Rescue and repair during photoreceptor cell renewal mediated by docosahexaenoic acid-derived neuroprotectin D1

Abstract

Retinal degenerative diseases result in retinal pigment epithelial (RPE) and photoreceptor cell loss. These cells are continuously exposed to the environment (light) and to potentially pro-oxidative conditions, as the retina's oxygen consumption is very high. There is also a high flux of docosahexaenoic acid (DHA), a PUFA that moves through the blood stream toward photoreceptors and between them and RPE cells. Photoreceptor outer segment shedding and phagocytosis intermittently renews photoreceptor membranes. DHA is converted through 15-lipoxygenase-1 into neuroprotectin D1 (NPD1), a potent mediator that evokes counteracting cell-protective, anti-inflammatory, pro-survival repair signaling, including the induction of anti-apoptotic proteins and inhibition of pro-apoptotic proteins. Thus, NPD1 triggers activation of signaling pathway/s that modulate/s pro-apoptotic signals, promoting cell survival. This review provides an overview of DHA in photoreceptors and describes the ability of RPE cells to synthesize NPD1 from DHA. It also describes the role of neurotrophins as agonists of NPD1 synthesis and how photoreceptor phagocytosis induces refractoriness to oxidative stress in RPE cells, with concomitant NPD1 synthesis.

Fig. 1.

RPE/photoreceptor interactions and NPD1 bioactivity. A: RPE control of the permeability of the outer blood-retinal barrier involving remodeling of the blood vessels and selective flux of nutrients and catabolites. B: NPD1 pro-survival enhances the expression of Bcl-2 proteins and decreases COX-2 expression. C: Shedding and phagocytosis of the photoreceptor outer segments.

Fig. 2.

Photoreceptor outer segment phagocytosis elicits protection in RPE cells subjected to oxidative stress. A: Quantitative analysis of Hoechst stained ARPE-19 cells indicates that photoreceptor outer segment phagocytosis significantly decreases the amount of apoptosis observed during oxidative stress. Phagocytosis of polystyrene microspheres during oxidative stress did not alter the amount of apoptosis observed during oxidative stress alone. Results represent averages ± SEM of repeats of two independent experiments. B: NPD1 changes as a function of time after photoreceptor outer segment phagocytosis or microspheres: effect of oxidative stress. NPD1 has been quantified in cells as well as in incubation media. Data represents average ± SEM of two independent studies. Statistical analysis is Student's t-test. NS, not statistically significant.

Fig. 3.

A: NPD1 biosynthesis. Representation of the oxygenation of DHA to form NPD1. PLA₂ releases DHA from the second carbon position of the phospholipids upon stimulation. 15-Lipoxygenase-1 catalyzes the synthesis of 17S-H(p)DHA, which is converted to a 16(17)- epoxide and then is enzymatically converted to NPD1. B: Comparison of NPD1/PD1 biosynthesis with that of 10S,17S-diHDHA isomer (see detailed discussion in the text).

Fig. 4.

Neurotrophins activate NPD1 synthesis in cultured primary human RPE cells. A: Zonula occludens-1 (ZO-1) antibody immunoreactivity (green) illustrates confluence of the monolayer polyhedric-shape of the cells. B: Differential ability of growth factors to selectively release NPD1 through the apical surface of the cell. Growth factors (20 ng/ml) were added to the apical medium. Apical and basal media were collected separately after 72 h and subjected to lipidomic analysis. Each bar is an average ± SEM of four or five independent wells. Values are averages ± SEM of five independent wells. Statistical analysis was performed using Student's t-test shows ^*P < 0.05. C: Schematic representation of the monolayer orientation within the insert. [Fig. 4 A, C, modified with permission from reference (40)].

Fig. 5.

NPD1 synthesis is mediated by 15-lipoxygenase-1. A: Immunocytochemistry showing localization of 15-LOX-1 in ARPE-19 cells. Right column shows normal cells and left column shows 15-LOX-1 silenced cells. The four upper panels depict the localization of 15-LOX1 (green) relative to the nuclei (blue) and Actin (red). The four lower panels display nuclear localization of 15-LOX-2 (red) in relationship with nuclei (blue) and Actin (green). B: Histograms showing the differential production of NPD1 upon different strength of oxidative stress treatment (0, 400, 600, 800 μM H₂O₂ and 10 ng/ml TNFα). In each cell, as in the medium, the production of NPD1 was almost completely abolished (^*P < 0.01). C: Apoptosis percentage measured by Hoechst staining of ARPE-19 cultures of control and silenced cells subjected to oxidative stress and treated with different metabolites of 15-LOX-1 and NPD1 precursor DHA. In silenced cells, apoptosis was augmented by oxidative stress. In normal cells, PEDF/DHA, NPD1, and lipoxin A4 did prevent apoptotic cell death, but in the silenced cells, neither DHA nor lipoxin A4 had any effect. Only NPD1 was able to rescue these cells from apoptosis. Data represents average ± SEM of two independent studies; statistical analysis is Student's t-test (^*P < 0.01, ^**P < 0.001, ^***P < 0.0001 and ^****P < 0.00001). Modified with permission from reference (91).

Fig. 6.

Diagram outlining 15-LOX-1 activity of NPD1 synthesis and bioactivity in RPE cells. Noxious stimuli activate 15-LOX-1, promoting the synthesis of NPD1 from DHA. NPD1 signaling modulates the activity and gene expression of proteins involved in pro- and anti-inflammatory signaling in apoptosis, ultimately fostering photoreceptor cell integrity and overall homeostasis.