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Keywords:

  • dopamine neurons;
  • extracellular signal-regulated kinase-1/2;
  • glial cells;
  • glutathione;
  • 12-lipoxygenase;
  • Parkinson's disease

Abstract

  1. Top of page
  2. Abstract
  3. References

To date, glutathione (GSH) depletion is the earliest biochemical alteration shown in brains of Parkinson's disease patients, but the role of GSH in dopamine cell survival is debated. In this study we show that GSH depletion, produced with GSH synthesis inhibitor, l-buthionine-(S,R)-sulfoximine (BSO), induces selectively neuronal cell death in neuron/glia, but not in neuronal-enriched midbrain cultures and that cell death occurs with characteristics of necrosis and apoptosis. BSO produces a dose- and time-dependent generation of reactive oxygen species (ROS) in neurons. BSO activates extracellular signal-regulated kinases (ERK-1/2), 4 and 6 h after treatment. MEK-1/2 and lipoxygenase (LOX) inhibitors, as well as ascorbic acid, prevent ERK-1/2 activation and neuronal loss, but the inhibition of nitric oxide sintase (NOS), cyclo-oxygenase (COX), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) does not have protective effects. Co-localization studies show that p-ERK-1/2 expression after BSO treatment increased in astrocytes and microglial cells, but not in neurons. Selective metabolic impairment of glial cells with fluoroacetate decreased ERK activation. However, blockade of microglial activation with minocycline did not. Our results indicate that neuronal death induced by GSH depletion is due to ROS-dependent activation of the ERK-1/2 signalling pathway in glial cells. These data may be of relevance in Parkinson's disease, where GSH depletion and glial dysfunction have been documented.

Abbreviations used
BSO

l-buthionine-(S,R)-sulfoximine

COX

cyclo-oxygenase

DA

dopamine

DMEM

Dulbecco's modified Eagle's medium

DTNB

5,5′-dithio-bis2-nitrobenzoic acid

EMEM

Eagle's minimal essential medium

ERK

extracellular signal-regulated kinase

FCS

fetal calf serum

FITC

Fluorescein isothiocyanate

GSH

glutathione

iNOS

inducible nitric oxide sintase

JNK

c-Jun N-terminal kinase

LDH

lactate dehydrogenase standard

l-NAME

N-nitro-l-arginine methyl ester

LOX

lipoxygenase

MAP-2

anti-microtubule-associated protein 2a + 2b

NDGA

nordihydroguaiaretic acid

NGS

normal goat serum

7NI

7-nitroindazole

NOS

nitric oxide sintase

p38 MAPK

p38 mitogen-activated protein kinase

PD

Parkinson's disease

ROS

reactive oxygen species

SDS

sodium dodecyl sulfate

TBS

Tris-buffered saline

TH

anti-tyrosine hydroxylase

U0126

1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene

An early and highly selective decrease in glutathione (GSH) in the substantia nigra is present in Parkinson's disease (PD) (Riederer et al. 1989), and low levels of GSH lead to the degeneration of cultured dopaminergic neurons (Jenner and Olanow 1996; Canals et al. 2001a, 2003a,b). Reactive oxygen species (ROS) have been implicated in a number of neurological disorders including PD (Coyle and Puttfarcken 1993; Di Monte 2001). Glutathione depletion induces accumulation of ROS, leading to cell death (Murphy et al. 1989).

Astrocytes play a very important function in the differentiation, survival, pharmacological properties and resistance to injury of dopamine (DA) neurons. The role of glia is mediated, at least in part, by the release of chemical substances to the media (Mena et al. 2002). Several studies suggest that glial cells may be important in the pathogenesis of PD, a common neurodegenerative disorder characterised by degeneration of the nigrostriatal DA system. In this disease, the role of glia could be because of excessive production of toxic products, such as nitric oxide (NO) or cytokines, characteristic of an inflammatory process (Hirsch et al. 1999; Liberatore et al. 1999; Hirsch and Hunot 2000; Le et al. 2001; Vila et al. 2001; Wu et al. 2002; Liu and Hong 2003), or related to a defective release of neuroprotective agents, such as small antioxidants with free radical scavenging properties or peptidic neurotrophic factors (Riederer et al. 1989; Muller et al. 1995; Engele et al. 1996; Mena et al. 2002; Kinor et al. 2003).

Although gliosis, and especially activated microglia, may sometimes be associated with beneficial effects, often gliosis appears to be deleterious. Blockade of microglial activation, inhibition of inducible nitric oxide sintase (iNOS) enzyme and deletion of iNOS gene, are neuroprotective in the MPTP mouse model of PD (Liberatore et al. 1999; Dehmer et al. 2000; Wu et al. 2002).

Mitogen-activated protein kinase (MAPK) family members, including extracellular signal-regulated kinases (ERK-1/2), respond to several extracellular stimuli. ERK-1/2 are activated by MAPK/ERK kinase 1/2 (MEK-1/2) by phosphorylating threonine and tyrosine residues (Seger and Krebs 1995). It is known that oxidative stress activates MAPK cascades. ERK-1/2 stimulation by ROS has been described in neurons (Samanta et al. 1998) and neuroprotection by MAPK/ERK kinase inhibition against oxidative stress in neuronal cell lines and in astrocytes (Satoh et al. 2000; Maher 2001; Rosenberger et al. 2001). Although numerous reports have implicated ERK in neuronal cell survival (Xia et al. 1995; Yujiri et al. 1998; de Bernardo et al. 2003), it has been proposed that the precise pattern of ERK-1/2 activation ultimately determines whether the kinase participates in cell death-promoting or cell survival pathway (Stanciu et al. 2000; Chang and Karin 2001; Stanciu and DeFranco 2002; Canals et al. 2003b). Furthermore, MAPK cascade may contribute to the regulation of dopamine signalling and synaptic strength (Moron et al. 2003).

We have previously described that DA neurons in neuronal-enriched midbrain cultures are highly resistant to GSH depletion. We show that low (non-toxic) doses of nitric oxide eliminate such tolerance to GSH depletion by a mechanism that includes ERK-1/2 and 12-lipoxygenase (12-LOX) activation (Canals et al. 2003a,b).

In the present work, we study the differential effect of BSO treatment in neuronal-enriched and mixed neuron/glia midbrain cultures, and demonstrate that the tolerance of DA cells to GSH depletion is also eliminated by glia. This effect, that is NO-independent, requires ROS-induced ERK-1/2 activation in glial cells.

