Oleoylethanolamide treatment reduces neurobehavioral deficits and brain pathology in a mouse model of Gulf War Illness

There are nearly 250,000 Gulf War (GW) veterans who suffer from Gulf War Illness (GWI), a multi-symptom condition that remains untreatable. The main objective was to determine if targeting peroxisomal function could be of therapeutic value in GWI. We performed a pilot study that showed accumulation of very long chain fatty acids (VLCFA), which are metabolized in peroxisomes, in plasma from veterans with GWI. We then examined if targeting peroxisomal β-oxidation with oleoylethanolamide (OEA) restores these lipids to the normal levels and mitigates neuroinflammation and neurobehavioral deficits in a well-established mouse model of GWI. In GWI mice, treatment with OEA corresponded with cognitive benefits and reduced fatigue and disinhibition-like behavior in GWI mice. Biochemical and molecular analysis of the brain tissue showed reduced astroglia and microglia staining, decreased levels of chemokines and cytokines, and decreased NFκB phosphorylation. Treatment with OEA reduced accumulation of peroxisome specific VLCFA in the brains of GWI mice. These studies further support the translational value of targeting peroxisomes. We expect that OEA may be a potential therapy for treating neurobehavioral symptoms and the underlying lipid dysfunction and neuroinflammation associated with GWI. Oleoylethanolamide is available as a dietary supplement, making it appealing for human translational studies.


Supplementary methods:
In vitro study: Murine macrophage RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC) and grown in Dulbecco's Modified Eagle Medium (DMEM) F12 medium (life science St. Louis, MO) containing 10% fetal bovine serum (FBS) and 1% mixture of antibiotics (penicillin, streptomycin sulfate) and antimycotics (amphotericin B). Cells were incubated in a humidified 5% CO2 atmosphere at 37°C and subsequently challenged with lipopolysaccharide (LPS) from E. coli (Sigma Aldrich) at 10ug/ml to induce NFkB activation. Oleoylethanolamide (OEA), docosahexaenoyl ethanolamide (DHEA), Prostaglandin E2 ethanol amide (PGE2EA) (Cayman chemical, Ann Arbor, MI) at 10μM, 1μM, 100nM each were used to investigate possible inhibition of NFkB phosphorylation by these compounds. Cells were incubated with either DMSO alone, with LPS and DMSO, or with each test compound in combination with LPS and DMSO. Conditioned media (CM) were collected and analyzed for the quantification of cytokines. The cells were lysed with mammalian protein extraction reagent (mPER) along with protease inhibitor to quantify NFkB phosphorylation and PPAR-alpha expression in these cells.

XTT and LDH assays
Viability of RAW cells in the presence of the CM for 24 hours was assessed by the XTT (Sigma-Aldrich), and cytotoxicity was determined by the presence of lactate dehydrogenase (LDH) (Roche) according to manufacturer's instructions.  Supplementary 1: Fatigue-like presentation is observed in GWI-exposed mice.
(A)FST data expressed as percent control of immobile time ± SEM (n = 24 per group). Total immobile time was significantly decreased in GWI mice at 3-months post-exposure, but no effect was seen at 1-month post-exposure. (B) At 3-months post-exposure, immobility was similar between control and GWI mice for the first 2 min, but the immobility increased in GWI mice with time. *p ≤ 0.05 Supplementary 2: There were no significant differences between any of the groups for the speed. On the EPM, there were no significant differences between any of the groups for the speed.
Supplementary 3: OEA treatment had no effect in locomotion and exploration behavior in GWI mice. On the OFT, there were no significant differences between any of the groups for the distance travelled, speed and time spend near the wall (in zone) or in the center zone among all groups. *p ≤ 0.05 Supplementary 4: Lipid profiles from the PB+PER mouse model at the 11month-chronic post-exposure timepoint. Result expressed as percentage to control shows all Free fatty acid detected by LC-MS.
Supplementary 5: Western blot analysis of PPAR- protein expression in the brain. There was significant increase in PPAR- level in the mice feed with OEA compared to non OEA controls. Histogram showing relative levels of each of the PPAR-alpha protein in all four groups.
Supplementary 6: Western blot analysis of PGC-1  protein expression in the brain. There was significant increase in PGC-1  level in the mice feed with OEA compared to non OEA controls. Histogram showing relative levels of each of the PGC-1 protein in all four groups.
Supplementary 7: Mice with GWI had elevated levels of pro-inflammatory cytokines compared to control mice. Among the cytokines examined in the brain, IFN-, and IL-1β were lower in OEA treated GWI mice plasma compared to GWI mice on normal chow.