Lipocalin 2 Plays an Important Role in Regulating Inflammation in Retinal Degeneration.

It has become increasingly important to understand how retinal inflammation is regulated because inflammation plays a role in retinal degenerative diseases. Lipocalin 2 (LCN2), an acute stress response protein with multiple innate immune functions, is increased in ATP-binding cassette subfamily A member 4 (Abca4) -/- retinol dehydrogenase 8 (Rdh8) -/- double-knockout mice, an animal model for Stargardt disease and age-related macular degeneration (AMD). To examine roles of LCN2 in retinal inflammation and degeneration, Lcn2-/-Abca4-/-Rdh8-/- triple-knockout mice were generated. Exacerbated inflammation following light exposure was observed in Lcn2-/-Abca4-/-Rdh8-/- mice as compared with Abca4-/-Rdh8-/- mice, with upregulation of proinflammatory genes and microglial activation. RNA array analyses revealed an increase in immune response molecules such as Ccl8, Ccl2, and Cxcl10 To further probe a possible regulatory role for LCN2 in retinal inflammation, we examined the in vitro effects of LCN2 on NF-κB signaling in human retinal pigmented epithelial (RPE) cells differentiated from induced pluripotent stem cells derived from healthy donors. We found that LCN2 induced expression of antioxidant enzymes heme oxygenase 1 and superoxide dismutase 2 in these RPE cells and could inhibit the cytotoxic effects of H2O2 and LPS. ELISA revealed increased LCN2 levels in plasma of patients with Stargardt disease, retinitis pigmentosa, and age-related macular degeneration as compared with healthy controls. Finally, overexpression of LCN2 in RPE cells displayed protection from cell death. Overall these results suggest that LCN2 is involved in prosurvival responses during cell stress and plays an important role in regulating inflammation during retinal degeneration.

A2E-associated cell death and inflammation in retinal pigmented epithelial cells from human induced pluripotent stem cells.

Accumulation of lipofuscin in the retinal pigmented epithelium (RPE) is observed in retinal degenerative diseases including Stargardt disease and age-related macular degeneration. Bis-retinoid N-retinyl-N-retinylidene ethanolamine (A2E) is a major component of lipofuscin. A2E has been implicated in RPE atrophy and retinal inflammation; however, mice with A2E accumulation display only a mild retinal phenotype. In the current study, human iPSC-RPE (hiPSC-RPE) cells were generated from healthy individuals to examine effects of A2E in human RPE cells. hiPSC-RPE cells displayed RPE-specific features, which include expression of RPE-specific genes, tight junction formation and ability to carry out phagocytosis. hiPSC-RPE cells demonstrated cell death and increased VEGF-A production in a time-dependent manner when they were cocultured with 10µM of A2E. PCR array analyses revealed upregulation of 26 and 12 pro-inflammatory cytokines upon A2E and H2O2 exposure respectively, indicating that A2E and H2O2 can cause inflammation in human retinas. Notably, identified gene profiles were different between A2E- and H2O2- treated hiPSC-RPE cells. A2E caused inflammatory changes observed in retinal degenerative diseases more closely as compared to H2O2. Collectively, these data obtained with hiPSC-RPE cells provide evidence that A2E plays an important role in pathogenesis of retinal degenerative diseases in humans.

Docosahexaenoic acid promotes differentiation of photoreceptor cells in three-dimensional neural retinas.

