Abstract
Purpose: :
In many retina neurodegenerative diseases, photoreceptors (PRs) progressively degenerate by apoptosis causing visual loss. We demonstrated that docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in the retina, promotes the survival of rat retina PRs during early development in vitro and upon oxidative stress by activating the ERK/MAP kinase signaling pathway. We have now investigated upstream this pathway, to establish whether DHA acts indirectly, modifying membrane properties to induce the activation of tyrosine kinase receptors (TRK) or directly, acting as a ligand for retinoid X receptors (RXR), as has been shown in the brain. We have also evaluated whether DHA has to be released from membrane phospholipids to exert its protective effect.
Methods: :
We prepared pure retinal neuronal cultures, supplemented or not with DHA, and incubated for 6 days or treated at day 3 with oxidants paraquat (PQ) or H2O2. To investigate activation of TRK and RXR, prior to DHA addition we treated the cultures with K252a, a TRK inhibitor and HX531 or PA452, RXR antagonists, respectively. We also studied if RXR agonists, HX630 or PA024 mimicked DHA protective effect on PRs in H2O2-treated cultures. To evaluate whether deacylation of DHA was required for its protective effects on PRs, DHA-supplemented cultures were treated with or without BEL, a phospholipase A2 inhibitor prior to H2O2 addition. Cellular viability was determined with propidium iodide and apoptosis by TUNEL assay and with the DNA probe DAPI.
Results: :
DHA reduced apoptosis of PRs during early development in vitro even in the presence of the TRK inhibitor. In contrast, this inhibitor blocked the protective effect of insulin on amacrine neurons, known to require TRK activation, increasing their apoptosis. RXR antagonists inhibited DHA protection during early development in vitro and upon PQ and H2O2-induced apoptosis. Moreover, RXR agonists decreased PR apoptosis in H2O2-treated cultures, as we have shown for DHA. Addition of BEL blocked DHA protection of PRs upon oxidative stress.
Conclusions: :
These results suggest a novel pathway for DHA effects in PRs, which involves its initial release from phospholipids upon cellular stress followed by its activation of RXR to promote PR survival.
Keywords: photoreceptors • lipids • retinal culture