Synthesis of docosahexaenoic acid from eicosapentaenoic acid in retina neurons protects photoreceptors from oxidative stress

Abstract

Oxidative stress is involved in activating photoreceptor death in several retinal degenerations. Docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in the retina, protects cultured retina photoreceptors from apoptosis induced by oxidative stress and promotes photoreceptor differentiation. Here, we investigated whether eicosapentaenoic acid (EPA), a metabolic precursor to DHA, had similar effects and whether retinal neurons could metabolize EPA to DHA. Adding EPA to rat retina neuronal cultures increased opsin expression and protected photoreceptors from apoptosis induced by the oxidants paraquat and hydrogen peroxide (H2 O2 ). Palmitic, oleic, and arachidonic acids had no protective effect, showing the specificity for DHA. We found that EPA supplementation significantly increased DHA percentage in retinal neurons, but not EPA percentage. Photoreceptors and glial cells expressed Δ6 desaturase (FADS2), which introduces the last double bond in DHA biosynthetic pathway. Pre-treatment of neuronal cultures with CP-24879 hydrochloride, a Δ5/Δ6 desaturase inhibitor, prevented EPA-induced increase in DHA percentage and completely blocked EPA protection and its effect on photoreceptor differentiation. These results suggest that EPA promoted photoreceptor differentiation and rescued photoreceptors from oxidative stress-induced apoptosis through its elongation and desaturation to DHA. Our data show, for the first time, that isolated retinal neurons can synthesize DHA in culture. Docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in retina photoreceptors, and its precursor, eicosapentaenoic acid (EPA) have multiple beneficial effects. Here, we show that retina neurons in vitro express the desaturase FADS2 and can synthesize DHA from EPA. Moreover, addition of EPA to these cultures protects photoreceptors from oxidative stress and promotes their differentiation through its metabolization to DHA.

Keywords: apoptosis; differentiation; fatty acid desaturase; omega-3 fatty acids; polyunsaturated fatty acid; retina.

Figure 1. Addition of EPA protected photoreceptors from Paraquat-induced apoptosis

Rat retina neuronal cultures were supplemented with 3 μM eicosapentaenoic acid (EPA) or with its vehicle, a bovine serum albumin (BSA) solution (−) at day 1 and treated with 48 μM Paraquat (PQ) at day 3. Cultures were fixed 24 h later. Phase contrast (a–d, i–l) and fluorescence photomicrographs (e–h, m–x) show the effect of EPA addition on apoptosis, evaluated by TUNEL assay (e–h) and by DAPI labeling (q-t; merge in u-x, blue labeling), to evidence pyknotic nuclei. Photoreceptors were identified by immunocytochemistry, using Rho4D2, anti-opsin, monoclonal antibody (m–p; merge in u–x, green labeling). Note that the amount of TUNEL-labeled, apoptotic cells (arrowheads in e–h) and cells showing pyknotic nuclei (s, w, thin arrows), including opsin-labeled photoreceptors (o, s, w, wide white arrow), increased in PQ-treated cultures (c, g, k, o, s, w) compared to controls and EPA-supplemented cultures. EPA addition prevented these increases (d, h, l, p, t, x) upon PQ treatment. Bars depict the effect of different EPA concentrations on cell viability (y), determined by propidium iodide (PI) labeling and apoptosis of photoreceptors (PhRs) (z), determined by analyzing the amount of pyknotic or fragmented nuclei. Three separate experiments with three dishes for each condition were analyzed and 10 fields per dish were counted blindly in all experiments. Bar, 20 μm. *p≤0.05; statistically significant differences determined by a one way-ANOVA test, followed by a Tukey’s post hoc test.

Figure 2. EPA addition protected photoreceptors from H₂O₂-induced apoptosis

Phase contrast (a–d, i–l) and fluorescence photomicrographs (e–h, m–t) of neuronal cultures supplemented with vehicle (−) or 3 μm EPA at day 1 in culture and treated at day 3 with 10 μM H₂O₂. Apoptosis was evaluated by TUNEL assay (e–h); large arrowheads (a–d) indicate the corresponding TUNEL-labeled (apoptotic) cells in e–h. Mitochondrial functionality (m–p) was determined with Mitotracker; small arrowheads show photoreceptors preserving their mitochondrial membrane polarization. Pyknotic or fragmented nuclei (q–t) were determined by DAPI labeling; arrows indicate apoptotic photoreceptors. Bars, 15 μm. Bars depict mean ± SD of the percentage of photoreceptors preserving their mitochondrial membrane polarization (u) and the ratio of increase in the number of apoptotic photoreceptors (v). Three different experiments with three dishes for each condition were analyzed and 10 fields per dish were counted blindly in all experiments. ** p≤0.01; statistically significant differences determined by a one way-ANOVA test followed by a Tukey’s test.