Materials and methods

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Dulbecco's modified Eagle's medium (DMEM) with high glucose (4.5 g/L), Ham's F12 nutrient mixture, Eagle's minimal essential medium (EMEM) with Earl's salts and Leibovitz's L-15 medium, all of which were supplemented with l-glutamine, fetal calf serum (FCS), sodium pyruvate and l-glutamine, were purchased from Gibco BRL (Paisley, Scotland, UK). Glucose 45%, insulin, putrescine, progesterone and sodium selenite were from Sigma (Madrid, Spain) and human transferrin, 30% iron-saturated, from Boehringer-Mannheim (Barcelona, Spain).

Anti-tyrosine hydroxylase (TH) antibodies made in mouse and rabbit were from Chemicon (Temecula, CA, USA); O1, O4 and A2B5 were obtained from hybridoma supernatants (Raff et al. 1979, 1983); polyclonal anti-GFAP antibody, raised in rabbit, was from Dako (Glostrup, Denmark); antibody against β-tubulin (βIII) raised in rabbit was from Babco (Richmond, CA, USA), isolectin B4 from Bandeiraea simplicifolia Fluorescein isothiocyanate (FITC) labelled, anti-mitogen activated protein kinase, anti-phospho-ERK1 and ERK2 antibody, anti-microtubule-associated protein 2a + 2b (MAP-2) antibody and anti-rabbit IgG conjugated with tetramethylrhodamine (TRITC) were purchased from Sigma, anti-OX-6 (specific for major histocompatibility complex class II antigens) were from Serotec (Oxford, UK); anti-mouse IgG fluorescein was from Jackson Immuno-Research (West Grove, PA, USA) and anti-mouse IgM Alexa Fluor® 594, anti-mouse IgG Alexa Fluor® 568 and anti-rabbit IgG Alexa Fluor® 488 were from Molecular Probes (Eugene, OR, USA).

Trypan blue, bovine serum albumin, poly d-lysine, p-phenylenediamine, bis-benzimide, l-buthionine-[S,R]-sulfoximine (BSO), pargyline, N-(1-napthyl)-ethylenediamine, sulfanilamide, dimethylsulfoxide (DMSO), 5,5′-dithio-bis2-nitrobenzoic acid (DTNB), reduced and oxidised forms of glutathione, 7-nitroindazole (7NI), N-nitro-l-arginine methyl ester (l-NAME), minocycline and fluoroacetate were from Sigma. NADPH, lactate dehydrogenase standard (LDH), the cytotoxicity detection kit for LDH, cell proliferation kit I (MTT) and GSH reductase (GR) were from Boehringer-Mannheim. SB20358 and PD98059 were from Alexis (Carlsbad, CA, USA). Baicalein, indomethacin, nordihydroguaiaretic acid (NDGA), SP600125 and 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene (U0126) were from Calbiochem (Darmstadt, Germany); CM-H2DCFDA from Molecular Probes (Eugene, OR, USA). The radiochemicals [3H]DA (70 Ci/mmol) and [3H]GABA (90 Ci/mmol) were obtained from Dupont NEN (Boston, MA, USA). The BCA protein assay kit was from Pierce (Rockford, IL, USA). All other reagents were of the highest purity commercially available from Merck or Sigma.

Mesencephalic neuron-enriched cultures

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Neuronal-enriched cultures from embryonic day 14 (E-14) rat midbrain were obtained and prepared as previously described (Mena et al. 1993; Pardo et al. 1997). The cells were seeded in DMEM with 15% fetal calf serum (DMEM-FCS) at a density of 105 cells/cm2 in multiwells or glass cover slides previously coated with poly d-lysine, 4.5 μg/cm2, in 0.1 m borate buffer, pH 8.4. The cultures were kept in a humidified chamber at 37°C in a 5% CO2 atmosphere. Twenty-four hours after plating, the cells were changed to serum-free defined medium (EF12) as reported elsewhere (Mena et al. 1993; Pardo et al. 1997). EF12 consisted of a 1 : 1 (v/v) EMEM and nutrient mixture of Ham's F-12, supplemented with d-glucose (6 mg/mL), insulin (25 μg/mL), transferrin (100 μg/mL), putrescine (60 μm), progesterone (20 nm) and sodium selenite (30 nm). With the above protocol, neuronal-enriched midbrain cultures consist of 85–90% neuronal cells (β-tubulin+ or MAP-2+ cells) of which around 3–5% are dopaminergic neurons (TH+ cells), and 10–15% glial cells composed mainly of astrocytes (GFAP+), glial progenitors (A2B5+) and oligodendrocytes (O1+ and O4+). Microglial cells are rarely present in the culture and when detected (isolectin B4 labelled cells and OX-6+ cells) they represent less than 0.05% of total cells.

Mesencephalic mixed neuron/glia cultures

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Primary rat ventral mesencephalic neuron/glia cultures from embryonic day 14 (E-14) rat midbrain were obtained and prepared as previously described (Mena et al. 1997; Pardo et al. 1997). The cells were seeded in DMEM-FCS at a density of 105 cells/cm2 in multiwells or glass cover slides previously coated with poly d-lysine, 4.5 μg/cm2, in 0.1 m borate buffer, pH 8.4. The cultures were kept in a humidified chamber at 37°C in a 5% CO2 atmosphere. Immunocytochemical analysis indicated that, at the time of treatment, the cultures were made of around 40% GFAP+ astrocytes, 12% microglia, 3% oligodendrocytes, 0.5% glial progenitors and 40% MAP2+ neurons, of which 1.8–3.4% were TH+ dopamine neurons. The rest of the cells are neuronal progenitors (nestin+).

Experimental treatments

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After 4 days in culture, cells were treated with 20 μm BSO or vehicle, for different time periods; 0.5–48 h for ERK-1/2 western blot, 6 h for ERK-1/2 immunocytochemistry analysis and 48 h for viability, cell type selectivity and neurotransmitter uptake assays. Enzyme inhibitors or their corresponding solvents, were routinely added 30 min before BSO treatment.

In order to selectively inactivate glial cells in the culture, we used two gliotoxins, minocycline and fluoroacetate (FA). Minocycline (20 μm) was added to the culture 30 min and FA (1 mm) 1 h before BSO treatment (Peters 1963; Keyser and Pellmar 1994; Tikka et al. 2001; Wu et al. 2002).