Retinal tissues generated from human pluripotent stem cells can be an excellent tool for investigating pathogenesis of retinal diseases and developing new pharmacologic therapies. Moreover, patient derived retinal tissues could allow for retinal transplantation therapy for degenerative retinal diseases. However, obtaining retinal tissues with matured photoreceptor outer segments, which are essential for photoreceptor functions, is currently challenging. Here we investigated the effects of docosahexaenoic acid (DHA) for maturation of photoreceptor outer segments at the late stage and visual chromophore analog, 9-cis-retinal for the early stage of differentiation to three-dimensional (3D)-retinal tissues from human embryonic stem cells (hESCs), respectively. In the presence of DHA, differentiated 3D-retinal tissues demonstrated improved maturation of photoreceptor outer segments and increased number of photoreceptor cells compared with tissues without DHA. Increased mRNA expression of mature photoreceptor markers was additionally documented in retinal tissues cultured with DHA. Conversely supplementation with 9-cis-retinal failed to improve differentiation of retinal tissues perhaps due to chronic aldehyde toxicity. The current study demonstrated that the addition of DHA to culture medium can help promote differentiation of photoreceptor outer segments in vitro and utilization of this methodology may lead to future therapies for patients with blinding diseases.

Receptor MER Tyrosine Kinase Proto-oncogene (MERTK) Is Not Required for Transfer of Bis-retinoids to the Retinal Pigmented Epithelium.

Accumulation of bis-retinoids in the retinal pigmented epithelium (RPE) is a hallmark of aging and retinal disorders such as Stargardt disease and age-related macular degeneration. These aberrant fluorescent condensation products, including di-retinoid-pyridinium-ethanolamine (A2E), are thought to be transferred to RPE cells primarily through phagocytosis of the photoreceptor outer segments. However, we observed by two-photon microscopy that mouse retinas incapable of phagocytosis due to a deficiency of the c-Mer proto-oncogene tyrosine kinase (Mertk) nonetheless contained fluorescent retinoid condensation material in their RPE. Primary RPE cells from Mertk-/- mice also accumulated fluorescent products in vitro Finally, quantification of A2E demonstrated the acquisition of retinal condensation products in Mertk-/- mouse RPE prior to retinal degeneration. In these mice, we identified activated microglial cells that likely were recruited to transport A2E-like condensation products to the RPE and dispose of the dying photoreceptor cells. These observations demonstrate a novel transport mechanism between photoreceptor cells and RPE that does not involve canonical Mertk-dependent phagocytosis.

Acute Stress Responses Are Early Molecular Events of Retinal Degeneration in Abca4-/-Rdh8-/- Mice After Light Exposure.

PURPOSE: Mice lacking ATP-binding cassette transporter 4 (ABCA4) and retinol dehydrogenase 8 (RDH8) mimic features of human Stargardt disease and age-related macular degeneration. RNA-sequencing of whole eyes was done to study early gene expression changes in Abca4-/-Rdh8-/- mice. METHODS: Abca4-/-Rdh8-/- mice at 4 weeks of age were exposed to intense light. Total RNA was extracted from whole eyes and used to generate RNA libraries that were paired-end sequenced on the Illumina HiSeq 2500 device. Differentially expressed genes were annotated using Gene set enrichment analysis (GSEA). Selected genes in enriched pathways exhibiting differential expression were validated using quantitative qRT-PCR and ELISA. RESULTS: Transcriptome analysis of the whole eye identified 200 genes that were differentially expressed 24 hours after light exposure compared to no light in Abca4-/-Rdh8-/- mice. Expression of several visual cycle and photoreceptor genes were decreased, indicative of photoreceptor/RPE cell death. Gene categories of early stress response genes, inflammatory cytokines, immune factors, and JAK STAT components were upregulated. Lipocalin 2 (Lcn2) was the most upregulated early stress response gene identified. Protein LCN2 was produced by RPE cells and the neural retina after intense light exposure as well as in cultured RPE cells from mice and humans incubated with lipopolysaccharide or photoreceptor outer segments. CONCLUSIONS: Identification of important mediators involved in the crosstalk between the acute stress response and immune activation in RPE cells and the neural retina, such as LCN2, provide novel molecular targets for reducing cellular stress during retinal degeneration.

Autophagy in light-induced retinal damage.

