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.

We previously demonstrated that the neuropeptide cocaine-and amphetamine-regulated transcript (CART) is protective against focal cerebral ischemia in vivo and against neuronal cell death in culture induced by oxygen-glucose deprivation (OGD). The mechanism of neuroprotection by CART is unknown, in part due to lack of knowledge regarding its putative receptor. Using a yeast two-hybrid system with CART's carboxy-terminal to screen a mouse brain cDNA library, we uncovered a potential direct interaction between CART and subunit B of the mitochondrial enzyme succinate dehydrogenase (SDHB). We confirmed CART/SDHB binding using in vitro pull-down assay, and tested the effects of CART peptide on SDH activity, Complex II (CII) activity and ATP production in primary cultured cortical neurons under basal conditions and after OGD. At concentrations between 0.2 and 4 nM, CART significantly increased SDH function, CII activity and ATP generation in purified mitochondria and intact neurons under baseline conditions. Furthermore, pretreatment with CART enhanced mitochondrial mechanisms of neuronal survival and prevented the decline in SDH and CII activities and ATP production after OGD. The findings suggest that CART's neuroprotective mechanism of action may be linked to preservation of mitochondrial function and prevention of energy failure after ischemia-reperfusion injury.

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Across mammalian species in the evolutionary ladder, a-synuclein protein is highly conserved, even though synucleinopathy develops selectively in humans. Only the human a-synuclein mRNA contains an IRE-like sequence in its 5 0 -UTR, and, of note, generation of the human a-synuclein transcript requires RNA splicing out of an intron that precisely interrupts the canonical 5 0 -CAGUG-3 0 motif of the predicted 5 0 UTR stem loop. 10 Thus, it is possible that a unique role for a-synuclein in presynaptic redox and iron metabolism rapidly evolved in primates with selection for RNA splicing that favored a gain of post-transcriptional regulation for a-synuclein protein production.If the novel stem-loop structure we describe in asynuclein mRNA functions to confer iron dependent post-transcriptional control, it would have implications for the central nervous system amyloidoses. It would immediately suggest a mechanism for induction of asynuclein by an age-related iron or redox pathophysiology upstream of neurodegeneration. Furthermore, it would provide an explanation for the well-characterized neuropathologic association of a-synuclein and the Alzheimer b-amyloid protein 3 since the Alzheimer bamyloid precursor protein mRNA also contains an IRE in its 5 0 -UTR. 8 If and how the IRE-like structure in asynuclein mRNA functions as a post-transcriptional regulator now awaits experimental clarification.