Immunocytochemistry

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DA neurons were characterized by immunostaining with a mouse monoclonal anti-TH antibody (1 : 100) or a rabbit polyclonal anti-TH antibody (1 : 500), astrocytes with a rabbit polyclonal anti-GFAP antibody (1 : 500), oligodendrocytes with monoclonal anti-O1 and anti-O4 antibodies (1 : 10) (Sommer and Schachner 1981), glial progenitors with anti-A2B5 (1 : 10) (Raff et al. 1979, 1983) and microglial cells were identified with isolectin B4 FITC-labelled, and anti-OX-6+ (Streit and Kreutzberg 1987; Ashwell 1991; Choi et al. 2003). To detect all neurons in the culture, a mouse monoclonal anti-MAP-2 antibody (1 : 250) or rabbit polyclonal anti-β-tubulin (βIII) (1 : 1000) were used. For TH, GFAP and MAP-2 immunostaining, cultures were fixed with 4% paraformaldehyde, washed in 0.1 m Tris-buffered saline, pH 7.4 (TBS), permeabilised with ethanol-acetic acid (19 : 1) and incubated overnight at 4°C with primary antibodies diluted in TBS containing 10% normal goat serum (NGS). Alexa Fluor®-conjugated secondary antibodies were employed to visualise positive cells under fluorescent or confocal microscopy. Active phosphorylated ERK-1/2 was immunocytochemically detected with the anti-phospho-ERK-1/2 antibody (1 : 200).

Co-localization of p-ERK-1/2 in neurons and glia was performed by double immunostaining in controls and BSO-treated cultures. Active ERKs with TH+ and GFAP+ cells were made as above but including anti-phospho-ERK-1/2 during overnight incubation. For β-tubulin and phospho-ERK co-localisation, paraformaldehyde fixed cultures were incubated overnight with appropriate antibodies in TBS containing 10% NGS and 0.01% Triton X-100, at 4°C. Different fixation protocols (paraformaldehyde + glutardialdehyde) and permeabilizations (higher Triton concentrations or ethanol-acetic acid) were also used with similar results. For O1 and A2B5 staining, antibodies were directly added to living cells and incubated for 15 min at room temperature (22°C), washed in DMEM and fixed with 4% paraformaldehyde previous to anti-mouse IgM Alexa Fluor® 488 development. For co-localization studies, after O1 or A2B5 staining had been completed, cells were incubated overnight with anti-phospho-ERK-1/2 antibody in TBS containing 10% NGS, 0.01% Triton X-100 and the next day developed with FITC-conjugated anti-mouse IgG (Jackson). Phospho-ERK-1/2 and microglial cells co-localisation was performed in 4% paraformaldehyde, 1% glutardialdehyde fixed cultures, by overnight incubation with 12.5 mg/L isolectin B4 and anti-phospho-ERK-1/2 in TBS with 1% Triton X-100, at 4°C. To eliminate glutardialdehyde auto-fluorescence, after fixation cover slides were treated with 4 mg/mL NaBH4 in TBS for 10 min at room temperature. The number of immunoreactive cells was counted in 1/7 of the total area of the cover slides by random sampling.

For co-localization of ROS production in neurons and glia, double staining with 10 μm DCF and antibodies for neurons and glial cells were performed.

Cell viability measurements

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Mitochondrial activity was measured with the MTT assay. Cells were grown on 24-well culture plates with 500 μL defined medium and treated with various reagents according to the experimental design. The MTT assay measures the ability of cells to metabolise 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). At the end of the treatment period, 300 μL of culture medium were removed from each well and 20 μL of MTT solution (5 mg/mL) were added and incubated for 1 h. At this time, 200 μL of solubilisation solution [10% sodium dodecyl sulfate (SDS) in HCl 0.01 m] were then added to the wells and after 24 h of incubation at 37°C, 100 μL were transferred into 96-well microtitre plates, and the absorption value at 540 nm was measured in an automatic microtiter reader (Spectra Fluor, Tecan).

Chromatin condensation and fragmentation were assessed by DNA staining with bis-benzimide (Hoechst 33342). Cells growing on cover slides were fixed in 4% paraformaldehyde and nuclei were stained with bis-benzimide added in the anti-fading solution (3 × 10−6 m final concentration) (Hilwig and Gropp 1975; Pardo et al. 1997).

For necrotic cell death determination, trypan blue dye exclusion assay and lactate dehydrogenase (LDH) activity were performed. LDH was measured in the culture medium by using a cytotoxicity detection kit (Decker and Lohmann-Matthes 1988), and expressed as a percentage versus detergent-extracted controls (100% cytotoxicity). In neuronal-enriched cultures, LDH release to the culture medium correlates with cell death measured by trypan blue dye exclusion assay (Canals et al. 2001b).

Western blot analysis

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Primary midbrain cultures were homogenized with a sonicator in buffer containing 20 mm Tris HCl, 10 mm potassium acetate (AcK), 1 mm dithiothreitol (DTT), 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride (PMSF), 1 mm benzamidine, leupeptin, aprotinin, pepstatin 5 μg/mL each, 10 mm sodium flouride (FNa), 2 mm sodium molibdate, 10 mmβ-glicerophosphate, 0.2 mm ortovanadate, 0.25% NP-40, pH 7.4, and then centrifuged at 12 000 g for 30 min at 4°C. The supernatant was used for protein determination by the BCA protein assay kit and for electrophoretical separation. Samples (30 μg) were added to SDS sample loading buffer, electrophoresed in 10% SDS-polyacrilamide gels and then electroblotted to 0.45 μm nitrocellulose membranes. For immunolabelling, the blots were blocked with TTBS (20 mm Tris-HCl pH 7.6, 137 mm NaCl plus 0.1% (v/v) Tween-20 and 5% dry skimmed milk) for 1 h at room temperature. After blocking non-specific binding, the membranes were incubated with mouse anti-phospho-ERK-1/2 (1 : 5000) in blocking solution overnight at 4°C. The blots were developed by chemiluminiscence detection using a commercial kit (Amersham) and quantified by computer-assisted videodensitometry. Rabbit anti-ERK-1/2 (1 : 20000) was employed as a control of charge after stripping nitrocellulose membrane.

For 12-LOX protein, the membranes were incubated with rabbit anti-12-LOX (1 : 2000). Mouse anti-β-actin (1 : 10000) as a control of charge.

Uptake studies

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[3H]DA uptake was measured after incubation of the cells with 10−8 m[3H]DA (70 Ci/mmol), in the presence of pargyline 10−5 m, and ascorbic acid 10−3 m, at 37°C for 30 min. Non-specific uptake/binding was calculated in the presence of 10−5 m mazindol and represented ≤ 5% (Beart and McDonald 1980;). [3H]GABA uptake was performed in the presence of 10−5 m aminooxyacetic acid and 10−3 m ascorbic acid and incubated for 4 min with 10 nm[3H]GABA (90 Ci/mmol). Non-specific uptake/binding was calculated by incubating cultures at 0°C and represented ≤ 7% of the total (Michel and Hefti 1990). Proteins were measured by the BCA protein assay kit.

