Characterization of iodoacetate-mediated neurotoxicity in vitro using primary cultures of rat cerebellar granule cells

Free Radical Biology & Medicine, Vol. 28, No. 1, pp. 102–107, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter

PII S0891-5849(99)00215-4

Original Contribution CHARACTERIZATION OF IODOACETATE-MEDIATED NEUROTOXICITY IN VITRO USING PRIMARY CULTURES OF RAT CEREBELLAR GRANULE CELLS CRAIG S. MALCOLM, KAREN R. BENWELL, HELEN LAMB, DAVID BEBBINGTON,

and

RICHARD H. P. PORTER

Cerebrus Ltd., Winnersh, Wokingham, UK (Received 13 July 1999; Revised 21 September 1999; Accepted 5 October 1999)

Abstract—The neuroprotective efficacy of antioxidant molecules against iodoacetate (IAA) neurotoxicity in rat cerebellar granule cell (CGC) cultures was investigated. Transient exposure to IAA caused a concentration-dependent decrease in cell viability (ED50 ⫽ 9.8␮M). Dizocilpine maleate (MK-801), and 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxaline-7-sulfonamide (NBQX), failed to prevent IAA toxicity. Certain antioxidant molecules were shown to be neuroprotective against IAA when combined with MK-801 but were ineffective when administered alone. (S)-(-)-Trolox, butylated hydroxytoluene (BHT), and U-83836E exhibited EC50 values of 78, 5.9, and 0.25 ␮M, respectively, in the presence of 10 ␮M MK-801. IAA also induced an increase in intracellular oxidative stress, which was quenched by the antioxidants (in the presence of MK-801) in cultures loaded with the oxidant sensitive dye 2⬘7⬘-dichlorodihydrofluorescein diacetate (DCFH-DA). © 2000 Elsevier Science Inc. Keywords—Free radicals, Cerebellar granule cells, Hypoglycemia, Antioxidants, MK-801

INTRODUCTION

neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (HD) [1,8 –11]. A variety of evidence suggests a role for impaired energy supply in the aetiopathogenesis of chronic neurodegenerative disorders. For example, it has been shown that the mitochondrial toxins 1-methyl4-phenyl-pyridine (MPP⫹) and 3-nitropropionic acid reproduce many of the pathological features of PD and HD, respectively [10,12]. Additionally, mitochondria may also be involved in acute neurodegenerative mechanisms, because their ability to take up excess cytosolic calcium can result in uncoupling of the electron transport chain and subsequent free radical generation [13,14]. As part of an ongoing investigation into the mechanisms of neurodegeneration, we have characterized the pharmacology of neuronal cell death elicited by the metabolic poison iodoacetate (IAA). IAA is a potent inhibitor of glycolysis, acting to alkylate the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Although glycolysis only accounts for a small proportion of the total energy available from glucose, it does supply the key intermediates for the tricarboxylic acid cycle and oxidative phosphorylation via the electron transport chain. Glycolysis is therefore a critical pathway in main-

Oxidative stress, and concomitant free radical production, is increasingly recognized as an important mediator of neuronal cell death [1]. Compared with other tissues, the brain is particularly prone to free radical damage because of its high content of polyunsaturated fatty acids and relatively low levels of endogenous antioxidants, such as glutathione, superoxide dismutase, and catalase. Although the generation of toxic free radicals may be a secondary event after cerebral ischemia or traumatic brain injury, the efficacy of a variety of antioxidant molecules in experimental models of acute neurodegeneration supports their importance as toxic mediators of tissue damage [2– 4]. Markers of oxidative stress have also been reported to be increased in various chronic neurodegenerative disease states [5–7]. Progressive impairment of neuronal energy metabolism occurring over many years is believed to be involved in the neuronal degeneration that is characteristic of certain Address correspondence to: Dr. R. H. P. Porter, Dept. of Molecular Pharmacology, Cerebrus Ltd., Oakdene Court, 613 Reading Road, Winnersh, Wokingham, RG41 5UA UK; Tel: ⫹44 (118) 977-3133; Fax: ⫹44 (118) 989-9300; E-mail: [email protected]. 102

