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. 2002 May;19(5):627-38.
doi: 10.1089/089771502753754091.

Cyclooxygenase-2 inhibition protects cultured cerebellar granule neurons from glutamate-mediated cell death

Kenneth I Strauss  1 , Ann M Marini
Affiliations

Cyclooxygenase-2 inhibition protects cultured cerebellar granule neurons from glutamate-mediated cell death

Kenneth I Strauss et al. J Neurotrauma. 2002 May.

Abstract

Primary insults to the brain can initiate glutamate release that may result in excitotoxicity followed by neuronal cell death. This secondary process is mediated by both N-methyl-D-aspartate (NMDA) and non-NMDA receptors in vivo and requires new gene expression. Neuronal cyclooxygenase-2 (COX2) expression is upregulated following brain insults, via glutamatergic and inflammatory mechanisms. The products of COX2 are bioactive prostanoids and reactive oxygen species that may play a role in neuronal survival. This study explores the role of neuronal COX2 in glutamate excitotoxicity using cultured cerebellar granule neurons (day 8 in vitro). Treatment with excitotoxic concentrations of glutamate or kainate transiently induced COX2 mRNA (two- and threefold at 6 h, respectively, p < 0.05, Dunnett) and prostaglandin production (five- and sixfold at 30 min, respectively, p < 0.05, Dunnett). COX2 induction peaked at toxic concentrations of these excitatory amino acids. Surprisingly, NMDA, L-quisqualate, and trans-ACPD did not induce COX2 mRNA at any concentration tested. The glutamate receptor antagonist NBQX (5 microM, AMPA/kainate receptor) completely inhibited kainate-induced COX2 mRNA and partially inhibited glutamate-induced COX2 (p < 0.05, Dunnett). Other glutamate receptor antagonists, such as MK-801 (1 microM, NMDA receptor) or MCPG (500 microM, class 1 metabotropic receptors), partially attenuated glutamate-induced COX2 mRNA. These antagonists all reduced steady-state COX2 mRNA (p < 0.05, Dunnett). To determine whether COX2 might be an effector of excitotoxic cell death, cerebellar granule cells were pretreated (24 h) with the COX2-specific enzyme inhibitor, DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl) phenyl-2((5)H)-furanone) prior to glutamate challenge. DFU (1 to 1000 nM) completely protected cultured neurons from glutamate-mediated neurotoxicity. Approximately 50% protection from NMDA-mediated neurotoxicity, and no protection from kainate-mediated neurotoxicity was observed. Therefore, glutamate-mediated COX2 induction contributes to excitotoxic neuronal death. These results suggest that glutamate, NMDA, and kainate neurotoxicity involve distinct excitotoxic pathways, and that the glutamate and NMDA pathways may intersect at the level of COX2.

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Figures

FIG. 1
FIG. 1
COX2 mRNA was induced in cultured cerebellar neurons after 3-h treatment with toxic concentrations of glutamate (100 μM) or kainate (500 μM). COX2 mRNA was not affected by toxic concentrations of NMDA (1 mM). COX2 levels were normalized both by the cyclophilin mRNA (CYC) and the total protein in each sample. In this experiment, COX2 mRNA (fg/μg protein) levels were control 1.65 ± 0.67; glutamate 4.88 ± 1.14*; NMDA 2.77 ± 0.98; kainate 8.22 ± 2.29* (p < 0.05, Dunnett). Results are mean ± SEM (n = 3 per point). Controls levels were above the limit of quantitation (LOQ) of the assay. The LOQ was determined using nulls (ø, treated in every way similar to samples except the hybridization step was performed on dry ice): LOQ = (mean null value + 2 SEM).
FIG. 2
FIG. 2
Concentration-response of COX2 mRNA to excitotoxic amino acids. COX2 mRNA levels, quantified by two methods of normalization, were increased after 6-h exposure to toxic concentrations of glutamate and kainate, but not NMDA (bars: fg mRNA/μg protein; boxes: molar ratio of COX2 to CYC mRNA). The 500-μM glutamate treatment did not significantly increase COX2 mRNA, due possibly to excessive toxicity at 6 h. Results are mean ± SEM (n = 2–4 per point), *p < 0.05, Dunnett.
FIG. 3
FIG. 3
Total prostaglandin levels rise acutely after excitotoxic amino acid treatment. The time course of total prost-aglandin changes was analyzed by polynomial regression. The quadratic curve had a corellation coefficient r2 = 0.62 (p < 0.0003). This is consistent with a steady rise and fall of prostaglandin levels over the time course described. Cerebellar granule cells were incubated in serum-free medium, small aliquots (5% total) were collected, frozen and replaced with an equal volume of serum free medium (±kainate). Total prostaglandins were measured using an EIA (Cayman Chemical). Unfortunately, the colorimetric screening assay used has been discontinued and no suitable replacement could be found. The glutamate time course though incomplete, showed a slower rise with a lower peak than those from the kainate time course (not shown). Results are presented as mean ± SEM (n = 2 per time point).
FIG. 4
FIG. 4
The COX2 inhibitor DFU protects rat cerebellar granule neurons from glutamate-mediated cell death. (A) Untreated neurons. (B) Neurons treated with 100 μM glutamate. (C) Neurons pretreated (24 h) with 10 nM DFU. (D) Neurons pretreated with DFU and treated with 100 μM glutamate. Cultures were pretreated with DFU (10 nM) for 24 h. An excitotoxic concentration of glutamate (100 μM) was added, and neuronal viability was assessed 24 h later with fluorescein diacetate. Photos are representative fields from replicates (n = 3 dishes per group). Experiment was repeated three times with the same outcome.
FIG. 5
FIG. 5
The COX2 inhibitor DFU protects cultured neurons against glutamate neurotoxicity. Cultured cerebellar granule cells (day 7 in vitro) were treated with various concentrations of DFU (0.01–1000 nM) for 24 h followed by the addition of glutamate (100 μM) for an additional 24 h. Neuronal viability was assessed using the fluorescein diacetate assay as described in Materials and Methods. Percent neuronal survival is the ratio of viable neurons in DFU treated to untreated cultures ± SD.
FIG. 6
FIG. 6
DFU blocks glutamate-mediated cell death in cerebellar granule neurons. Cultures were pretreated with 10 nM DFU and exposed to 100 μM glutamate as described in Materials and Methods. Neuronal viability was determined with fluorescein diacetate as described in Figure 4. Results presented as percent survival (mean ± SEM).
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