Glutathione measurements

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Total glutathione levels were measured by the method of Tietze (1969). Briefly, 1 × 105 cells were washed with PBS, lysed in 100 μL of 3% perchloric acid (PCA) for 30 min at 4°C, centrifuged, and the supernatants were neutralised with 4 vol. of 0.1 m NaH2PO4, 5 mm EDTA, pH 7.5. Fifty microlitres of resulting supernatants were mixed with DTNB (0.6 mm), NADPH (0.2 mm) and glutathione reductase (1 U) and the reaction monitored in a P96 automatic microtitre reader at 412 nm for 6 min. Oxidised glutathione (GSSG) was measured in the cells by the method of Griffith (1980). Briefly, after PCA extraction and pH neutralisation, GSH was derivatized with 2-vinylpyridine at room temperature for 1 h and the reaction carried out as above. GSH was obtained by subtracting GSSG levels from total glutathione levels.

DCF assay

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Intracellular ROS were detected using an intracellular ROS dye. The non-fluorescence DCF is oxidized by intracellular ROS to form the highly fluorescence DCF. The cells were incubated with 5 μm DCFH-DA 30 min before BSO treatments and after washing the excess of DCF with a medium free of phenol red (PBS with 1 mm glucose), the fluorescence was measured in a fluorescence microscope or in an automatic microtitre reader (Spectra Fluor) at excitation and emission of 485 and 582 nm, respectively.

For co-localization of ROS production in neurons and glia, the cells were incubated with DCF 30 min before BSO treatment (1 mm × 1 h) and after gentle fixation, neurons were characterized by immunostaining with anti-MAP-2, astrocytes with anti-GFAP and microglia with isolectin B4.

Determination of H2O2 concentration

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The concentration of H2O2 in the culture media 1 h after BSO treatment were estimated with a colorimetric assay. The media were centrifuged at 10 000 g for 2 min, and 100 μL of the supernatants were added to 50 μL of 3,3′-dimethoxybenzidine (2 mm) and 50 μL of horseradish peroxidase (240 IU/mL). 3,3′-dimethoxybenzidine, which is colourless in its reduced form, is oxidized in the presence of H2O2 and peroxidase into a red-coloured product. Optical density was estimated at 490 nm. The concentration of H2O2 in the culture media were determined using standard solutions.

Nitrite measurement

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NO production was routinely quantified by measuring nitrite, a stable oxidation end product of NO (Green et al. 1982). Briefly, 400 μL of culture medium were mixed with 800 μL of Griess reagent (1.5% sulfanilamide in 1 N HCl plus 0.15%N-(1-naphtyl)-ethylenediamine dihydrochloride in distilled water, v : v). After 10 min of incubation at room temperature, the absorbance at 540 nm was determined in an automatic microtitre reader, by means of sodium nitrite as standard.

Statistical analysis

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The results were statistically evaluated for significance with one-way analysis of variance followed by the Newman–Keuls multiple comparison test as a post-hoc evaluation. Differences were considered statistically significant when p < 0.05.

Differential effects of GSH depletion in neuronal-enriched and mixed neuron/glia mesencephalic cultures

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Neuronal-enriched cultures grown in serum-free defined medium for 7 days in vitro consist of 85–90% neurons, 3–5% of which are dopaminergic, and 10–15% of which are glial cells consisting of astrocytes, glial progenitors and oligodendrocytes. Microglia are rarely present in the culture, or in proportions no greater than 0.05% of the total population. Mixed neuron/glia mesencephalic cultures grown in DMEM with serum for 7 days in vitro consist of 40% astrocytes, 12% microglia, 3% oligodendrocytes, 0.5% glial progenitors and 40% neurons, of which 1.8–3.4% were dopamine neurons. The rest of the cells are neuronal progenitors.

In neuronal-enriched midbrain cultures, reduction of GSH levels to 20% of baseline by treatment with the GSH inhibitor, BSO (20 μm for 48 h) did not induce cell death. However, in mixed neuron/glial midbrain cultures, BSO treatment (20 μm for 48 h) induced astrocyte morphological changes and selective neuronal death (Fig. 1a). Cell death was evident by an increase in apoptotic and necrotic markers; the number of apoptotic and necrotic cells increased and mitochondrial activity decreased in the BSO-treated cultures versus their respective controls (Fig. 1b). In order to test the relationship between GSH depletion and neuronal death, we measured GSH levels in both culture models. GSH levels were higher in neuron/glia than in the neuronal-enriched cultures and BSO treatment depleted to lower GSH levels in the neuronal-enriched than in mixed cultures (Fig. 1c).

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Figure 1. Effects of BSO treatment on cell viability and glutathione homeostasis in neuronal-enriched and mixed neuron/glia mesencephalic cultures. (a) Mixed neuron/glia cultures; MAP2+ and GFAP+ cells photomicrographs showing controls and cultures treated on the 4th day in vitro with 20 μm BSO for 48 h. A great reduction of neurons and activation of astrocytes is observed in cultures treated with BSO. Scale bar = 25 μm. (b) Effects of treatment with BSO 20 μm for 48 h on cell viability parameters in mixed neuron/glia mesencephalic cultures. (c) Glutathione levels in neuronal-enriched and mixed neuron/glia mesencephalic cultures treated on the 4th day in vitro with solvent or 20 μm BSO treatment for 48 h. Values are the mean ± SEM from six replicates of two independent experiments. Statistical analysis was performed by anova followed by Newman–Keuls multiple comparison test. **p < 0.01, ***p < 0.001 versus their respective controls; and +p < 0.05, +++p < 0.001 mixed neuron/glia versus neuronal-enriched cultures.

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In the mixed neuron/glial cultures, BSO treatment decreased neuron markers (MAP-2+ cells and 3H-GABA uptake, Figs 2b and d) and DA cell markers (TH+ cells and 3H-DA uptake, Figs 2a and c) and increased apoptotic cell death (Fig. 2f) and astrocyte activation (Fig. 2e). In contrast, BSO treatment did not affect neuronal or glial population in neuronal-enriched midbrain cultures (Figs 2a–f).

