Abstract
The cyclopentenone prostaglandin (CyPG) J&sub2; series, including prostaglandin J&sub2; (PGJ&sub2;), Δ¹²-PGJ&sub2;, and 15-deoxy-∆¹²,¹&sup4;-prostaglandin J&sub2; (15d-PGJ&sub2;), are active metabolites of PGD&sub2;, exerting multiple effects on neuronal function. However, the physiologic relevance of these effects remains uncertain as brain concentrations of CyPGs have not been precisely determined. In this study, we found that free PGD&sub2; and the J&sub2; series CyPGs (PGJ&sub2;, Δ¹²-PGJ&sub2;, and 15d-PGJ&sub2;) were increased in post-ischemic rat brain as detected by UPLC-MS/MS with 15d-PGJ&sub2; being the most abundant CyPG. These increases were attenuated by pre-treating with the cyclooxygenase (COX) inhibitor piroxicam. Next, effects of chronic exposure to 15d-PGJ&sub2; were examined by treating primary neurons with 15d-PGJ&sub2;, CAY10410 (a 15d-PGJ&sub2; analog lacking the cyclopentenone ring structure), or vehicle for 24 to 96 h. Because we found that the concentration of free 15d-PGJ&sub2; decreased rapidly in cell culture medium, freshly prepared medium containing 15d-PGJ&sub2;, CAY10410, or vehicle was changed twice daily to maintain steady extracellular concentrations. Incubation with 2.5 μM 15d-PGJ&sub2;, but not CAY10410, increased the neuronal cell death without the induction of caspase-3 or PARP cleavage, consistent with a primarily necrotic mechanism for 15d-PGJ&sub2;-induced cell death which was further supported by TUNEL assay results. Ubiquitinated protein accumulation and aggregation was observed after 96 h 15d-PGJ&sub2; incubation, accompanied by compromised 20S proteasome activity. Unlike another proteasome inhibitor, MG132, 15d-PGJ&sub2; treatment did not activate autophagy or induce aggresome formation. Therefore, the cumulative cytotoxic effects of increased generation of CyPGs after stroke may contribute to delayed post-ischemic neuronal injury.