Jonathan W. Nelson

Mechanism of the sex difference in neuronal ischemic cell death

BACKGROUND Stroke risk and outcome are different in men and women. We hypothesized that this is partly due to an inherent difference in susceptibility to ischemia between neurons from male vs. female brains. We tested whether neurons from male rodents are more susceptible to in-vitro ischemia than cells from females, and if this is related to increased expression of soluble epoxide hydrolase (sEH). sEH contributes to neuronal cell death by inactivating neuroprotective epoxyeicosatrienoic acids (EETs). METHODS Rodent cortical neurons were cultured, and exposed to oxygen-glucose deprivation (OGD); then cell death was measured. EETs levels were determined by LC-MS/MS. Expression of sEH-encoding ephx2 was determined by qRT-PCR. Western blotting, immunocytochemistry, and hydrolase activity assay assessed protein expression and activity. RESULTS Cell death after OGD was higher in neurons from males vs. females, which correlated with higher ephx2 mRNA and stronger sEH immunoreactivity. However, EETs levels were similar in both sexes and pharmacological inhibition of the hydrolase domain of sEH did not abolish the sex difference in cell death. Genetic knockout of sEH in mice abolished the sex difference observed in neurons isolated from these mice after OGD. CONCLUSIONS Cultured cortical neurons from females are more resistant to ischemia than neurons from males. Neurons from females have less sEH activity compared to neurons from males at baseline, although sEH levels were not measured after OGD. While pharmacological inhibition of the hydrolase domain of sEH does not affect cell death, knockout of the gene encoding sEH eradicates the sex difference seen in wild-type neurons, suggesting a role for further study of the lesser-known phosphatase domain of sEH and its role in sexual dimorphism in neuronal sensitivity to ischemia.

Soluble Epoxide Hydrolase: Sex Differences and Role in Endothelial Cell Survival

Objective—Sex differences in cerebral ischemic injury are, in part, arrtibutable to the differences in cerebrovascular perfusion. We determined whether the brain microvascular endothelial cells (ECs) isolated from the female brain are more resistant to ischemic injury compared with male ECs, and whether the difference is attributable to lower expression of soluble epoxide hydrolase and higher levels of vasoprotective epoxyeicosatrienoic acids (EETs). We also determined whether protection by EETs is linked to the inhibition of rho-kinase (ROCK). Methods and Results—EC ischemic damage was measured after oxygen–glucose deprivation (OGD) using propidium iodide (PI) and cleaved caspase-3 labeling. Expression of soluble epoxide hydrolase was determined by quantitative polymerase chain reaction and immunocytochemistry, EETs levels by liquid chromatography-tandem mass spectrometry, and ROCK activity by ELISA. EC damage was higher in males compared with females, which correlated with higher soluble epoxide hydrolase mRNA, stronger immunoreactivity, and lower EETs compared with female ECs. Inhibition of soluble epoxide hydrolase abolished the sex difference in EC damage. ROCK activity was higher in male versus female ECs after OGD, and sex differences in EC damage and ROCK activity were abolished by 14,15-EET and ROCK inhibition. Conclusion—Sex differences in ischemic brain injury are, in part, attributable to differences in EET-mediated inhibition of EC ROCK activation after ischemia.

