Abstract
Purpose :
Elovanoids (ELVs) are cell-specific neuroprotective novel lipid mediators derived from 32:6n3 and 34:6n3 that contribute to photoreceptor cell integrity and survival in response to uncompensated oxidative stress induced inflammation. The ω-3 essential fatty acid (FA) docosahexaenoic acid (DHA, 22:6) and its Very Long Chain Polyunsaturated Fatty Acid derivatives (VLC-PUFAs, ≥28 carbons), are concentrated in photoreceptors (PRC) and necessary for vision. The purpose of this work was to find the ELV receptors that elict survival in RPE cells.
Methods :
To identify the receptor targets responsible for the activity of ELVs 32:6 and 34:6, we employed preliminary high throughput screening using PathHunter β-arrestin assay system to monitor GPCR activity (DiscoverX). This system detects the interaction of ELVs and candidate GPCRs and transduce the signal by producing chemiluminescence. The positive candidates were validated using siRNA to knock them down to test the involvement of the ELVs. The silenceing were confirmed using droplet digital PCR and capillary western blots (Jess/ProteinSimple). The activity of the ELVs were testedin primary human RPE cells in culture using real time imaging platform (Incucyte SX5).
Results :
GPCR Screening by DiscoverX showed that ELV-N32 and ELV-N34 were agonoists of CNR2 and GPR120 respectively. Silencing of GPR120 resulted in 90% of protein reduction. The knoxkdown of GPR120 abrogated the survival activity of ELV-N34 but not ELV-N32. Contrarily, the silencing of CNR2 prevented ELV-N32 to elicit its protective effects. In both cases, these was a decrease in the phosphorylation of AKT.
Conclusions :
Our results show that ELV-N32 and ELV-34 promote survival in human RPE cells by activating CNR2 and GPR120 which results in the activation of AKT.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.