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. 2013 Aug;54(8):2236-46.
doi: 10.1194/jlr.M039040. Epub 2013 May 30.

Retinoid X Receptor Activation Is Essential for Docosahexaenoic Acid Protection of Retina Photoreceptors

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Free PMC article

Retinoid X Receptor Activation Is Essential for Docosahexaenoic Acid Protection of Retina Photoreceptors

Olga L German et al. J Lipid Res. .
Free PMC article

Abstract

We have established that docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in the retina, promotes survival of rat retina photoreceptors during early development in vitro and upon oxidative stress by activating the ERK/MAPK signaling pathway. Here we have investigated whether DHA turns on this pathway through activation of retinoid X receptors (RXRs) or by inducing tyrosine kinase (Trk) receptor activation. We also evaluated whether DHA release from phospholipids was required for its protective effect. Addition of RXR antagonists (HX531, PA452) to rat retinal neuronal cultures inhibited DHA protection during early development in vitro and upon oxidative stress induced with Paraquat or H2O2. In contrast, the Trk inhibitor K252a did not affect DHA prevention of photoreceptor apoptosis. These results imply that activation of RXRs was required for DHA protection whereas Trk receptors were not involved in this protection. Pretreatment with 4-bromoenol lactone, a phospholipase A2 inhibitor, blocked DHA prevention of oxidative stress-induced apoptosis of photoreceptors. It is noteworthy that RXR agonists (HX630, PA024) also rescued photoreceptors from H2O2-induced apoptosis. These results provide the first evidence that activation of RXRs prevents photoreceptor apoptosis and suggest that DHA is first released from phospholipids and then activates RXRs to promote the survival of photoreceptors.

Keywords: RXR agonists; apoptosis; oxidative damage; photoreceptor survival.

Figures

Fig. 1.
Fig. 1.
Expression of RXRs in retina photoreceptors in vitro. A: Phase (V and IX) and fluorescence (I–IV, VI–VIII) micrographs of 6 day retina neuronal cultures immunolabeled with pan-RXR antibody (blue staining in I, IV, and VI), Rho4D2 (II, green), MitoTracker (III and VIII, red staining), merge (IV), and HPC-1 (VII, green staining). Note that opsin (+) cells (photoreceptors) express RXRs mainly in their nuclei. In contrast, HPC-1 (+) cells (amacrine neurons) express RXRs mainly in the cytoplasm, with only scarce aggregates in their nuclei (n). The scale bar in I–V represents 20 μm and in VI–IX, 10 μm. B: Proteins obtained from lysates from 3 day neurons (NEU) and from human retinal pigment epithelial cells (ARPE-19, used as a positive control) were analyzed by Western blot with a pan-RXR antibody.
Fig. 2.
Fig. 2.
Effect of RXR antagonists on DHA prevention of photoreceptor apoptosis. A: Phase (left) and fluorescence (right) micrographs showing TUNEL in 4 day cultures without (I, VI; BSA) or with PQ (II, VII; BSA+PQ) treatment, and supplemented with DHA, without (III, VIII) or with pretreatment with RXR antagonists HX531 (IV, IX) and PA452 (V, X) before PQ addition. The scale bar represents 10 μm. B: Day 1 retinal neurons were preincubated with vehicle (control) or with either RXR antagonist for 1 h, and then supplemented without (BSA) or with DHA (DHA). The cultures were finally treated or not treated at day 3 with PQ for 24 h. The percentage of apoptotic photoreceptors was determined by analyzing nuclear fragmentation with DAPI. C: Retinal neurons were preincubated with vehicle (control) or with the RXR antagonist for 1 h, then supplemented without (BSA) or with DHA (DHA) and finally treated or not treated with H2O2 for 5.5 h at day 3. The percentage of apoptotic photoreceptors was determined with DAPI. D: Retinal neurons were cultured for 6 days without (BSA) or with DHA (DHA) in cultures incubated without (control) or with the RXR antagonists (1 μM HX531 or 1 μM PA452). The percentage of apoptotic photoreceptors was determined by TUNEL assay. Each value represents the mean of three experiments ± SD. *P < 0.05, ***P < 0.001.
Fig. 3.
Fig. 3.
RXR agonists prevented photoreceptor death upon oxidative stress. Retinal neurons without (control) or with RXR agonists, 100 nM HX630 or 10 nM PA024, added at day 1, were treated or not treated with H2O2 at day 3. A: Micrographs show: left, phase contrast; middle, PI (+) cells; right, nuclei labeling with DAPI. Arrows indicate photoreceptors showing fragmented or pycnotic nuclei. The scale bar represents 10 μm. B: The percentage of dead cells was determined by PI labeling and (C) of apoptotic photoreceptors with DAPI. Bars are means ± SD. *Statistical significance was calculated using two-way ANOVA test for each graphic. At the 0.05 level, each RXR agonist+H2O2 mean was significantly different compared with H2O2. Mean values were compared using Tukey statistics (P < 0.05).
Fig. 4.
Fig. 4.
Inhibition of Trks did not affect DHA prevention of photoreceptor death during development in vitro. A: Retinal neurons were cultured for 6 days without (−) or with DHA (+) in cultures treated without (−) or with (+) different concentrations of K252a, a Trk inhibitor. The percentage of apoptotic photoreceptors was determined with DAPI. B: Retinal neurons were cultured for 6 days without (control) or with insulin in cultures incubated either without (−) or with (+) different concentrations of K252a. The percentage of apoptotic amacrine neurons was determined with DAPI. Bars represent means ± SD. Statistically significant differences compared with control: *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 5.
Fig. 5.
Inhibition of iPLA2 blocked DHA protection of photoreceptors from oxidative stress-induced apoptosis. Retinal neurons, supplemented without (BSA) or with DHA (DHA) at day 1 for 24 h were preincubated or not preincubated (control) with iPLA2 inhibitor BEL (5 μM) at day 3 for 30 min and finally treated or not treated with H2O2 for 5.5 h at day 3. A: Micrographs show: left column, phase contrast; right column, TUNEL (+) cells. The scale bar represents 10 μm. B: The percentage of apoptotic photoreceptors in the different experimental conditions was determined with DAPI. Bars represent means ± SD. ***P < 0.001.

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