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taining metabolic function in neurons. Studies using cultured cortical and hippocampal neurons have previously demonstrated that IAA induces a delayed form of neuronal death that can be partially prevented by the free radical scavenger vitamin E [15]. Additionally, in vivo studies have shown that IAA produces striatal excitotoxic lesions that are accompanied by an increase in the production of hydroxyl radicals [16]. The present study extends the investigation of IAA-induced neurotoxicity to rat cerebellar granule cell (CGC) cultures and examines the neuroprotective efficacy of various antioxidants and examines the role of excitotoxicity and oxidative stress after transient exposure of cultures to this glycolytic inhibitor. MATERIALS AND METHODS

Cell culture Cerebellar granule cell cultures were prepared using a modification of a method described previously [17]. Briefly, cerebella from 6 – 8-d-old Sprague Dawley rat pups were dissected, finely chopped, and incubated for 15 min at 37°C in a 0.025% trypsin solution in a supplemented Earle’s balanced salt solution (Sigma Chemical Co., St. Louis, MO, USA). Proteolysis was inhibited by the addition of type I-S soybean trypsin inhibitor (Sigma) with deoxyribonuclease I (Sigma), at final concentrations of 0.03 and 0.004%, respectively. The cells were then triturated to dissociate cells into a suspension, which was then underlayed with 4% bovine serum albumin, centrifuged at 250 ⫻ g and resuspended in culture media (Dulbecco’s modified Eagle’s medium supplemented with final concentrations of 2 mM glutamine, 33 M glucose, 5% donor horse serum, 10% fetal calf serum, 25 M KCl and 1% penicillin/streptomycin). Cells were plated in 96-well poly-L-lysine coated tissue culture plates (Corning Costar, High Wycombe, UK) at a density of 2.5 ⫻ 105 cells per well and placed in an atmosphere of 5% CO2 and 95% humidity. The mitotic inhibitor cytosine arabinoside (10 ␮M) was added 16 h later to prevent proliferation of non-neuronal cells. Cell death assays After 6 – 8 d in culture, the cultures were prepared for hypoglycemia. Iodoacetate was made up in a balanced salt solution (BSS) (154 mM NaCl, 5.6 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, 10 mM N-[2-hydroxyethyl]piperazine-N⬘-[2-ethanesulfonic acid], 5.6 mM D-glucose, pH 7.4) and any neuroprotective agents were made up in prewarmed tissue culture media and allowed to equilibriate to 37°C in a controlled environment (5% CO2, 95% humidity) before being added to cultures. The

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assay was initiated by aspiration of the maintenance media, which was replaced by IAA in BSS in the absence or presence of other agents as indicated in the legends to each figure. The exposure to IAA was for 30 min at 37°C, after which time the well contents were aspirated and replaced with fresh, pre-equilibriated maintenance media containing compounds as indicated. All conditions were performed in quadruplicate in each 96-well plate. For the mechanistic studies, each negative control (no IAA) and positive control (30 ␮M iodoacetate) were repeated at least twice in the same plate. The final volume for each well was always 200 ␮l. After a 24 h incubation, visual inspection of the cells was followed by quantification of neuronal cell death by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT)-reductase activity as described previously [18]. Briefly, cultures were washed with BSS, and 50 ␮l/well MTT solution (200 ␮g/ml) added. After a 10-min incubation of plates at 37°C, 125 ␮l acidified isopropanol/ triton X-100 (10% triton X-100 in isopropanol/0.04 M HCl) was added to terminate the reduction of MTT and facilitate solubilization of the colored MTT–formazan complex. After an overnight incubation at 4°C to allow full solubilization of the MTT-formazan complex, absorption at 570 nm (A570) was measured using a spectrophotometric microplate reader. Data are expressed as the percentage absorbance (A570) of control cultures after subtraction of background absorbance from cultures pretreated for 20 min with 2% triton before MTT assay.