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Figure 2. Pattern of cellular effects of GSH depletion in neuronal-enriched (white squares) and mixed neuron/glia (black squares) mesencephalic cultures. (a) Number of DA neurons expressed as TH+ cells/well. (b) Area of total neurons (MAP-2+) in the cultures. (c) High-affinity 3H-DA uptake. (d) High affinity 3H-GABA uptake. (e) Area of glial cells (GFAP+) in the cultures. (f) Chromatin condensed nuclei stained with bis-benzimide were counted and expressed as a percentage of apoptotic cells with respect to the total cell number. Values are the mean ± SEM from six replicates of two independent experiments. Statistical analysis was performed by anova followed by Newman–Keuls multiple comparison test. **p < 0.01, ***p < 0.001 versus their respective controls; and ++p < 0.01, +++p < 0.001 mixed neuron/glia versus neuronal-enriched cultures.

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ROS generation by GSH depletion in neuron/glia cultures

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BSO treatment induced a dose- and time-dependent increase in intracellular ROS levels (Figs 3a, b and e). The generation of intracellular ROS takes place in neurons (Fig. 3c) and the concentration of H2O2 in the culture media highly increased 1 h after BSO treatment (Fig. 3d). Furthermore, pre-treatment with 200 μm ascorbic acid 30 min before 20 μm BSO prevented the ROS generation (Fig. 3f).

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Figure 3. Dose- and time-dependently ROS generation after BSO treatment in mixed neuron/glia cultures. (a) DCF+ cells photomicrographs showing controls and cultures treated on the 6th day in vitro with 1 mm BSO for 3 h. Scale bar = 25 μm. (b) DCF fluorescence area for the cultures showing in (a). (c) Generation of ROS (DCF) in neurons (MAP-2) and negative co-localization in astrocytes (GFAP) and microglia (isolectin B4) cells. All double staining are from the same field. DCF signals localized in neurons (in yellow in the merge image). Scale bar = 25 μm. (d) Levels of H2O2 in the culture media 1 h after BSO treatment. (e) Time-dependently ROS generation-nduced by 20 μm BSO treatment. DCF fluorescence was measured with a reader at 485 nm of excitation and 582 nm emission. (f) Pre-treatment with 200 μm ascorbic acid 30 min before BSO prevented the ROS generation. The results are expressed as mean ± SEM from six replicates. Statistical analysis was performed by anova followed by Newman–Keuls multiple comparison test. *p < 0.05, ***p < 0.001 versus controls; +++p < 0.001 versus BSO-treated groups.

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Pattern of cell phenotype affected by GSH depletion-induced toxicity in mixed neuron/glia cultures

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Immunocytochemical characterization of cell death in cultures treated for 48 h with 20 μm BSO, revealed that all neuronal types in the cultures (TH+ and MAP-2+) are strongly affected by the treatment, but TH+ cells remain more preserved (Figs 4a, c and d). TH+ cells represent a minor proportion of the total neurons in the control cultures (Fig. 4a, top panels) but after treatment with BSO, TH+ cells became the most abundant population of surviving neurons (Fig. 4a, middle panels co-staining for TH and MAP-2). DA neurons therefore are more resistant than other neurons to GSH-depletion-induced toxicity in midbrain mixed neuron/glia cultures. We have described previously the same phenomena in neuronal-enriched midbrain cultures (Canals et al. 2001a) and Nakamura et al. (2000) reported that the preferential resistance of DA neurons to the toxicity of GSH depletion was independent of cellular glutathione peroxidase (GPx) and was mediated by tetrahydrobiopterin (BH4).

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Figure 4. Role of ERK-1/2 activation in the cell death induced by GSH-depletion in mixed neuron/glia cultures. After 4 days in vitro, the culture was treated with 20 μm BSO or vehicle for 48 h and, 30 min before treatment, the pre-established groups received the p-ERK-1/2 inhibitors, PD 98059 (25 μm) or U0126 (10 μm). (a) Photomicrographs show cells with double immunostaining for DA neurons (TH+), left panel and total neurons (MAP-2+), right panel. Scale bar: 50 μm. (b) Number of microglial cells, expressed as percentage of total cells. (c) Number of DA neurons expressed as TH+ cells/well. (d) Area of total neurons (MAP-2+) expressed as percentage versus control. (e) Area of glial cells (GFAP+) in the culture. (f) Chromatin condensed and fragmented nuclei, stained with bis-benzimide, were counted and expressed as a percentage of apoptotic cells with respect to the total cell number. (g) Mitochondrial activity measured by MTT assay is presented as viability percentage versus controls. The results are expressed as mean ± SEM from six replicates of three independent experiments. The number of TH+ and percentage apoptotic cells in the control group were 2058 ± 302.9 and 6.8 ± 0.05%, respectively. Statistical analysis was performed by anova followed by Newman–Keuls multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001 versus controls; +p < 0.05, ++p < 0.01, +++p < 0.001 versus BSO-treated groups.

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With respect to glia, only astrocytes were affected, changing their morphology to more protoplasmic (Fig. 1a) and with increased expression of GFAP (Fig. 4e). However, microglia (isolectin B4) and oligodendrocytes (O1+ and O4+ cells) were unaffected by BSO-treatment (Fig. 4b and data not shown). Markers of activated microglia as assessed by immunocytochemistry or western blot were not found.

MEK-1/2 inhibitors, U0126 and PD98059, block GSH depletion-induced cell death in neuron/glia cultures

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  2. Abstract
  3. References

We and others have previously demonstrated that a GSH depletion of 80–90% versus controls for up to 72 h, did not induce cell death in neuron enriched primary midbrain cultures (Marshall et al. 1999; Canals et al. 2001a,b). Nevertheless, these GSH depleted conditions in mixed neuron/glia cultures result in selective neuronal death (Figs 1 and 2). To investigate the role of ERK activation in the cell death process, we used U0126 and PD98059, two selective inhibitors of the ERK-activating kinases, MEK-1 and MEK-2 (MEK-1/2). As shown in Figs 4 and 5, these inhibitors prevent both ERK-1/2 activation and neuronal cell death induced by GSH-depleted conditions. In contrast, when SB203580, a specific inhibitor of p38 MAPK, was used at concentrations of up 20 μm, or SP600125, an inhibitor of Jun N-terminal kinase (JNK) at doses of (0.25, 0.5, 5 and 10 μm) no protection from cell death was observed (data not shown).

