Cyclooxygenase-2 in Ischemia Neuronal Injury

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Secondary prostaglandin metabolites produced by activation of cyclooxygenase 2 (COX2) after ischemia such as the cyclopentenone prostaglandins (cyPGs) may produce neuronal cell death. In our preliminary studies we find that concentrations of the cyPG 15-deoxy-?12,14-PGD2 are increased in brain after ischemia. We found that 15-deoxy-?12,14-PGD2 induces cell death in neuron-enriched primary cultures independent of its effects on the PPAR? and DP2 receptors. We found that cyPGs bind to ubiquitin C-terminal hydrolase-L1 (UCH-L1), a key enzyme in the protein ubiquitination pathway important in removing unfolded or misfolded proteins from the cell. Furthermore, treatment with cyPGs inhibits UCH-L1 and induces accumulation of ubiquitinated proteins (Ub-proteins) in primary neurons. Based on these and other data we hypothesize that concentrations of cyPGs are increased in brain after ischemia and that cyPGs increase accumulation of Ub-proteins, endoplasmic reticulum (ER) stress and protein aggregation after ischemia by covalent modification and inhibition of UCH-L1 activity. The following specific aims are proposed: 1. Determine the site where cyPGs covalently bind to UCH-L1. Determine the effect of cyPG adduct formation on UCH-L1 function, accumulation of Ub-proteins and production of ER stress in primary neuronal cultures. 2. Identify the specific cyPGs found in rat brain after temporary focal ischemia. Determine if these cyPGs directly induce cell death or modify the susceptibility of neurons to hypoxic ischemic cell death in vitro. 3. Characterize the role of cyPG inhibition of UCH-L1 after ischemia in vivo. Determine if cyPgs bind to UCH-L1 in vivo. Determine if cyPgs produced by COX2 activity after temporary focal ischemia bind to UCH-L1, and produce accumulation of Ub-proteins, ER stress and protein aggregation. ER stress, accumulation of Ub-proteins and protein aggregation are important etiologies of delayed neuronal death in ischemia and neurodegenerative diseases. These studies aim to elucidate a new mechanism by which ER stress and accumulation of Ub-proteins are induced after ischemia that may be relevant to the pathogenesis of stroke and neurodegenerative diseases such as Parkinson¿s and Alzheimer¿s disease.