Figure 3. Oxidative stress-induced apoptosis of photoreceptors was prevented only by EPA addition

Neuronal cultures were supplemented at day 1 with either 3 μM EPA, or 4 μM oleic (OLA), palmitic (PAM), or arachidonic (ARA) acid, or vehicle (−) and treated with PQ at day 3. Bars depict the percentage of PI-labeled cells (a) and of photoreceptors with fragmented or pyknotic (apoptotic) nuclei (b). Three experiments with three dishes for each condition were analyzed and 10 fields per dish were counted blindly in all experiments. *p≤0.05, statistically significant differences determined by a one-way ANOVA test followed by a Tukey’s test.

Figure 4. EPA addition stimulated opsin expression in photoreceptors

Phase contrast (a, b) and fluorescence (c, d) photomicrographs of control (a, c) and 3 μM EPA-supplemented (b, d) 6 day neuronal cultures, show opsin expression (c, d) determined by immunocytochemistry. Note that EPA supplementation increased the number of opsin-expressing photoreceptors (arrows) and led to the formation of apical processes (arrowheads in d). Bar, 10 μm. Bars (e) depict the fold-change in the amount of photoreceptors expressing opsin in EPA-supplemented, 4 and 6 day cultures compared to controls, determined by immunocytochemistry. Bars (f) depict the fold-change in the level of opsin mRNA, determined by qRT-PCR in 6-day cultures supplemented with EPA compared to controls. Three experiments with three dishes for each condition were analyzed and 10 fields per sample were counted blindly in all experiments. *p≤0.05; ** p≤0.01, statistically significant differences compared to controls, determined by a Student’s t-test.

Figure 5. Addition of EPA increased docosahexaenoic acid (DHA) levels in retina neurons

Retina neurons were supplemented with vehicle (−) or with 3 μM EPA added at day 1 in vitro and the fatty acid composition of neuronal phospholipids was determined by GLC at day 4. Bars (a) depict mean ± SD of the mole % content of EPA, docosapentaenoic acid (DPA) and DHA in neuronal phospholipids. Three different experiments with two dishes for each condition were analyzed. * p≤0.05, statistically significant differences, compared to the respective controls, determined by a Student’s t-test. Sequence of metabolic reactions (b) leading to 22:6 n-3 (DHA) synthesis from 18:3 n-3 that involves its desaturation by FADS2, the further elongation and desaturation catalyzed by ELOVL5,2 and FADS1 to synthesize 20:5 n-3 (EPA), which is successively elongated by ELOVL5,2 and ELOVL2 to 24:5 n-3, which is then desaturated by FADS2 to 24:6 n-3, which after a (peroxisomal) β-oxidation gives rise to DHA.

Figure 6. Retina neurons expressed FADS2

Fluorescence confocal photomicrographs (a–j) of 6 day neuronal cultures (a–d), and neuroglial mixed cultures (e–g) and epifluorescence photomicrographs of ARPE-19 cell cultures (h–j) showing expression of opsin (green in a, d), FADS2 (red in b, d, e, g, h, j), vimentin (green in g) and nuclei labeled with TOPRO (c, d, f, g) or DAPI (i, j). Note that glial cell nuclei (arrowheads in f) were much larger than those of neurons (arrows in f). FADS2 expression was observed in nuclei in opsin-labeled photoreceptors (arrows in a–d), neurons (arrows in e, g) and vimentin-labeled glial cells (arrowheads in e, g). In contrast, in ARPE-19 cells expression of FADS2 was mainly observed in the cytoplasm (h, j). Bars, 20 μm. Electrophoresis of FADS2 mRNAs after RT-PCR (k) in 4 day neuronal cultures supplemented with vehicle (−) or 3 μM EPA; a specific 167 bp band was observed in samples from neuronal cultures, which was absent in Reverse Transcriptase negative control (RT negative) and No Template Control (NTC) conditions. Bars (l) depict relative quantification of FADS2 mRNA levels by RT-PCR in lysates prepared from 4 day neuronal cultures (n=3) supplemented with vehicle (−) or 3 μM EPA, from 13 day pure glial cultures (n=3) from rat retina (glia) and from PN10 rat brain (n=1).

Figure 7. Inhibition of DHA synthesis blocked EPA effects on photoreceptor apoptosis and differentiation

Neuronal cultures were treated with or without CP, a FADS1/FADS2 inhibitor, before supplementation with vehicle or with 3 μM EPA at day 1. Bars (a) depict mean ± SD of EPA, DPA and DHA mole % content in total phospholipids extracted from 4 day neuronal cultures supplemented with vehicle (−), CP, EPA and EPA+CP. Three separate experiments with two dishes for each condition were analyzed. Bars (b) depict mean ± SD of the percentage of photoreceptors (PhRs) showing pyknotic or fragmented (apoptotic) nuclei in neuronal cultures treated without (white bars) or with H₂O₂ (grey bars) at day 3. Bars (c) depict the mean ± SD of the percentage of photoreceptors expressing opsin after 6 days in culture in each experimental condition. Three separate experiments with three dishes for each condition were analyzed and 10 fields per sample were counted blindly in (b) and (c). *p≤0.05, **p≤0.01, statistically significant differences determined by a one way-ANOVA test followed by a Tukey’s test.