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Figure 5. Time-dependent activation of p-ERK-1/2 in GSH-depleted mixed neuron/glia cultures. (a) Western blot and (b) densitometric analysis show the time-course activation of p-ERK-1/2. After 6 days in vitro, the cells were treated with 20 μm BSO or vehicle for the indicated times. The active and total ERK-1/2 were assayed in the cell lysate. (c) Active and total ERK-1/2 visualized by western blot corroborating the P-ERK-1/2 inhibition by PD 98059 (25 μm) or U0126 (10 μm). (d) Densitometric analysis of (c) expressed as percentage versus control. After 6 days in vitro, pre-established groups were treated with the ERK-1/2 inhibitors, PD 98059 (25 μm) or U0126 (10 μm) 30 min before BSO treatment for 6 h. Statistical analysis was performed by anova followed by Newman–Keuls multiple comparison test. *p < 0.05, **p < 0.01 versus controls; ++p < 0.01 versus BSO-treated groups.

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Added to the culture 30 min before BSO treatment, U0126 preserves all cell viability parameters (chromatin condensation and DNA fragmentation, MTT activity and trypan blue exclusion assay) studied, being 10 μm the concentration required for maximal protection (Fig. 4f and data not shown). In the same way, PD98059 preserves cell viability in the culture, with maximal protection observed at 25 μm (Figs 4f and g). Furthermore, MEK inhibitors protect all cell types in the culture and abolish the astroglia activation (Figs 4a–e).

Glutathione depletion activates ERK-1/2 in midbrain mixed neuron/glia cultures

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  2. Abstract
  3. References

To investigate in this model, the activation of ERK-1/2 in response to GSH depletion, we first studied the phosphorylation state of these proteins by western blot analysis. We analyzed the kinetic of ERK activation in response to BSO or vehicle-treated cultures and the phosphorylation state of ERK-1/2 was analyzed 30 min, 1, 4, 6, 8, 16, 24, 32 and 48 h after BSO addition. The maximal ERK activation was observed 4 and 6 h after BSO treatment (Figs 5a–b). Next, we showed that MEK-1/2 inhibitors, U0126 and PD98059, added 30 min before BSO treatment for 6 h, blocked or reduced the ERK activation induced by GSH depletion in mixed cultures (Figs 5c–d).

Nitric oxide sintase activation is not implicated in the glutathione depletion-induced cell death

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  3. References

We have shown that NO precipitates neuronal death and activation of glia in BSO-treated neuronal cultures (Canals et al. 2001a) and NO overproduction has been implicated in DA cell death and in PD (Hirsch et al. 1999; Du et al. 2001; Le et al. 2001; Wu et al. 2002; Canals et al. 2003a,b). Furthermore, GSH depletion may activate NOS (Heales et al. 1996; Ibi et al. 1999) and iNOS co-localizates with activated microglia (Ryu et al. 2002; Choi et al. 2003) but not with astrocytes in an in vivo model of nigral DA dysfunction (Choi et al. 2003). To investigate the role of NO in the BSO-induced cell death, we used l-NAME 1 mm, a selective and wide spectrum NOS inhibitor. As shown in Fig. 6, this inhibitor, added 30 min before BSO treatment, did not prevent the neuronal death process and neither did 7NI (0.5 and 1 mm), another NOS inhibitor. In addition, nitrite concentration in the culture media did not increase after BSO treatment (data not shown).

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Figure 6. Nitric oxide sintase (NOS) is not implicated in the neurotoxicity induced by GSH-depletion in mixed neuron/glia cultures. After 4 days in vitro, the cells were treated with the NOS inhibitor, l-NAME (1 mm) or vehicle 30 min before the BSO treatment (20 μm for 48 h). (a) Number of DA neurons expressed as TH+ cells/well. (b) Area of total neurons (MAP-2+) expressed as percentage versus control. (c) Chromatin condensed nuclei stained with bis-benzimide were counted and expressed as a percentage of apoptotic cells with respect to the total cell number. The results are expressed as mean ± SEM from six replicates of three independent experiments. The number of TH+ and percentage apoptotic cells in the control group were 1876 ± 28 and 6.8 ± 0.05%, respectively. Statistical analysis was performed by anova followed by Newman–Keuls multiple comparison test. **p < 0.01 versus controls.

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Inhibitors of lipoxygenase block cell death in GSH-depleted neuron/glia cultures

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GSH depletion activates 12-LOX in nerve cell death caused by glutamate (Li et al. 1997a) and 12-LOX inhibitors block cell death induced by GSH-depletion in PC12 cells (Le Foll and Duval 2001) and in midbrain neuronal-enriched cultures (Canals et al. 2003a).

To investigate the possible role of lipoxygenase in cell death in the above conditions, given that 12-LOX is the predominant brain isoform of this enzyme (Piomelli and Greengard 1990), we tested baicalein, a selective 12-LOX inhibitor, in cell viability assays. Baicalein at the dose of 10 μm rescues TH+ cells (Figs 7a–b) as well as the BSO-induced apoptosis (Fig. 7d); However, indomethacin, 25 μm, a cyclo-oxygenase (COX) inhibitor, did not protect (data not shown). Baicalein blocked the increased GFAP expression induced by BSO (Fig. 7e). NDGA, another flavonoid LOX inhibitor and ascorbic acid, a classic antioxidant, also protected from the BSO-induced neuronal cell death (Fig. 7f). Furthermore, baicalein and ascorbic acid blocked the BSO-induced activation of ERK-1/2 proteins (Fig. 8). LOX inhibitor effects on neuronal death protection because of their antioxidant properties may not be discarded, as has been described in other cell death models (Suk et al. 2003).

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Figure 7. Role of 12-lipoxygenase in the cellular death induced by GSH-depletion in mixed neuron/glia cultures. After 4 days in vitro, the cells were treated with 20 μm BSO or vehicle for 48 h, and 30 min before treatment, the pre-established groups received the 12-lipoxygenase inhibitor, baicalein (5–10 μm). (a) Photomicrographs of DA neurons (TH+), left panel and total neurons (MAP-2+), right panel. Scale bar: 50 μm. (b) Number of DA neuron expressed as TH+ cells/well. (c) Area of total neurons (MAP-2+) expressed as percentage versus control; pre-established groups received the 12- LOX inhibitor, baicalein (10 μm). (d) Chromatin condensed nuclei stained with bis-benzimide were counted and expressed as a percentage of apoptotic cells with respect to the total cell number. (e) Area of glial cells (GFAP+) in the culture expressed as percentage versus control. (f) Area of total neurons (MAP-2+) expressed as percentage versus control; pre-established groups received the LOX inhibitor NDGA (0.5 μm) or the antioxidant, ascorbic acid (200 μm) 30 min before the BSO treatment. The results are expressed as mean ± SEM from six replicates of three independent experiments. Statistical analysis was performed by anova followed by Newman–Keuls multiple comparison test. *p < 0.05, ***p < 0.001 versus controls; +p < 0.05, ++p < 0.01, +++p < 0.001 versus BSO-treated groups.