Measurement of oxidative stress Intracellular oxidative stress was measured using the oxidant-sensitive fluorescent dye 2⬘,7⬘-dichlorodihydrofluorescein diacetate (DCFH-DA) (Molecular Probes, Eugene, OR, USA). The nonfluorescent DCFH-DA readily crosses cell membranes into the cytoplasm, whereupon it is deacetylated and trapped as its nonmembrane permeable form, dichlorodihydrofluorescein (DCFH). Upon oxidation, DCFH yields the highly fluorescent product dichlorofluorescein (DCF). Briefly, growth medium was aspirated from cultures and replaced with 200 ␮l BSS (plus 10 ␮M MK-801) containing 30 ␮M IAA alone or in combination with various concentrations of antioxidants. After incubation at 37°C for 30 min, cultures were aspirated once more and 10 ␮M DCFH-DA in BSS (200 ␮l) added to all wells. After a further 20 min incubation at 37°C, fluorescence was measured using a Cytofluor II fluorescent plate reader (␭ex ⫽ 485 nm; ␭em ⫽ 530 nm). Background fluorescence values from wells treated with 2% triton X-100 were subtracted from both basal (BSS alone) and stimulated fluorescence values and the effects of antioxidants ex-

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Fig. 1. Dose-dependence of IAA-induced neurotoxicity in rat CGC cultures. Cultures were exposed to IAA (1 ␮M–1 mM) in BSS for 30 min followed by recovery in growth medium for 24 h, after which time viability was assessed by measurement of MTT-reductase activity. Data are the mean ⫾ SEM of three independent experiments performed in quadruplicate.

pressed as a percent inhibition of the IAA-stimulated fluorescence increase over basal.

Fig. 2. Effect of EAA receptor antagonists MK-801 and NBQX on IAA-induced neurotoxicity. Cultures were exposed to BSS or 30 ␮M IAA alone or in combination with either 10 ␮M MK-801 or 100 ␮M NBQX (as shown in the figure) and incubated at 37°C for 30 min. After a 24 h recovery in maintenance growth medium (plus or minus MK801 or NBQX as indicated), viability was assessed by MTT assay. Data are the mean ⫾ SEM of three independent determinations.

Neuroprotection with antioxidants Statistical analysis

Effect of EAA receptor blockade

Three different antioxidant molecules (Fig. 3) were tested for their ability to protect CGC cultures against IAA-induced neurotoxicity. In the absence of MK-801, butylated hydroxytoluene (BHT), U-83836E (a vitamin E/21-aminosteroid analogue) and (S)-(-)-Trolox (a water-soluble analogue of vitamin E) displayed marginal (but significant) neuroprotective effects (maximal recovery to 10 –20% of control) (Fig. 4; open symbols). In the presence of MK-801 (10 ␮M), however, the neuroprotective efficacy of these antioxidants was greatly enhanced (synergised), giving rise to a recovery in MTTreductase activity equivalent to 80 –100% of control cultures (Fig 4; closed symbols). The compounds displayed EC50 values of 5.9, 0.25, and 78 ␮M, for BHT, U-83836E, and (S)-(-)-Trolox, respectively. The concentration response curve for each of the antioxidant compounds tested was very steep, with effectively all activity evident within one log unit of concentration. In the case of (S)-(-)-Trolox, an inverted U-shape curve was observed with maximal neuroprotection at 300 ␮M and a

In an attempt to clarify the mechanism of IAA-induced cell death, the effect of various neuroprotective agents were investigated. The results (Fig. 2) show that neither the N-methyl-D-aspartate (NMDA) receptor channel blocker MK-801 (at a concentration of 10 ␮M) or the non-NMDA receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX) (100 ␮M), had any effect on the degree of cell death caused by 30 ␮M IAA.

Fig. 3. Structures of the antioxidant molecules used in the present study.

