The retinal pigment epithelium (RPE) serves a crucial role in the maintenance of healthy photoreceptors and, consequently, vision. The basolateral membrane of the RPE is in contact with the choriocapillaris, thereby allowing nutrients to pass from the blood stream into the eye. This semi-permeable epithelium allows all-trans retinol (Vitamin A), DHA, and other molecules to be captured and delivered to photoreceptors. The apical aspect of the RPE is in contact with the tip of the outer segments of photoreceptor cells. During photoreceptor outer segment renewal, the RPE phagocytizes the old disks discarded by the distal outer segment. These old disks once housed the phototransduction machinery for the eye, as well as DHA, and get recycled back to the RPE ().
NPD1 also cytoprotects against the apoptotic effects of the accumulated bispyridinium bisretinoid A2E. A2E is a byproduct of phototransduction that becomes toxic when it accumulates in RPE cells during aging, in age-related macular degeneration, and in Stargardt macular dystrophy. It is interesting to note that there is a six hour window in which these neuroprotective effects can be seen. NPD1 also neuroprotects against oxidative stress generated by serum starvation/H2
(600μM)/TNFα (10 ng/ml), and does so for an eight-hour time frame. In looking at the pathway for cytoprotection, NPD1 (50 nM) did not affect the conversion of A2E to A2E oxiranes. The action of NPD1 was found to occur at the premitochondrial stage of the apoptosis pathway via decreasing caspase-3 cleavage, decreasing Bax expression, and increasing Bcl-2 expression [10
Neurotrophins stimulated production and release of NPD1 from the RPE. Human RPE cells were used in monolayers to define whether the NPD1 was secreted on the apical or the basal side of the RPE. Glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), neurotrophin-3 (NT3), ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), fibroblast growth factor (FGF), brain-derived neurotropic factor (BDNF), Persephin, and pigment epithelium-derived factor (PEDF) all produced an increase of NPD1 through the apical surface. PEDF elicited the largest synthesis and release of NPD1. Increasing concentrations of PEDF or ciliary neurotrophic factor (CNTF), when added to the basal medium, induced less apical incubation of NPD1, whereas adding the neurotrophins to the apical medium yielded a concentration-dependent increase in apical NPD1 levels [10
DHA potentiates PEDF-induced release of NPD1 through the apical surface of RPE cells. NPD1 concentration is dependent on whether the PEDF was placed in the media on the apical or basolateral side, with the apical side resulting in more NPD1 production. The addition of DHA to either side of the monolayer produced a PEDF-potentiated increase of NPD1 only on the apical side. Increasing concentrations of DHA on both sides of the monolayer yielded a stepwise increase in apical NPD1 concentration up to a point (50 nM), whereas the basolateral NPD1 concentration had the opposite effect. The addition of PEDF (50 ng/ml) to the increasing DHA concentration synergistically augmented apical NPD1 concentration.
When presented with oxidative stress (serum starvation/H2O2 (600 μM)/TNFα (10 ng/ml)), DHA and PEDF synergistically act to protect human RPE cells in culture. The concentration of NPD1 is greater when DHA and PEDF are added together than when either DHA or PEDF are added alone. Moreover, DHA and PEDF synergistically increased the expression of antiapoptotic proteins Bcl-2 and Bfl-1 while concomitantly decreasing the proapoptotic proteins Bid, Bax, and Bad during oxidative stress conditions. DHA alone, when increased from 10 to 50 nM, amplified Bcl-2 and Bfl-1 production. Though PEDF by itself does not modify expression of the Bcl-2 family of proteins, PEDF synergistically acts with DHA to increase antiapoptotic protein expression and decrease proapoptotic protein expression. Caspase-3 cleavage increases with increased apoptosis (as seen by Hoechst-positive RPE cells) under oxidative stress. Caspase-3 decreases with the addition of 10 to 50 nM DHA, and this effect is potentiated by the addition of PEDF.