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Figure 8. Baicalein and ascorbic acid blocked p-ERK-1/2 expression induced by GSH-depletion in mixed neuron/glia cultures. (a) Western blot and (b) densitometric analysis show the baicalein effect on activation of p-ERK-1/2. (c) Western blot and (d) densitometric analysis show the ascorbic acid effect on activation of p-ERK-1/2. After 6 days in vitro, pre-established groups were treated with the 12-LOX inhibitor, baicalein (10 μm) or the antioxidant, ascorbic acid (200 μm) 30 min before BSO treatment for 6 h. Statistical analysis was performed by anova followed by Newman–Keuls multiple comparison test. *p < 0.05, **p < 0.01 versus controls; +p < 0.05, ++p < 0.01 versus BSO-treated groups.

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Effect of BSO treatment on 12-LOX protein levels

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  2. Abstract
  3. References

Li et al. (1997b) have previously shown that GSH depletion induced by glutamate treatment in primary immature cortical cultures, induces a 2- to 3-fold increase in 12-LOX protein. To test this possibility in mixed neuron/glia midbrain cultures depleted of GSH, we performed western blot analysis of 12-LOX protein. GSH depletion for up to 48 h (30 min; 1, 4, 6, 8, 16, 24, 32 and 48 h), did not vary 12-LOX protein levels (data not shown), in accordance with our previous data in neuronal-enriched cultures (Canals et al. 2003a).

ERK activation in GSH-depleted midbrain mixed cultures is restricted to glial cells

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To investigate if BSO-induced ERK phosphorylation occurs in concrete cell types of the culture, we performed double immunocytochemical techniques for fluorescent microscopy and confocal microscopy examination. In control conditions, we found less than 7% of cells showing a very light phospho-ERK positive (p-ERK+) staining (Figs 9a and b). In contrast, after BSO treatment, p-ERK+ staining become more evident and there was a 2.5-fold increase in the number of positive cells (Figs 9a and b). Phospho-ERK labelled cells were diverse in morphology and size. BSO treatment greatly increased the intensity of p-ERK+ staining (Fig. 9).

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Figure 9. GSH depletion selectively activates ERK-1/2 in glial cells in neuron/glia midbrain cultures. (a) There is a subpopulation of cells in the culture in which GSH depletion is able to activate ERK-1/2. After 6 days in vitro, the cultures were treated with 20 μm BSO; after 6 h, the cultures were fixed and immunostained using an anti-phospho (p)-ERK antibody. (b) Labelled cells were counted and presented as a percentage versus control. Values are expressed as the mean ± SEM from n = 6 replicates. Similar results were obtained in two independent experiments. (c) Co-localization of p-ERK+ cells with astrocytes (GFAP+ cell) and microglia (isolectin+ cell) in BSO-treated cultures. (d) Double-stained cells in control and BSO-treated groups were quantified and expressed as a percentage versus total cells. Values are expressed as the mean ± SEM from n = 6 replicates of independent experiments. Similar results were obtained in two independent experiments. Statistical analysis was performed by anova followed by the Newman–Keuls multiple comparison test. *p < 0.05 versus controls. Scale bar in all photomicrographs = 50 μm.

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To identify the midbrain culture cell types, in which BSO-treatment activates ERK, co-localization studies were performed. The basal levels of p-ERK+ cells in control conditions were identified meanly as astrocytes and microglia (Fig. 9c). After 6 h of BSO treatment, the number of astrocytes and microglia co-staining with p-ERK greatly increased (Figs 9c and d). In contrast, GSH depletion did not increase the number of p-ERK co-staining neurons (β-Tub+ nor TH+ cells).

If ERK-1/2 activation in response to BSO occurs selectively in glial cells, then selective metabolic impairment of these cells from the culture must prevent BSO-dependent ERK phosphorylation. To test this hypothesis, we used the specific gliotoxic drug, fluoroacetate (FA), an aconitase inhibitor that is selectively uptaken by glial cells and rapidly depletes intracellular ATP levels (Peters 1963; Keyser and Pellmar 1994; Waniewski and Martin 1998). Pre-treatment with 1 mm FA (1 h) decreased ERK-1/2 activation induced by BSO treatment (Figs 10a and b). However, blockade of microglial activation with minocycline, 20 μm added 30 min before BSO treatment, did not block ERK-1/2 protein activation (Figs 10c and d).

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Figure 10. Role of glial cells in the ERK-1/2 activation in GSH-depleted mixed neuron/glia cultures. After 6 days in vitro, the cells were treated with 20 μm BSO or vehicle for 6 h; 1 h and 30 min before, pre-established groups received the gliotoxin fluoroacetate (FA, 1 mm) or minocycline (Mino, 20 μm), respectively. Cells were then lysed and both active and total ERK-1/2 were assayed in the cell lysate by western blot (a and c). (b and d) show the densitometric analysis of (a and c), respectively. The results are expressed as mean ± SEM of three independent experiments. Statistical analysis was performed by anova followed by Newman–Keuls multiple comparison test. *p < 0.05, **p < 0.01 versus controls; +p < 0.5 versus BSO-treated groups.

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Viability studies, co-localization experiments and gliotoxins studies, altogether, demonstrate that in mixed neuron/glia midbrain cultures, ROS generation-induced by GSH depletion specifically activates ERK-1/2 in glial cells, and that such activation induces selective neuronal death.

Discussion

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  2. Abstract
  3. References

The effect of GSH on neuronal survival has been widely studied in several experimental models of neurodegenerative diseases. PD models have received special attention as a severe GSH depletion in the SN of PD patients has been described (Perry et al. 1982; Riederer et al. 1989; Sian et al. 1994). Experimental GSH depletion in vivo does not cause nigral degeneration by itself in the rat, but it renders DA neurons more susceptible to concomitant insults (Pileblad et al. 1989; Wullner et al. 1996; Toffa et al. 1997).