Vision is reliant upon converting photon signals to electrical information which is interpreted by the brain and therefore allowing us to receive information about our surroundings. However, when exposed to excessive light, photoreceptors and other types of cells in the retina can undergo light-induced cell death, termed light-induced retinal damage. In this review, we summarize our current knowledge regarding molecular events in the retina after excessive light exposure and mechanisms of light-induced retinal damage. We also introduce works which investigate potential roles of autophagy, an essential cellular mechanism required for maintaining homeostasis under stress conditions, in the illuminated retina and animal models of light-induced retinal damage.

Di-retinoid-pyridinium-ethanolamine (A2E) Accumulation and the Maintenance of the Visual Cycle Are Independent of Atg7-mediated Autophagy in the Retinal Pigmented Epithelium.

Autophagy is an evolutionarily conserved catabolic mechanism that relieves cellular stress by removing/recycling damaged organelles and debris through the action of lysosomes. Compromised autophagy has been implicated in many neurodegenerative diseases, including retinal degeneration. Here we examined retinal phenotypes resulting from RPE-specific deletion of the autophagy regulatory gene Atg7 by generating Atg7(flox/flox);VMD2-rtTA-cre+ mice to determine whether autophagy is essential for RPE functions including retinoid recycling. Atg7-deficient RPE displayed abnormal morphology with increased RPE thickness, cellular debris and vacuole formation indicating that autophagy is important in maintaining RPE homeostasis. In contrast, 11-cis-retinal content, ERGs and retinal histology were normal in mice with Atg7-deficient RPE in both fasted and fed states. Because A2E accumulation in the RPE is associated with pathogenesis of both Stargardt disease and age-related macular degeneration (AMD) in humans, deletion of Abca4 was introduced into Atg7(flox/flox);VMD2-rtTA-cre+ mice to investigate the role of autophagy during A2E accumulation. Comparable A2E concentrations were detected in the eyes of 6-month-old mice with and without Atg7 from both Abca4(-/-) and Abca4(+/+) backgrounds. To identify other autophagy-related molecules involved in A2E accumulation, we performed gene expression array analysis on A2E-treated human RPE cells and found up-regulation of four autophagy related genes; DRAM1, NPC1, CASP3, and EIF2AK3/PERK. These observations indicate that Atg7-mediated autophagy is dispensable for retinoid recycling and A2E deposition; however, autophagy plays a role in coping with stress caused by A2E accumulation.

Conditional Ablation of Retinol Dehydrogenase 10 in the Retinal Pigmented Epithelium Causes Delayed Dark Adaption in Mice.

Regeneration of the visual chromophore, 11-cis-retinal, is a crucial step in the visual cycle required to sustain vision. This cycle consists of sequential biochemical reactions that occur in photoreceptor cells and the retinal pigmented epithelium (RPE). Oxidation of 11-cis-retinol to 11-cis-retinal is accomplished by a family of enzymes termed 11-cis-retinol dehydrogenases, including RDH5 and RDH11. Double deletion of Rdh5 and Rdh11 does not limit the production of 11-cis-retinal in mice. Here we describe a third retinol dehydrogenase in the RPE, RDH10, which can produce 11-cis-retinal. Mice with a conditional knock-out of Rdh10 in RPE cells (Rdh10 cKO) displayed delayed 11-cis-retinal regeneration and dark adaption after bright light illumination. Retinal function measured by electroretinogram after light exposure was also delayed in Rdh10 cKO mice as compared with controls. Double deletion of Rdh5 and Rdh10 (cDKO) in mice caused elevated 11/13-cis-retinyl ester content also seen in Rdh5(-/-)Rdh11(-/-) mice as compared with Rdh5(-/-) mice. Normal retinal morphology was observed in 6-month-old Rdh10 cKO and cDKO mice, suggesting that loss of Rdh10 in the RPE does not negatively affect the health of the retina. Compensatory expression of other retinol dehydrogenases was observed in both Rdh5(-/-) and Rdh10 cKO mice. These results indicate that RDH10 acts in cooperation with other RDH isoforms to produce the 11-cis-retinal chromophore needed for vision.

