play an important role in antioxidative processes in the brain. J. Neurochem. 62, 4553. Maragos C. M., Morley D., Wink D. A., Dunams T. M., Saavedra J. E., Hoffman A., Bove A. A., Isaac L., Hrabie J. A. and Keefer L. K. (1991) Complexes of NO with nucleophiles as agents for the controlled biological release of nitric oxide. Vasorelaxant effects. J. Med. Chem. 34, 32423247. Mizui T., Kinouchi H. and Chan P. H. (1992) Depletion of brain glutathione by buthionine sulfoximine enhances cerebral ischemic injury in rats. Am. J. Physiol. 262, H313H317. Moncada S., Palmer R. M. and Higgs E. A. (1991) Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43, 109142. Morley D., Maragos C. M., Zhang X. Y., Boignon M., Wink D. A. and Keefer L. K. (1993) Mechanism of vascular relaxation induced by the nitric oxide (NO) /nucleophile complexes, a new class of NO-based vasodilators. J. Cardiovasc. Pharmacol. 21, 670676. Padgett C. M. and Whorton A. R. (1998) Cellular responses to nitric oxide: role of protein S-thiolation/dethiolation. Arch. Biochem. Biophys. 358, 232242. Peters A., Palay S. L. and Webster H. D. F. (1976) The Fine Structure of the Nervous System: the Neurons and Supporting Cells. W.B. Saunders, Philadelphia. Petroski R. E. and Geller H. M. (1994) Selective labeling of embryonic neurons cultured on astrocyte monolayers with 5 (6)-carboxyuorescein diacetate (CFDA). J. Neurosci. Methods 52, 2332. Rauhala P., Lin A. M. and Chiueh C. C. (1998) Neuroprotection by S-nitrosoglutathione of brain dopamine neurons from oxidative stress. FASEB J. 12, 165173. Robinson M. B. and Dowd L. A. (1997) Heterogeneity and functional properties of subtypes of sodium-dependent glutamate transporters in the mammalian central nervous system. Adv. Pharmacol. 37, 69115. Rosenberg P. A. and Aizenman E. (1989) Hundred-fold increase in neuronal vulnerability to glutamate toxicity in astrocyte-poor cultures of rat cerebral cortex. Neurosci. Lett. 103, 162168. Rosenberg P. A., Amin S. and Leitner M. (1992) Glutamate uptake disguises neurotoxic potency of glutamate agonists in cerebral cortex in dissociated cell culture. J. Neurosci. 12, 5661. Sagara J., Makino N. and Bannai S. (1996) Glutathione efux from cultured astrocytes. J. Neurochem. 66, 18761881. Samdani A. F., Dawson T. M. and Dawson V. L. (1997) Nitric oxide synthase in models of focal ischemia. Stroke 28, 12831288. Singh R. J., Hogg N., Joseph J. and Kalyanaraman B. (1996) Mechanism of nitric oxide release from S-nitrosothiols. J. Biol. Chem. 271, 1859618603. Skerritt S. H. and Johnston G. A. (1981) Uptake and release of N-methyl-d-aspartate by rat brain slices. J. Neurochem. 36, 881885. Slivka A., Mytilineou C. and Cohen G. (1987) Histochemical evaluation of glutathione in brain. Brain Res. 409, 275284. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J. and Klenk D. C. (1985) Measurement of protein using bicinchoninic acid (published erratum appears in Anal. Biochem. 1987, 163, 279). Anal Biochem. 150, 7685. Squadrito G. L. and Pryor W. A. (1998) Oxidative chemistry of nitric oxide: the roles of superoxide, peroxynitrite, and carbon dioxide. Free Radic. Biol. Med. 25, 392403. Stamler J. S., Singel D. J. and Loscalzo J. (1992) Biochemistry of nitric oxide and its redox-activated forms [see comments]. Science 258, 18981902. Swanson R. A. and Choi D. W. (1993) Glial glycogen stores affect

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 16011610

1610 Y. Chen et al.

neuronal survival during glucose deprivation in vitro. J. Cereb. Blood Flow Metab. 13, 162169. Szabo C. and Dawson V. L. (1998) Role of poly (ADP-ribose) synthetase in inammation and ischaemia-reperfusion. Trends Pharmacol. Sci. 19, 287298. Tanaka K. (1993) Expression cloning of a rat glutamate transporter. Neuroscience Res. 16, 149153. Tanaka J., Toku K., Zhang B., Ishihara K., Sakanaka M. and Maeda N. (1999) Astrocytes prevent neuronal death induced by reactive oxygen and nitrogen species. Glia 28, 8596. Vibulsreth S., Hefti F., Ginsberg M. D., Dietrich W. D. and Busto R. (1987) Astrocytes protect cultured neurons from degeneration induced by anoxia. Brain Res. 422, 303311.

Wilson J. X. (1997) Antioxidant defense of the brain: a role for astrocytes. Can. J. Physiol. Pharmacol. 75, 11491163. Xu L., Lee J. E. and Giffard R. G. (1999) Overexpression of bcl-2, bcl-XL or hsp70 in murine cortical astrocytes reduces injury of co-cultured neurons. Neurosci. Lett. 277, 193197. Ye Z. C. and Sontheimer H. (1998) Astrocytes protect neurons from neurotoxic injury by serum glutamate. Glia 22, 237248. Ying W., Han S.-K., Miller J. W. and Swanson R. A. (1999) Acidosis potentiates oxidative neuronal death by multiple mechanisms. J. Neurochem. 73, 15491556. Yudkoff M., Pleasure D., Cregar L., Lin Z., Nissim I., Stern J. and Nissim I. (1990) Glutathione efux from cultured astrocytes: studies with [15N]glutamate. J. Neurochem. 55, 137145.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 16011610