In cell culture, different susceptibility to GSH depletion has been reported according to the cell type investigated and the brain region used for primary cultures. Cell death in primary immature cortical neurons, and HT22 hippocampal nerve cell line, is dose-dependently induced by BSO-mediated GSH depletion and involves activation of 12-LOX and GC, but not of PKG (Li et al. 1997b). In contrast, neuronal enriched midbrain cultures and the CSM14.1.4 rat midbrain cell line are resistant to depletion of GSH as severe as 95% of baseline, induced by treatment with BSO for 4 days (Marshall et al. 1999; Mytilineou et al. 1999; Canals et al. 2001a, 2003a).

We have previously described that GSH synthesis inhibition in neuronal-enriched midbrain cultures lasting for 4 days causes the death of the neurons. LOX, GC and PKG inhibitors prevent the neuronal death. Despite the differences in susceptibility, cell death induced by GSH depletion seems to be tightly linked with 12-LOX and GC activation in different culture models (Canals et al. 2003a).

In this study, we show that GSH depletion, 48 h after treatment with BSO, induces cell death in neuron/glia but not in neuronal-enriched midbrain cultures, indicating that glial cells abrogate DA neuron tolerance to GSH depletion. The mechanism for this effect includes generation of intracellular ROS in neurons and selective activation of ERK pathway in glial cells. Glutathione depletion activates p-ERK-1/2 proteins 4 and 6 h after BSO treatment. MEK-1/2 and 12-LOX inhibitors, as well as ascorbic acid, block the induced cell death and the p-ERK activation. However, NOS, COX, JNK and p38 MAPK inhibitors did not protect from neuronal death. As shown, increased p-ERK-1/2 induced by BSO is expressed mainly in astrocytes and microglial cells, but not in neurons. Selective metabolic impairment of glia with fluoroacetate decreased ERK activation, but pre-treatment with minocycline did not. Our results indicate that ROS-dependent activation of ERK in glia may be the critical factor for the neuronal death induced by GSH depletion.

The gliotoxins used, fluoroacetate and minocycline, have been widely employed to discriminate glial-cell mediated actions in different models, including mixed neuron-glia cultures (O'Malley et al. 1994; Kenigsberg and Mazzoni 1995; Krieglstein and Unsicker 1997; Canals et al. 2003b), brain slice preparations (Keyser and Pellmar 1994; Alvarez-Maubecin et al. 2000) and in vivo studies (Largo et al. 1996; Wu et al. 2002). In all these reports, as well as in the present study, FA and minocycline at the doses used, have demonstrated high specific glial actions with low or no effect on the neuronal population.

p38 MAPK has been implicated in dopamine cell death in oxidative stress experimental models of parkinsonism and in patients with PD (Ferrer et al. 2001; Zawada et al. 2001; Jeohn et al. 2002; Choi et al. 2003). Activated microglia stimulates p38-MAPK and neuronal cell death (Bhat et al. 1998; Li et al. 2003). In a model of nigral dopamine neurons degeneration in vivo, activated microglia via MAPKs such as ERK-1/2 and p38 MAPK, increased expression of iNOS and COX-2 (Choi et al. 2003). Our results, however, suggest that microglia and NO play an irrelevant role in the neuronal death induced by GSH depletion in neuronal/glial midbrain cultures, as there are not activated microglia markers, nor increased iNOS and COX-2 activities or stimulated p38 MAPK pathway in this model. Furthermore, minocycline at doses previously tested to inactivate microglia, did not block p-ERK activation.

LOX inhibitors provide full protection from BSO toxicity in neuronal midbrain cultures (Mytilineou et al. 1999; Canals et al. 2003a; Kramer et al. 2004), indicating that arachidonic acid metabolism, through the LOX pathway and the generation of ROS, plays a role in the loss of cell viability. Phospholipase A2, the principal enzyme that stimulates the release of arachidonic acid from membrane phospholipids (Piomelli 1993) is found predominantly in astrocytes (Farooqui et al. 2003). We have shown that LOX inhibitors, but not ascorbic acid, block NO triggered cell death in GSH-depleted neuronal-enriched midbrain cultures (Canals et al. 2003a). In this study with neuron/glial cultures, we show that NDGA and baicalein, flavonoids and LOX inhibitors as well as the classic antioxidant ascorbic acid, prevent ERK activation and protect from DA cell death induced by GSH depletion. Overall, these results do not allow us to exclude that LOX inhibitors' neuroprotection in the mixed cultures is as a result of their antioxidant properties.

In PD, one of the earliest chemical findings is a drop out of GSH levels in the substantia nigra which supports the oxidative stress hypothesis for the pathogenesis of this disease (Riederer et al. 1989). The loss of GSH also renders cells unable to efficiently remove the ROS, even though the levels of GSH peroxidase are normal. A final common pathway in neurodegenerative diseases and the regulatory role of the glutathione cycle have been proposed. A candidate-signalling pathway in this regard is characterized by the cascade arachidonic acid/HPETE/cGMP followed by activation of cGMP-dependent kinase and phosphorylation of NF-kB proteins and possibly CREB (Weber 1999).

Our results indicate that the MEK pathway plays a central role in the neuronal death caused by the GSH depletion in presence of glial cells. Recent accumulating evidence suggests that MAPK activation and its functional significance are specific to cell type and/or stimulus (Chang and Karin 2001). ERK may have a dual role in the regulation of cell death and survival. Strong and persistent activation of ERK leads to cell death (Stanciu et al. 2000; Schulz and Gerhardt 2001; Seo et al. 2001; Canals et al. 2003b), whereas a short-lived activation of ERK is associated with survival (Xia et al. 1995; Fukunaga and Miyamoto 1998; Mena et al. 2002; de Bernardo et al. 2003). MAPK inhibitors block stress-induced nerve cell death in an immortalized hippocampal cell line (Maher 2001) and peroxynitrite-induced apoptosis in human dopaminergic neuroblastoma cells (Oh-hashi et al. 1999). Finally, MEK inhibition by U0126 was protective against oxidative glutamate injury in a mouse neuronal cell line, but not against death caused by TNFα or staurosporine (Satoh et al. 2000).

In conclusion, our data show that GSH depletion induces selective neuronal cell death in co-cultures of neuron/glia, but not in neuronal-enriched midbrain cultures, via a free-radical producing cascade which activates ERK/MAP kinase specifically in glial cells. This may have relevance to the pathogenesis of PD and the possible pharmacological manipulation of this signalling pathway may open new therapeutic targets in this disease.

Acknowledgements

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This work was supported by the Spanish Government Grant, FIS 2002/PI20265 and CAM 8.5/49/2001. JM and SB were recipients of FIS pre-doctoral fellowships; SC and RMS of postdoctoral fellowships (MCYT and CAM, respectively). The authors thank R. Villaverde for her kind technical assistance.

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