Photoreceptor Shedding Research Articles

Annexin A5 regulates surface αvβ5 integrin for retinal clearance phagocytosis.

Diurnal clearance phagocytosis by the retinal pigment epithelium (RPE) is a conserved efferocytosis process whose binding step is mediated by αvβ5 integrin receptors. Two related annexins, A5 (ANXA5) and A6 (ANXA6), share an αvβ5 integrin-binding motif. Here, we report that ANXA5, but not ANXA6, regulates the binding capacity for spent photoreceptor outer segment fragments or apoptotic cells by fibroblasts and RPE. Similar to αvβ5-deficient RPE, ANXA5-/- RPE in vivo lacks the diurnal burst of phagocytosis that follows photoreceptor shedding in wild-type retina. Increasing ANXA5 in cells lacking αvβ5 or increasing αvβ5 in cells lacking ANXA5 does not affect particle binding. Association of cytosolic ANXA5 and αvβ5 integrin in RPE in culture and in vivo further supports their functional interdependence. Silencing ANXA5 is sufficient to reduce levels of αvβ5 receptors at the apical phagocytic surface of RPE cells. The effect of ANXA5 on surface αvβ5 and on particle binding requires the C-terminal ANXA5 annexin repeat but not its unique N-terminus. These results identify a novel role for ANXA5 specifically in the recognition and binding step of clearance phagocytosis, which is essential to retinal physiology.This article has an associated First Person interview with the first author of the paper.

CaV1.3 L-type channels, maxiK Ca2 +-dependent K+ channels and bestrophin-1 regulate rhythmic photoreceptor outer segment phagocytosis by retinal pigment epithelial cells

Phagocytosis of shed photoreceptor outer segments by the retinal pigment epithelium (RPE) is critical for maintenance of visual function. Because changes in intracellular Ca2+ regulate phagocytosis, we studied in vitro the impact of different ion channels in addition to mice deficient for Cav1.3 L-type Ca2+ channels (Ca1.3−/−) and maxiK Ca2+-dependent K+ channels (BK−/−). The knockdown of Bestrophin-1 protein, a regulator of intracellular Ca2+ homeostasis, affected phagocytosis in porcine RPE cultures. Blockage of voltage-gated L-type channels by (+)BayK8644 inhibitor reduced phagocytosis in vitro, in contrast L-type activation by (−)BayK8644 had no impact. The expression rate of Cav1.3, the predominant L-type Ca2+ channel in RPE cells, varied at different times of day. CaV1.3−/− RPE lacked peak phagocytic activity following morning photoreceptor shedding in wild-type RPE and retained a higher number of phagosomes at a later time of day. The BK-channel blocker paxilline lowered phagocytosis in RPE cultures in a concentration-dependent manner. BK−/− RPE in vivo retained phagocytic capability but this activity, which is normally well synchronized with circadian photoreceptor shedding, shifted out of phase. Retinae of older BK−/− mice showed shortened photoreceptor outer segments and diminished rhodopsin content. Store-operated Ca2+ channels Orai-1 did not affect phagocytosis in cultured RPE. TRPV channel inhibition by ruthenium-red reduced phagocytosis, whereas activation at high concentrations of 2-APB increased phagocytosis. Our data demonstrate essential roles for bestrophin-1, BK, TRPV and L-type channels in regulating retinal phagocytosis. These data indicate further the importance of BK and CaV1.3 for rhythmic phagocytic activity synchronized with photoreceptor shedding.

Lack of αvβ5 Integrin Receptor or Its Ligand MFG-E8: Distinct Effects on Retinal Function

Background/Aims: Diurnal phagocytosis of spent photoreceptor outer segment fragments by the retinal pigment epithelium (RPE) is critical for vision. We recently identified an important role for αvβ5 integrin receptors and their ligand Milk fat globule-EGF factor 8 (MFG-E8) in RPE phagocytosis. Methods: We compared RPE phagocytosis and retinal function between mice deficient in αvβ5 integrin receptors and mice deficient in the secreted integrin ligand MFG-E8. Results: Both β5^–/– and MFG-E8^–/– mice exhibit the same phagocytic defect: RPE cells retain basal uptake activity but completely lack the burst of phagocytic activity as well as the rhythmic activation of Mer tyrosine kinase that follow circadian photoreceptor shedding in wild-type RPE. Strikingly, electroretinogram photoresponses decline with age only in β5^–/– but not in MFG-E8^–/– retina. Conclusion: These results identify a critical role of αvβ5 integrin receptors and their ligand MFG-E8 in synchronizing retinal phagocytosis. Additionally, we show that lack of αvβ5 receptors and MFG-E8 ligand have distinct consequences for retinal function. These intriguing results suggest that loss of phagocytic rhythm is not solely responsible for the age-related blindness of β5^–/– mice.