Soluble epoxide hydrolase dimerization is required for hydrolase activity

Background: Soluble epoxide hydrolase (sEH) forms a homodimer that metabolizes a neuroprotective class of lipids termed epoxyeicosatrienoic acids. Results: Mutations on the dimerization interface of sEH eliminate hydrolase enzymatic activity. Conclusion: Dimerization is required for sEH enzymatic activity. Significance: Dimerization is a novel target for sEH inhibition. Soluble epoxide hydrolase (sEH) plays a key role in the metabolic conversion of the protective eicosanoid 14,15-epoxyeicosatrienoic acid to 14,15-dihydroxyeicosatrienoic acid. Accordingly, inhibition of sEH hydrolase activity has been shown to be beneficial in multiple models of cardiovascular diseases, thus identifying sEH as a valuable therapeutic target. Recently, a common human polymorphism (R287Q) was identified that reduces sEH hydrolase activity and is localized to the dimerization interface of the protein, suggesting a relationship between sEH dimerization and activity. To directly test the hypothesis that dimerization is essential for the proper function of sEH, we generated mutations within the sEH protein that would either disrupt or stabilize dimerization. We quantified the dimerization state of each mutant using a split firefly luciferase protein fragment-assisted complementation system. The hydrolase activity of each mutant was determined using a fluorescence-based substrate conversion assay. We found that mutations that disrupted dimerization also eliminated hydrolase enzymatic activity. In contrast, a mutation that stabilized dimerization restored hydrolase activity. Finally, we investigated the kinetics of sEH dimerization and found that the human R287Q polymorphism was metastable and capable of swapping dimer partners faster than the WT enzyme. These results indicate that dimerization is required for sEH hydrolase activity. Disrupting sEH dimerization may therefore serve as a novel therapeutic strategy for reducing sEH hydrolase activity.

Role of soluble epoxide hydrolase in age-related vascular cognitive decline.

P450 eicosanoids are important regulators of the cerebral microcirculation, but their role in cerebral small vessel disease is unclear. We tested the hypothesis that vascular cognitive impairment (VCI) is linked to reduced cerebral microvascular eicosanoid signaling. We analyzed human brain tissue from individuals formerly enrolled in the Oregon Brain Aging Study, who had a history of cognitive impairment histopathological evidence of microvascular disease. VCI subjects had significantly higher lesion burden both on premortem MRI and postmortem histopathology compared to age- and sex-matched controls. Mass spectrometry-based eicosanoid analysis revealed that 14,15-dihydroxyeicosatrienoic acid (DHET) was elevated in cortical brain tissue from VCI subjects. Immunoreactivity of soluble epoxide hydrolase (sEH), the enzyme responsible for 14,15-DHET formation, was localized to cerebral microvascular endothelium, and was enhanced in microvessels of affected tissue. Finally, we evaluated the genotype frequency of two functional single nucleotide polymorphisms of sEH gene EPHX2 in VCI and control groups. Our findings support a role for sEH and a potential benefit from sEH inhibitors in age-related VCI.

Soluble Epoxide Hydrolase in Hydrocephalus, Cerebral Edema, and Vascular Inflammation After Subarachnoid Hemorrhage

Background and Purpose— Acute communicating hydrocephalus and cerebral edema are common and serious complications of subarachnoid hemorrhage (SAH), whose causes are poorly understood. Using a mouse model of SAH, we determined whether soluble epoxide hydrolase (sEH) gene deletion protects against SAH-induced hydrocephalus and edema by increasing levels of vasoprotective eicosanoids and suppressing vascular inflammation. Methods— SAH was induced via endovascular puncture in wild-type and sEH knockout mice. Hydrocephalus and tissue edema were assessed by T2-weighted magnetic resonance imaging. Endothelial activation was assessed in vivo using T2*-weighted magnetic resonance imaging after intravenous administration of iron oxide particles linked to anti–vascular cell adhesion molecule-1 antibody 24 hours after SAH. Behavioral outcome was assessed at 96 hours after SAH with the open field and accelerated rotarod tests. Results— SAH induced an acute sustained communicating hydrocephalus within 6 hours of endovascular puncture in both wild-type and sEH knockout mice. This was followed by tissue edema, which peaked at 24 hours after SAH and was limited to white matter fiber tracts. sEH knockout mice had reduced edema, less vascular cell adhesion molecule-1 uptake, and improved outcome compared with wild-type mice. Conclusions— Genetic deletion of sEH reduces vascular inflammation and edema and improves outcome after SAH. sEH inhibition may serve as a novel therapy for SAH.

The START App: a web-based RNAseq analysis and visualization resource.