Supplementation with vitamin a derivatives to rescue vision in animal models of degenerative retinal diseases.

The perception of light begins when photons reach retinal tissue located at the back of the eye and photoisomerize the visual chromophore 11-cis-retinal to all-trans-retinal within photoreceptor cells. Isomerization of 11-cis-retinal activates the protein rhodopsin located in photoreceptor outer segments, thereby inducing a phototransduction cascade leading to visual perception. To maintain vision, 11-cis-retinal is regenerated in the retinal pigmented epithelium (RPE) via the visual cycle and delivered back to the photoreceptor cells where it may again bind to rhodopsin. Distinct pathological mechanisms have been observed to contribute to inherited retinal degenerative diseases including severe delay in 11-cis-retinal regeneration and delayed clearance of all-trans-retinal, which leads to the accumulation of harmful retinoid by-products. In the last decade, our group has conducted several proof-of-concept (POC) studies with retinoid derivatives aimed at developing treatments for retinal degenerative diseases caused by an impaired visual cycle. Here, we will introduce experimental procedures, which have been developed for POC studies involving retinoid biology.

All-trans-retinal induces Bax activation via DNA damage to mediate retinal cell apoptosis.

The current study investigates the cellular events which trigger activation of proapoptotic Bcl-2-associated × protein (Bax) in retinal cell death induced by all-trans-retinal (atRAL). Cellular events which activate Bax, such as DNA damage by oxidative stress and phosphorylation of p53, were evaluated by immunochemical and biochemical methods using ARPE-19 cells, 661 W cells, cultured neural retinas and a retinal degeneration model, Abca4(-/-)Rdh8(-/-) mice. atRAL-induced Bax activation in cultured neural retinas was examined by pharmacological and genetic methods. Other Bax-related cellular events were also evaluated by pharmacological and biochemical methods. Production of 8-OHdG, a DNA damage indicator, and the phosphorylation of p53 at Ser46 were detected prior to Bax activation in ARPE-19 cells incubated with atRAL. Light exposure to Abca4(-/-)Rdh8(-/-) mice also caused the above mentioned events in conditions of short term intense light exposure and regular room lighting conditions. Incubation with Bax inhibiting peptide and deletion of the Bax gene partially protected retinal cells from atRAL toxicity in cultured neural retina. Necrosis was demonstrated not to be the main pathway in atRAL mediated cell death. Bcl-2-interacting mediator and Bcl-2 expression levels were not altered by atRAL in vitro. atRAL-induced oxidative stress results in DNA damage leading to the activation of Bax by phosphorylated p53. This cascade is closely associated with an apoptotic cell death mechanism rather than necrosis.

CCL3 production by microglial cells modulates disease severity in murine models of retinal degeneration.

Many degenerative retinal diseases illustrate retinal inflammatory changes that include infiltration of microglia and macrophages into the subretinal space. In this study, we examined the role of chemokines in the Abca4(-/-)Rdh8(-/-) mouse model of Stargardt disease and the Mertk(-/-) mouse model of retinitis pigmentosa. PCR array analysis of 84 chemokines and related molecules revealed 84.6-fold elevated expression of Ccl3 (MIP-1a) 24 h after light exposure in Abca4(-/-)Rdh8(-/-) mice. Only MIP-1 chemokines, including Ccl3 and Ccl4, displayed peak expression 24 h after light exposure, and peaked earlier than the other chemokines. Secretion of Ccl3 was documented only in microglia, whereas both microglia and retinal pigment epithelium cells produced Ccl2. Exposure of Cx3Cr1(gfp/Δ)Abca4(-/-)Rdh8(-/-) mice to intense light resulted in the appearance of Cx3Cr1GFP(+) monocytes in the subretinal space. To address the in vivo role of CCL3 in retinal degeneration, Ccl3(-/-)Abca4(-/-)Rdh8(-/-) mice and Ccl3(-/-)Mertk(-/-) mice were generated. Following intense light exposure, Ccl3(-/-)Abca4(-/-)Rdh8(-/-) mice displayed persistent retinal inflammation with appearance of Iba-1(+) cells in the subretinal space, severe photoreceptor cell death, and increased Ccl4 expression compared with Abca4(-/-)Rdh8(-/-) mice. In contrast, Ccl3(-/-)Abca4(-/-)Rdh8(-/-) mice exhibited a milder retinal inflammation and degeneration than Abca4(-/-)Rdh8(-/-) mice did in age-related chronic retinal degeneration under room light conditions. The deficiency of Ccl3 also attenuated the severity of retinal degeneration in Mertk(-/-) mice. Taken together, our results indicate that Ccl3 has an essential role in regulating the severity of retinal inflammation and degeneration in these mouse models.