Novel role for αvβ5-integrin in retinal adhesion and its diurnal peak

αvβ5-Integrin is the sole integrin receptor at the retinal pigment epithelium (RPE)-photoreceptor interface and promotes RPE phagocytic signaling to the tyrosine kinase Mer tyrosine kinase (MerTK) once a day in response to circadian photoreceptor shedding. Herein we identify a novel role for αvβ5-integrin in permanent RPE-photoreceptor adhesion that is independent of αvβ5's function in retinal phagocytosis. To compare retinal adhesion of wild-type and β 5 -integrin −/− mice, we mechanically separated RPE and neural retina and quantified RPE protein and pigment retention with the neural retina. Lack of αvβ5-integrin with normal expression of other RPE integrins greatly weakened retinal adhesion in young mice and accelerated its age-dependent decline. Unexpectedly, the strength of wild-type retinal adhesion varied with a diurnal rhythm that peaked 3.5 h after light onset, after the completion of phagocytosis, when integrin signaling to MerTK is minimal. Permanent αvβ5 receptor deficiency attenuated the diurnal peak of retinal adhesion in β 5 -integrin −/− mice. These results identify αvβ5-integrin as the first RPE receptor that contributes to retinal adhesion, a vital mechanism for long-term photoreceptor function and viability. Furthermore, they indicate that αvβ5 receptors at the same apical plasma membrane domain of RPE cells fulfill two separate functions that are synchronized by different diurnal rhythms.

Loss of Synchronized Retinal Phagocytosis and Age-related Blindness in Mice Lacking αvβ5 Integrin

Daily phagocytosis by the retinal pigment epithelium (RPE) of spent photoreceptor outer segment fragments is critical for vision. In the retina, early morning circadian photoreceptor rod shedding precedes synchronized uptake of shed photoreceptor particles by RPE cells. In vitro, RPE cells use the integrin receptor αvβ5 for particle binding. Here, we tested RPE phagocytosis and retinal function in β5 integrin–deficient mice, which specifically lack αvβ5 receptors. Retinal photoresponses severely declined with age in β5−/− mice, whose RPE accumulated autofluorescent storage bodies that are hallmarks of human retinal aging and disease. β5−/− RPE in culture failed to take up isolated photoreceptor particles. β5−/− RPE in vivo retained basal uptake levels but lacked the burst of phagocytic activity that followed circadian photoreceptor shedding in wild-type RPE. Rhythmic activation of focal adhesion and Mer tyrosine kinases that mediate wild-type retinal phagocytosis was also completely absent in β5−/− retina. These results demonstrate an essential role for αvβ5 integrin receptors and their downstream signaling pathways in synchronizing retinal phagocytosis. Furthermore, they identify the β5−/− integrin mouse strain as a new animal model of age-related retinal dysfunction.

Docosahexaenoic acid increases in frog retinal pigment epithelium following rod photoreceptor shedding

The vertebrate retina conserves docosahexaenoic acid (22:6n-3) during n-3 fatty acid deficiency. The mechanism of conservation is not known, although recycling of this fatty acid between the retinal pigment epithelium (RPE) and retina is one possibility. We examined the role of the RPE in conservation of 22:6n-3 by quantitating the fatty acids and phospholipid molecular species (PLMS) in frog RPE before and after light-stimulated shedding of rod outer segments (ROS). RPE cells were dissociated with brush agitation and purified by a discontinuous ficoll density gradient. One hour after the light-induced shedding of ROS, the phagocytosed ROS tip and opsin content of RPE had increased. Simultaneously, the levels of 22:6n-3 and 22:6(n-3)-containing PLMS were increased in the RPE. Within 8 hr following the shedding event, 22:6n-3 in the RPE had returned to the dark level. These findings indicate that the phagocytosed ROS tips contain 22:6n-3 and that the RPE metabolizes these ROS tips and eliminates 22:6n-3 from the cell. Thus, the RPE is intimately involved in the metabolism of 22:6n-3 in the retina. The recycling of 22:6n-3 from the RPE to the retina is a possible means of conserving this important fatty acid in the retina.

Increased levels of leukotriene C4in retinal pigment epithelium are correlated with early events in photoreceptor shedding inXenopus laevis

The levels of 5-lipoxygenase products of arachidonic acid, leukotriene (LT) B4 and LTC4 in retinal pigment epithelia (RPE) from Xenopus laevis were measured by radioimmunoassay (RIA). RPE were isolated during various stages of photoreceptor renewal to determine possible alterations in 5-lipoxygenase activity concurrent with photoreceptor detachment and phagocytosis. Both LTC4 and LTB4 were released to RPE incubation media, although levels of LTB4 in unstimulated RPE were close to the limits of detection by RIA. Incubation of RPE with the calcium ionophore A23187 increased the levels of both LTB4 and LTC4. When animals were maintained on a cycle of 12 hr light/dark, normal photoreceptor shedding, as measured by histological quantitation of the appearance of phagosomes in the RPE, occurred 1 hr after light onset. Levels of LTC4 in RPE were lower 1 hr after light onset, as compared to 1 hr prior to light onset. Due to the low levels of LTB4, no significant differences could be detected. However, when LTB4 levels were elevated with A23187, LTB4 also declined 1 hr after light onset. When animals were maintained in constant light for 5 days, then exposed to 2 hr dark and 2 hr light, a massive shedding response occurred. Levels of LTC4 were stimulated 5 min after light onset (prior to detectable shedding) and declined below dark levels as shedding progressed. These data suggest a correlation between 5-lipoxygenase activity and the events of photoreceptor shedding and phagocytosis.