Statistical analysis was performed by two-tailed paired t-test or Tukey’s test for multiple comparisons in combination with one-way analysis of variance (ANOVA). Statistical significance is defined as p ⬍ .05. RESULTS

Dose-dependent neurotoxicity of IAA IAA caused a dose-dependent decrease in MTT-reductase activity (used as an index of cell death) in CGC cultures with an EC50 of 9.8 ␮M (Fig. 1). The steepness of the IAA dose-response curve is probably indicative of the “all or nothing” nature of neuronal cell death triggered by this exposure paradigm. Thirty micromolar IAA reliably killed ⱖ95% of the cells present and was used to test the efficacy of neuroprotective agents.

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Fig. 5. Inhibition of IAA-induced oxidative stress in rat cerebellar granule cells. CGCs were exposed to IAA (30 ␮M) alone or in combination with various antioxidants (plus 10 ␮M MK-801) at the concentrations shown and incubated for 30 min at 37°C, after which time the cultures were loaded with DCFH-DA as described in Materials and Methods. IAA induced a 3– 4-fold increase in fluorescence compared to control cultures (data not shown). Inhibitory effects of antioxidants are shown as a percent inhibition of IAA-stimulated fluorescence. Data are the mean ⫾ SEM of three independent determinations. *Significantly lower than fluorescence from control cultures (p ⬍ .05).

Inhibition of IAA-induced oxidative stress

Fig. 4. Neuroprotective efficacy of antioxidants against IAA-induced toxicity is enhanced by MK-801. CGCs were exposed to BSS or 30 ␮M IAA for 30 min at 37°C before a 24 h recovery in maintenance growth medium containing the antioxidants BHT (A), U-83836E (B), and (S)-(-)-Trolox (C). Experiments were performed in the absence (open symbols) or presence (closed symbols) of 10 ␮M MK-801 during both exposure (30 min) and recovery periods (24 hours). Data are the mean ⫾ SEM of three independent determinations. *Significantly different from same concentration of antioxidant in the absence of MK-801 (p ⬍ .05). #Signicantly different from treatment with 30 ␮M IAA alone (p ⬍ .05).

complete loss of neuroprotection at 1 mM (Fig. 4C; closed symbols). A similar trend was observed for U-83836E (Fig. 4B; closed symbols), but compound solubility limitations precluded investigation of concentrations higher than 3 ␮M.

Previous studies have shown that cultured rat cortical and hippocampal CA1 cultures undergo delayed neuronal death after transient exposure to IAA. That this neuronal death can be partially prevented by the antioxidant vitamin E, suggests a role for free radicals in IAA-mediated neuronal cell death [15]. In order to further test the involvement of a free radical–mediated mechanism for IAA-induced cell death in CGC cultures, the ability of BHT, U-83836E, and (S)-(-)-Trolox to inhibit IAA-induced fluorescence in cultures loaded with the oxidant-sensitive dye DCFH-DA was investigated. IAA alone caused a 3– 4-fold increase in fluorescence as compared with control cultures (data not shown). Fig. 5 shows that the fluorescence increase in IAA-stimulated DCFH-DA-loaded cultures was inhibited in a dose-dependent manner by the antioxidants BHT, U-83836E, and (S)-(-)-Trolox with IC50 values of 2.9, 0.38, and 97 ␮M, respectively. At higher concentrations, both BHT (⬎10 ␮M) and U-83836E (ⱖ1 ␮M) inhibited fluorescence to levels that were below those obtained with control cultures (Fig. 5). DISCUSSION

We have shown, by measurement of MTT-reductase activity, that cerebellar granule cells subjected to a transient exposure to IAA undergo dose-dependent delayed cell death over a period of 24 h after a transient (30 min) exposure to IAA. IAA toxicity was not inhibited by the excitatory