Vitamin A derivatives as treatment options for retinal degenerative diseases.

The visual cycle is a sequential enzymatic reaction for vitamin A, all-trans-retinol, occurring in the outer layer of the human retina and is essential for the maintenance of vision. The central source of retinol is derived from dietary intake of both retinol and pro-vitamin A carotenoids. A series of enzymatic reactions, located in both the photoreceptor outer segment and the retinal pigment epithelium, transform retinol into the visual chromophore 11-cis-retinal, regenerating visual pigments. Retina specific proteins carry out the majority of the visual cycle, and any significant interruption in this sequence of reactions is capable of causing varying degrees of blindness. Among these important proteins are Lecithin:retinol acyltransferase (LRAT) and retinal pigment epithelium-specific 65-kDa protein (RPE65) known to be responsible for esterification of retinol to all-trans-retinyl esters and isomerization of these esters to 11-cis-retinal, respectively. Deleterious mutations in these genes are identified in human retinal diseases that cause blindness, such as Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). Herein, we discuss the pathology of 11-cis-retinal deficiency caused by these mutations in both animal disease models and human patients. We also review novel therapeutic strategies employing artificial visual chromophore 9-cis-retinoids which have been employed in clinical trials involving LCA patients.

Dietary 9-cis-ß,ß-carotene fails to rescue vision in mouse models of leber congenital amaurosis.

Synthetic 9-cis-stereoisomers of vitamin A (all-trans-retinol) are especially promising agents for the fight against blinding diseases. Several studies suggested that 9-cis-ß,ß-carotene (9-cis-BC), a natural and abundant ß-carotene isomer in the diet, could be the precursor of 9-cis-retinoids and thus could have therapeutic applications. Here we showed that 9-cis-BC is metabolized both in vitro and in vivo by two types of mouse carotenoid oxygenases, ß,ß-Carotene monooxygenase 1 (BCMO1), and ß,ß-carotene dioxygenase 2 (BCDO2). In the symmetric oxidative cleavage reaction at C15,C15' position by BCMO1, part of the 9-cis-double bond was isomerized to the all-trans-stereoisomer, yielding all-trans-retinal and 9-cis-retinal in a molar ratio of 3:1. The asymmetric cleaving enzyme BCDO2 preferentially removed the 9-cis-ring site at the C9,C10 double bond from this substrate, providing an all-trans-ß-10'-apocarotenal product that can be further metabolized to all-trans-retinal by BCMO1. Studies in knockout mouse models confirmed that each carotenoid oxygenase can metabolize 9-cis-BC. Therefore, treatment of mouse models of Leber congenital amaurosis with 9-cis-BC and 9-cis-retinyl-acetate, a well established 9-cis-retinal precursor, showed that the cis-carotenoid was far less effective than the cis-retinoid in rescuing vision. Thus, our in vitro and in vivo studies revealed that 9-cis-BC is not a major source for mouse 9-cis-retinoid production but is mainly converted to all-trans-retinoids to support canonical vitamin A action.