Summary: Transcriptional profiling using RNA sequencing (RNAseq) has emerged as a powerful methodology to quantify global gene expression patterns in various contexts from single cells to whole tissues. The tremendous amount of data generated by this profiling technology presents a daunting challenge in terms of effectively visualizing and interpreting results. Convenient and intuitive data interfaces are critical for researchers to easily upload, analyze and visualize their RNAseq data. We designed the START (Shiny Transcriptome Analysis Resource Tool) App with these requirements in mind. This application has the power and flexibility to be resident on a local computer or serve as a web‐based environment, enabling easy sharing of data between researchers and collaborators. Availability and Implementation: Source Code for the START App is written entirely in R and can be freely available to download at https://github.com/jminnier/STARTapp with the code licensed under GPLv3. It can be launched on any system that has R installed. The START App is also hosted on https://kcvi.shinyapps.io/START for researchers to temporarily upload their data. Contact: minnier@ohsu.eduSummary Transcriptional profiling using RNA sequencing (RNAseq) has emerged as a powerful methodology to quantify global gene expression patterns in various contexts from single cells to whole tissues. The tremendous amount of data generated by this profiling technology presents a daunting challenge in terms of effectively visualizing and interpreting results. Convenient and intuitive data interfaces are critical for researchers to easily upload, analyze and visualize their RNAseq data. We designed the START (Shiny Transcriptome Analysis Resource Tool) App with these requirements in mind. This application has the power and flexibility to be resident on a local computer or serve as a web-based environment, enabling easy sharing of data between researchers and collaborators. Availability and Implementation Source Code for the START App is written entirely in R and can be freely available to download at https://github.com/jminnier/STARTapp with the code licensed under GPLv3. It can be launched on any system that has R installed. The START App is also hosted on https://kcvi.shinyapps.io/START for researchers to temporarily upload their data. Contact minnier@ohsu.edu

Genetic variation in soluble epoxide hydrolase: association with outcome after aneurysmal subarachnoid hemorrhage

OBJECT Patients with aneurysmal subarachnoid hemorrhage (SAH) are at high risk for delayed cerebral ischemia (DCI) and stroke. Epoxyeicosatrienoic acids (EETs) play an important role in cerebral blood flow regulation and neuroprotection after brain injury. Polymorphisms in the gene for the enzyme soluble epoxide hydrolase (sEH), which inactivates EETs, are associated with ischemic stroke risk and neuronal survival after ischemia. This prospective observational study of patients with SAH compares vital and neurologic outcomes based on functional polymorphisms of sEH. METHODS Allelic discrimination based on quantitative real-time polymerase chain reaction was used to differentiate wild-type sEH from K55R heterozygotes (predictive of increased sEH activity and reduced EETs) and R287Q heterozygotes (predictive of decreased sEH activity and increased EETs). The primary outcome was new stroke after SAH. Secondary outcomes were death, Glasgow Outcome Scale score, and neurological deterioration attributable to DCI. RESULTS Multivariable logistic regression models adjusted for age at admission and Glasgow Coma Scale scores revealed an increase in the odds of new stroke (OR 5.48 [95% CI 1.51-19.91]) and death (OR 7.52 [95% CI 1.27-44.46]) in the K55R group, but no change in the odds of new stroke (OR 0.56 [95% CI 0.16-1.96]) or death (OR 3.09 [95% CI 0.51-18.52]) in patients with R287Q genotype, compared with wild-type sEH. The R287Q genotype was associated with reduced odds of having a Glasgow Outcome Scale score of ≤ 3 (OR 0.23 [95% CI 0.06-0.82]). There were no significant differences in the odds of neurological deterioration due to DCI. CONCLUSIONS Genetic polymorphisms of sEH are associated with neurological and vital outcomes after aneurysmal SAH.

