Polymorphisms in the human soluble epoxide hydrolase gene EPHX2 linked 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.

Figure 1.

Genetic polymorphisms and localization of soluble epoxide hydrolase (sEH). A, Colabeling with dendritic marker MAP-2 (red) localizes sEH immunoreactivity (green) in cytoplasm (yellow) and axons (arrowhead) but not dendrites (arrow) of primary cultured cortical neurons. B, Amino acid changes associated with EPXH2 mutations and their localization within the amino acid sequence (1–555) and structural domains (1–4) of human sEH. C, Cloning strategy for TAT-hsEH fusion protein. Variants of human EPXH2 were inserted downstream of TAT (47–57) protein transduction domain in pTAT2.1 vector. KanR, Kanamycin resistance.

Figure 2.

Efficient transduction and cytoplasmic localization of TAT-hsEH fusion protein in primary cultured cortical neurons. A–C, neurons at 6 h after transduction with 1 μm wild-type TAT-hsEH. A is stained with anti-MAP2 antibody; B is stained with anti-HIS-tag antibody; and C is an overlay of A and B, illustrating cytoplasmic localization of HIS-tagged TAT-hsEH fusion protein in transduced neurons. D, Untreated cells; E is a confocal image through C. F, Western blot with anti-sEH antibody showing dose- and time-dependent increase in sEH level after transduction with wild-type TAT–hsEH fusion protein.

Figure 3.

TAT-mediated protein transduction produces high levels of functionally active sEH in primary cultured neurons. A, Detection of 14,15-DHET by selected reaction monitoring results in extracted ion chromatograms showing enhanced conversion of 14,15-EET (1 μm) to 14,15-DHET (peak at 12.28 min) in neurons transduced with wild-type TAT-hsEH (bottom middle panel) compared with untreated cells (bottom left panel). 14,15-EET conversion in Arg287Gln TAT-hsEH transduced neurons (bottom right panel) was not different from untreated cells. Top, Internal standard extracted ion chromatograms for each assay. B, Summary graph depicting changes in 14,15-EET metabolism when neurons are transduced with various sEH variants compared with wild-type (WT) sEH.

Figure 4.

Variants of soluble epoxide hydrolase differentially affect neuronal survival after oxygen-glucose deprivation. A, B, Cell death after oxygen-glucose deprivation was measured using the LDH (A) and MTT assays (B). A, Cell death was increased in neurons transduced with Arg103Cys TAT-hsEH and decreased in neurons transduced with Arg287Gln TAT-hsEH compared with untreated cells (p = 0.05 and 0.024, respectively). Only the Arg287Gln variant was statistically different compared with wild-type (WT) sEH variant (p = 0.003). B, Similar trends were observed when mitochondrial mechanisms of cell death were assessed by the MTT assay, although differences among groups were not statistically significant (p = 0.11).

Figure 5.

Neuronal rescue from TAT-hsEH induced cell death by sEH substrate addition and pharmacological inhibition. A, Excess 14,15-EET, the substrate for soluble epoxide hydrolase, prevents the increase in cell death induced by TAT-hsEH. B, Inhibition of soluble epoxide hydrolase by 4-PCO prevents the increase in OGD-induced cell death caused by TAT-hsEH. WT, Wild type.