Epoxyeicosatrienoic acids enhance axonal growth in primary sensory and cortical neuronal cell cultures

Abstract

It has recently been reported that soluble epoxide hydrolase (sEH), the major enzyme that metabolizes epoxyeicosatrienoic acids (EETs), is expressed in axons of cortical neurons; however, the functional relevance of axonal sEH localization is unknown. Immunocytochemical analyses demonstrate predominant axonal localization of sEH in primary cultures of not only cortical but also sympathetic and sensory neurons. Morphometric analyses of cultured sensory neurons indicate that exposure to a regioisomeric mixture of EETs (0.01-1.0 μM) causes a concentration-dependent increase in axon outgrowth. This axon promoting activity is not a generalized property of all regioisomers of EETs as axonal growth is enhanced in sensory neurons exposed to 14,15-EET but not 8,9- or 11,12-EET. 14,15-EET also promotes axon outgrowth in cultured cortical neurons. Co-exposure to EETs and either of two structurally diverse pharmacological inhibitors of sEH potentiates the axon-enhancing activity of EETs in sensory and cortical neurons. Mass spectrometry indicates that sEH inhibition significantly increases EETs and significantly decreases dihydroxyeicosatrienoic acid metabolites in neuronal cell cultures. These data indicate that EETs enhance axon outgrowth and suggest that axonal sEH activity regulates EETs-induced axon outgrowth. These findings suggest a novel therapeutic use of sEH inhibitors in promoting nerve regeneration.

Figure 1. Soluble epoxide hydrolase (sEH) is selectively localized to axons in primary neuronal cell cultures

Neuronal cell cultures derived from the neocortex of embryonic rat pups (A, B) or the superior cervical ganglia (C, D) were co-labeled with antibodies specific for the somatodendritic cytoskeletal protein MAP-2 (A, C) and sEH (B, D). Comparison of MAP-2 (A) versus sEH (B) patterns in cultured cortical neurons suggests that only a subset of cortical neurons express sEH. In those neurons that are immunopositive for sEH, fluorescence is significantly more intense in axons relative to dendrites. Comparison of MAP-2 (C) versus sEH (D) immunostaining in cultured sympathetic neurons suggests that all neurons are immunopositive for sEH. Consistent with results in cultured cortical neurons, sEH immunofluorescence is more intense in axonal than in dendritic processes. Intense sEH immunofluorescence was also observed in the axons of sensory neurons cultured from dorsal root ganglia of embryonic rat pups (F) relative to control cultures reacted with secondary antibody only (E). Bar = 50 μm.

Figure 2. Epoxyeicosatrienoic acids (EETs) enhance axon outgrowth in cultured sensory neurons

Representative fluorescence micrographs of sensory neurons derived from embryonic rat dorsal root ganglia exposed to vehicle (ethanol and DMSO) or 14, 15-EET in the absence or presence of the sEH inhibitor TUPS. 14, 15-EET (0.3 μM) and TUPS (0.1 μM) were added 1h and 12h after plating. At 20 to 24h in vitro, cultures were fixed and immunostained for the peripheral neuronal cell marker PGP9.5. Bar = 100 μm.

Figure 3. Epoxyeicosatrienoic acids (EETs) enhance axon outgrowth in cultured sensory neurons

Sensory neurons derived from embryonic rat dorsal root ganglia were exposed to vehicle (ethanol and DMSO) or EETs in the absence or presence of sEH inhibitors (sEHi). EETs ± sEHi were added 1h and 12h after plating. At 20 to 24h in vitro, cultures were fixed and immunostained for the peripheral neuronal cell marker PGP9.5. Exposure to a mixture of EET isomers caused a dose-dependent enhancement of axon outgrowth (A). Co-exposure to the sEHi TUPS further enhanced the effects of EETs on axon outgrowth (A). Addition of TUPS enhanced axon outgrowth even in the absence of exogenous EETs (B). Morphometric analyses of sensory neurons exposed to purified EET isomers indicated that relative to vehicle control cultures, EET 14,15-EET, but not 8,9- or 11,12-EET significantly enhanced axon outgrowth at concentrations ranging from 0.01 to 0.3 μM (C). Addition of TUPS further enhanced axonal outgrowth in cultures co-exposed to 11,12- and 14,15-EET at 0.01 μM, but not 8,9-EET at 0.01 μM (C). Exposure to 14,15-EET at even higher concentrations (1 to 3 μM) also enhanced axon outgrowth and these effects were further enhanced by co-exposure to 4-PCO at 2 or 6 μM (D). Data presented as the mean ± S.E.M. (n = 100 neurons per experimental condition); ***p < 0.001 relative to vehicle control (One-way ANOVA with post-hoc Student Newman-Keuls analysis).

Figure 4. Epoxyeicosatrienoic acids (EETs) enhance axon outgrowth in cultured cortical neurons

Cortical neurons derived from embryonic rat neocortex were exposed to vehicle (ethanol and DMSO) or 14,15-EET at concentrations ranging from 0.1 to 3 μM in the absence or presence of the sEH inhibitor (sEHi) 4-PCO. EETs ± sEHi were added 1h and 12h after plating. On day 4 in vitro, cultures were fixed and immunostained for neurofilaments. 14,15-EET significantly enhanced axon outgrowth at 1.0 μM and this effect was further enhanced by co-exposure to 4-PCO (A). Exposure to 4-PCO alone did not cause a significant increase in axon outgrowth relative to neurons exposed only to vehicle (B). Data presented as the mean ± S.E.M. (n = 50 neurons per experimental condition); *p < 0.05 and **p< 0.01 (One-way ANOVA with post-hoc Student Newman-Keuls analysis). An asterisk above bar of a treated group indicates a significant difference from vehicle control; whereas an asterisk above a horizontal line indicates a significant difference between cultures grown in the absence or presence of sEHi at the same EETs concentration.

Figure 5. Pharmacologic inhibition of soluble epoxide hydrolase (sEH) significantly decreases hydration of 14,15-epoxyeicosatrienoic acid (EET) in neuronal cell cultures

Sensory neurons derived from embryonic rat DRG were exposed to vehicle (ethanol and DMSO) or 14,15-EET (free fatty acid, 0.3 μM) in the absence or presence of the sEH inhibitor TUPS (0.1μM) added 1h and 12h after plating. At 24h in vitro, conditioned medium and cell pellets were collected. The concentration of 14,15-EET and its metabolite, 14,15-dihydroxyeicosatrienoic acid (DHET), were determined in both culture medium and cells using liquid chromatography-mass spectroscopy. Addition of 14,15-EET significantly increased the concentration of 14,15-EET in the cells and media (* p<0.05 vehicle versus 14,15-EET ) and the concentration of 14,15-DHET in the cells and media (*p<0.001 vehicle versus 14,15-EET). Simultaneous treatment with TUPS further increased the levels of 14,15-EET in cells and media (+ p<0.05 14,15-EET versus 14,15-EET+TUPS) and decreased the levels of 14,15-DHET in cells and media (Δ p<0.002 14,15-EET versus 14,15-EET+TUPS). Data are presented as the mean ± S.E.M. (n = 3 per experimental condition); statistical significance was determined by One-way ANOVA followed by Student-Newman-Keuls post hoc analysis.