Functional screening for G protein-coupled receptor targets of 14,15-epoxyeicosatrienoic acid

Epoxyeicosatrienoic acids (EETs) are potent vasodilators that play important roles in cardiovascular physiology and disease, yet the molecular mechanisms underlying the biological actions of EETs are not fully understood. Multiple lines of evidence suggest that the actions of EETs are in part mediated via G protein-coupled receptor (GPCR) signaling, but the identity of such a receptor has remained elusive. We sought to identify 14,15-EET-responsive GPCRs. A set of 105 clones were expressed in Xenopus oocyte and screened for their ability to activate cAMP-dependent chloride current. Several receptors responded to micromolar concentrations of 14,15-EET, with the top five being prostaglandin receptor subtypes (PTGER2, PTGER4, PTGFR, PTGDR, PTGER3IV). Overall, our results indicate that multiple low-affinity 14,15-EET GPCRs are capable of increasing cAMP levels following 14,15-EET stimulation, highlighting the potential for cross-talk between prostanoid and other ecosanoid GPCRs. Our data also indicate that none of the 105 GPCRs screened met our criteria for a high-affinity receptor for 14,15-EET.

Peroxisomal biogenesis in ischemic brain.

AIMS Peroxisomes are highly adaptable and dynamic organelles, adjusting their size, number, and enzyme composition to changing environmental and metabolic demands. We determined whether peroxisomes respond to ischemia, and whether peroxisomal biogenesis is an adaptive response to cerebral ischemia. RESULTS Focal cerebral ischemia induced peroxisomal biogenesis in peri-infarct neurons, which was associated with a corresponding increase in peroxisomal antioxidant enzyme catalase. Peroxisomal biogenesis was also observed in primary cultured cortical neurons subjected to ischemic insult induced by oxygen-glucose deprivation (OGD). A catalase inhibitor increased OGD-induced neuronal death. Moreover, preventing peroxisomal proliferation by knocking down dynamin-related protein 1 (Drp1) exacerbated neuronal death induced by OGD, whereas enhancing peroxisomal biogenesis pharmacologically using a peroxisome proliferator-activated receptor-alpha agonist protected against neuronal death induced by OGD. INNOVATION This is the first documentation of ischemia-induced peroxisomal biogenesis in mammalian brain using a combined in vivo and in vitro approach, electron microscopy, high-resolution laser-scanning confocal microscopy, and super-resolution structured illumination microscopy. CONCLUSION Our findings suggest that neurons respond to ischemic injury by increasing peroxisome biogenesis, which serves a protective function, likely mediated by enhanced antioxidant capacity of neurons.

Disrupting Dimerization Translocates Soluble Epoxide Hydrolase to Peroxisomes

The epoxyeicosatrienoic acid (EET) neutralizing enzyme soluble epoxide hydrolase (sEH) is a neuronal enzyme, which has been localized in both the cytosol and peroxisomes. The molecular basis for its dual localization remains unclear as sEH contains a functional peroxisomal targeting sequence (PTS). Recently, a missense polymorphism was identified in human sEH (R287Q) that enhances its peroxisomal localization. This same polymorphism has also been shown to generate weaker sEH homo-dimers. Taken together, these observations suggest that dimerization may mask the sEH PTS and prevent peroxisome translocation. In the current study, we test the hypothesis that dimerization is a key regulator of sEH subcellular localization. Specifically, we altered the dimerization state of sEH by introducing substitutions in amino acids responsible for the dimer-stabilizing salt-bridge. Green Fluorescent Protein (GFP) fusions of each of mutants were co-transfected into mouse primary cultured cortical neurons together with a PTS-linked red fluorescent protein to constitutively label peroxisomes. Labeled neurons were analyzed using confocal microscopy and co-localization of sEH with peroxisomes was quantified using Pearson’s correlation coefficient. We find that dimer-competent sEH constructs preferentially localize to the cytosol, whereas constructs with weakened or disrupted dimerization were preferentially targeted to peroxisomes. We conclude that the sEH dimerization status is a key regulator of its peroxisomal localization.