Cone photoreceptor shedding in the tree shrew (Tupaia belangerii)

Tree shrews were sacrificed at various times during a 12 h light-12 h dark cycle and the retinal pigment epithelium (RPE) was examined for phagosomes. Analysis of photoreceptor densities showed that the tree-shrew retina consists of approximately 96% cone photoreceptors. Therefore, phagosomes in the RPE were assumed to be mostly those of cones. A peak in the number of RPE phagosomes was found about one hour after the onset of light. The number of phagosomes/mm RPE during the light cycle varied from 17.02 at the peak to 2.49 ten hours after light onset. During the dark cycle, values ranged from 0.10 to 0.61 phagosomes/mm RPE. Size profiles of phagosomes showed that large phagosomes peak in number 1/2 h after light onset, while smaller sizes peak at about 1 h after light onset. This may indicate that maximal shedding and phagocytotic activity occurs sometime before the peak in the total number of phagosomes is reached. Statistical corrections for phagosome size, section thickness and phagosomal degradation time were applied to the data in order to assess outer segment renewal time for tree shrew cones.

Photoreceptor shedding can be initiated within the eye

IN the vertebrate retina the light-sensitive rod outer segments (ROS) are composed of stacks of double membrane ‘disks’ surrounded by a plasma membrane. Throughout life, the ROS membranes are renewed in a highly ordered manner which has made this cell type the model of choice for many studies on membrane synthesis and turnover. Autoradiographical studies have shown that new membrane precursors are synthesised within the rod inner segment, then assembled into new disks at the base of the ROS, displacing previously formed disks away from the cell body1,2. An optimum ROS length is maintained by the periodic shedding of groups of older disks from the ROS tip. These discarded disk packets are then engulfed and degraded by the adjacent pigment epithelium3,4. The shed ROS fragments persist for several hours within the cytoplasm of the pigment epithelial cells and are referred to as phagosomes.3 LaVail5 has reported that ROS in the rat retina shed their apical tips shortly after the onset of light in animals maintained under cyclic lighting (12h light (L):12h dark (D)). We have shown that a similar pattern of rod outer segment shedding occurs in the amphibian retina6–8, and similar patterns have also been observed in the goldfish9, rabbit10 and chick11. In all these animals, a nearly synchronous burst of ROS shedding occurs within two hours of the onset of light, and minimal amounts of rod shedding occur during the remainder of the day. Also, in amphibians, constant light inhibits daily rod shedding, and the outer segments elongate due to continued outer segment disk addition7,12,13. In the rat5 and Xenopus7, shedding occurs at approximately the same time each day in animals maintained for several days in the dark, although in Rana the onset of light is required to initiate shedding, and dark-maintained animals do not shed for several days6. Taken together, these results have suggested that ROS shedding follows a circadian rhythm related to the onset of the light cycle. The photoreceptive nature of the amphibian pineal gland, and the well-documented circadian rhythm in serotonin–melatonin metabolism in the rat pineal, made this organ a particularly good candidate for controlling photoreceptor shedding, although several other extraocular systems have been suggested14. However, several laboratories have demonstrated that pinealectomy, hypophysectomy, superior cervical ganglionectomy, and cutting of the optic nerves have no effect on the diurnal pattern of ROS shedding in either the rat or the frog retina12,15. Furthermore, we have been unable to affect rod shedding in the frog retina by direct injection of melatonin, serotonin or the β-adrenergic effectors propranolol and iso-prenaline. Thus, even though rod outer segment shedding consistently occurs in both eyes and seems to follow a circadian rhythm in darkness, suggesting some form of central regulation, the intraocular control of this response remained a possibility. We report here experiments which strongly suggest that rod shedding in frogs can be initiated within the eye and is not secondarily controlled by a gland or organ elsewhere in the body.

Photoreceptor shedding is initiated by light in the frog retina.

Frogs maintained on a diurnal light-dark cycle (14 hours light and 10 hours darkness) shed their rod photoreceptor outer segment tips shortly after the onset of light. Shedding is synchronous and occurs in about 25 percent of the rod photoreceptors each day. Prolonged exposure to total darkness decreases the amount of shedding, after which exposure to light results in a large burst of synchronous shedding. Thus in the frog retina, the synchronous shedding of rod outer segment tips is shown to be directly related to light stimulation.