Polymorphisms in the Human Soluble Epoxide Hydrolase GeneEPHX2Linked to Neuronal Survival after Ischemic Injury

Abstract

Single nucleotide polymorphisms (SNPs) in the human EPHX2 gene have recently been implicated in susceptibility to cardiovascular disease, including stroke. EPHX2 encodes for soluble epoxide hydrolase (sEH), an important enzyme in the metabolic breakdown of arachidonic acid-derived eicosanoids referred to as epoxyeicosatrienoic acids (EETs). We previously demonstrated that EETs are protective against ischemic cell death in culture. Therefore, we tested the hypothesis that polymorphisms in the human EPHX2 gene alter sEH enzyme activity and affect neuronal survival after ischemic injury in vitro. Human EPHX2 mutants were recreated by site-directed mutagenesis and fused downstream of TAT protein transduction domain. Western blot analysis and immunocytochemistry staining revealed high-transduction efficiency of human TAT-sEH variants in rat primary cultured cortical neurons, associated with increased metabolism of 14,15-EET to corresponding 14,15-dihydroxyeicosatrienoic acid. A human variant of sEH with Arg103Cys amino acid substitution, previously demonstrated to increase sEH enzymatic activity, was associated with increased cell death induced in cortical neurons by oxygen-glucose deprivation (OGD) and reoxygenation. In contrast, the Arg287Gln mutation was associated with reduced sEH activity and protection from OGD-induced neuronal cell death. We conclude that sequence variations in the human EPHX2 gene alter susceptibility to ischemic injury and neuronal survival in a manner linked to changes in the hydrolase activity of the enzyme. The findings suggest that human EPHX2 mutations may in part explain the genetic variability in sensitivity to ischemic brain injury and stroke outcome.

Role of Soluble Epoxide Hydrolase in Exacerbation of Stroke by Streptozotocin-Induced Type 1 Diabetes Mellitus

“…15,36 In line with this, we have previously shown that brain EETs protect neurons against ischemia by mechanism(s) separate from cerebrovascular dilation in the absence of hyperglycemia. 40 Our current study supports a neuroprotective role of brain EETs that is likely independent of cerebral perfusion, this time in the context of hyperglycemia, as STZ decreased brain EET concentrations, while increasing infarct size without altering cerebral perfusion; effects that were reversed by sEH inhibition.…”

Peroxisomal Translocation of Soluble Epoxide Hydrolase Protects against Ischemic Stroke Injury

“…4 PCRmediated deletion was used to remove the PTS of TAT-sEH-WT to create translocation deficient TAT-sEH-ΔPTS ( Figure 1A). Deletion of the PTS was verified by sequencing.…”

“…Recombinant proteins were expressed and purified as previously described. 4 Concentrations of purified fusion proteins were measured with a Bradford assay (Bio-Rad, Hercules, CA, USA). Protein purity and specificity was confirmed by immunoblotting.…”

“…2,3 The interest in the role of sEH in stroke was further enhanced by the observation that common human singlenucleotide polymorphisms (SNPs) of sEH affect its hydrolase activity and ability to inactivate protective EET. 4 A human sEH variant that exhibits reduced hydrolase activity in vitro, the R287Q SNP, is associated with a decrease in stroke incidence. 5 The same R287Q variant improves neuronal survival after in vitro ischemia, 4 further emphasizing the relevance for sEH in stroke injury.…”

“…4 A human sEH variant that exhibits reduced hydrolase activity in vitro, the R287Q SNP, is associated with a decrease in stroke incidence. 5 The same R287Q variant improves neuronal survival after in vitro ischemia, 4 further emphasizing the relevance for sEH in stroke injury.…”