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amino acid antagonists MK-801 or NBQX, which is in agreement with previous reports in which MK-801 did not inhibit cell death caused by IAA in cultured cortical and hippocampal cells [15]. In addition, MK-801 did not inhibit hypoxia-mediated cell death in an organotypic culture model [19]. In contrast, previous reports using an isolated retina preparation have suggested that IAA-mediated cell death is mediated via an NMDA receptor mediated mechanism [20]. This discrepancy may be due to the dissociated preparations of cells used by ourselves and others [15] being more susceptible to the NMDA receptor-independent toxic effects of IAA, such as oxidative stress. Alternatively, a lower level of inhibition of energy metabolism in the previous studies [20], and the assay of more immediate markers of cell death may be the reason for the conflicting reports. Our results suggest that free radical production is a principal mediator of IAA-induced neuronal injury. This is in agreement with the study by Uto and colleagues where vitamin E (but not MK-801) was shown to be partially neuroprotective against IAA exposure in primary cultures of rat cortical and hippocampal cultures [15]. Furthermore, comparison of the IC50 values for antioxidant inhibition of IAA-stimulated fluorescence in DCFH-DA loaded cultures (Fig. 5) with the corresponding EC50 values for antioxidant neuroprotection (Fig. 4) reveals that for each antioxidant the IC50 and EC50 values are virtually identical. These data suggest that in these CGC cultures IAA-induced cell death is mediated by free radical production, although an additional MK801-sensitive (excitotoxic) component is involved as demonstrated by the requirement for MK-801 (Fig. 4). Similarly, inhibition of GAPDH in vivo by intrastriatal injection of IAA has also been shown to induce striatal lesions that are associated with increased hydroxyl radical and peroxynitrite formation but are also significantly attenuated by removal of the corticostriatal glutamatergic input to the striatum, which supports an excitotoxic mechanism [16]. The degree of lipophilicity in an antioxidant molecule influences its ability to associate with and cross cell membranes to inactivate intracellular free radicals. Thus, hydrophobic antioxidants, such as vitamin E, readily associate with cell membranes, and act as chain-breaking antioxidants to prevent the vicious cycle of lipid peroxidation, to which neuronal membranes are particularly vulnerable [20]. Although only a small sample, it is interesting to note that the potency of the various antioxidants with respect to neuroprotection decreases in the order U-83836E ⬎ BHT ⬎ (S)-(-)-Trolox. This is the same order seen for the decreasing lipophilicity of these antioxidants. This suggests that the different neuroprotective profiles of these antioxidants may in part be attributable to their differences in physicochemical prop-

erties, as well as differences and selectivities in their free radical trapping ability. In the case of (S)-(-)-Trolox, a U-shaped curve was observed (Fig. 4C; closed symbols) with maximal neuroprotection occurring at 300 ␮M. The significance of this U-shaped response is unclear but may be owing to a loss of neuroprotection or a pro-oxidant action of the antioxidant at high concentrations. The present study demonstrates that transient inhibition of the glycolytic enzyme GAPDH triggers a toxic process involving both excitotoxicity and free radical– mediated mechanisms. Interestingly, it has been suggested that the neurodegenerative disorder HD may be mediated by inhibition of GADPH caused by binding to the polyglutamine huntingtin protein, resulting in a subtle chronic impairment of energy metabolism [22]. Such a mechanism would clearly be different to that used in this study, as the impairment in energy supply would be expected to be far more subtle, occurring gradually over many years, and as such may be selectively NMDA receptor mediated. Stimulation of NMDA receptors has previously been shown to trigger formation of superoxide (O•2⫺) radicals and induce neuronal death in CGC cultures that were attenuated by the nitrone spin trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) [23]. It is therefore conceivable that neurotoxicity induced by metabolic inhibition may comprise an NMDA receptormediated component (probably involving O•2⫺) that is exacerbated by reduced cellular energy levels [11] in addition to a more general increase in oxidative stress due to metabolic inhibition. Thus, the requirement for NMDA receptor blockade in order to facilitate antioxidant neuroprotection against toxicity induced by GADPH inhibition in the present study suggests that neuronal cell death can involve multiple mechanisms, and that more than one neuroprotective agent may be required.

CONCLUSION

The present study demonstrates that under certain conditions, the antioxidants BHT, U-83836E, and (S)(-)-Trolox demonstrate potent and specific neuroprotective actions in an in vitro model of neurodegeneration induced by inhibition of the glycolytic enzyme GAPDH. Such an action may be of relevance to the design of therapeutic agents and further promotes the use of antioxidants for the treatment of neurodegenerative diseases [1,21,24].

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