Many degenerative retinal diseases illustrate retinal inflammatory changes that include infiltration of microglia and macrophages into the subretinal space. In the current 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 RPE cells produced Ccl2. Exposure of Cx3Cr1gfp/ΔAbca4-/-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-positive 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 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.

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 () retinol dehydrogenase 8 () 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, triple-knockout mice were generated. Exacerbated inflammation following light exposure was observed in mice as compared with mice, with upregulation of proinflammatory genes and microglial activation. RNA array analyses revealed an increase in immune response molecules such as, , and 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 HO 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 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.

The current study investigates the cellular events which trigger activation of proapoptotic Bcl-2-associated X 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, 661W 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 Ser 46 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.

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.

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-␤,␤-carotene (9-cis-BC), a natural and abundant ␤-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, ␤,␤-Carotene monooxygenase 1 (BCMO1), and ␤,␤-carotene dioxygenase 2 (BCDO2). In the symmetric oxidative cleavage reaction at C15,C15Ј 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-␤-10Ј-apocarotenal product that can be further metabolized to alltrans-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 ciscarotenoid 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. IntroductionAn enzyme-based cyclic pathway for trans-to-cis isomerization of the visual pigment chromophore all-trans-retinal is intrinsic to mammalian retinal rod and cone vision. This pathway, called the visual or retinoid cycle, involves two cellular compartments, both rod and cone outer segments and closely associated retinal pigmented epithelium (RPE) . In addition, cones can be supported by a pathway involving Mü ller cells (Fleisch and Neuhauss, 2010;Wang and Kefalov, 2011). Genetic disruption of the visual cycle in mice results in rapid or slowly progressive death of rods and cones (Travis et al., 2007). For example, inactivating mutations in lecithin:retinol acyltransferase (LRAT) and retinoid isomerase [also known as RPE protein of 65 kDa (RPE65) that converts all-trans-retinyl esters to 11-cis-retinol] are associated with severe forms of retinitis pigmentosa (RP) including LCA in humans (Thompson and Gal, 2003). In contrast, perhaps because of redundancy in the redox system, mutations in retinol dehydrogenase 5, an enzyme responsible for oxidation of 11-cis-retinol to 11-cis-retinal, cause a mild form of retinal dysfunction called fundus albipunctatus and mild RP with slow dark adaptation (recovery of vision after illumination) and cone and rod degeneration (Travis et al., 2007;den Hollander et al., 2008).Substantial efforts have been undertaken to establish therapies for patients who have blinding diseases affecting the retina (for review see Palczewski 2010;den Hollander et a...

Background: RDH10 is a candidate 11-cis-retinol dehydrogenase in the retinal pigmented epithelium (RPE) capable of regenerating the visual chromophore, 11-cis-retinal. Results: Loss of Rdh10 in RPE cells caused delayed regeneration of 11-cis-retinal. Conclusion: Rdh10 is an NAD ϩ -dependent 11-cis-retinol dehydrogenase localized to RPE cells.

Background: Atg7 is an essential autophagic enzyme. Disrupted autophagy has been implicated in retinal degeneration. Results: Mice with RPE-specific Atg7 deletion exhibit normal retinoid recycling, histology, and A2E accumulation, but display hypertrophy and cytosolic debris. Conclusion: Atg7 deficiency does not severely affect the health of RPE cells in mice. Significance: A2E accumulation and retinoid recycling are independent of Atg7-mediated autophagy in RPE cells.

PurposeMice 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.MethodsAbca4−/−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.ResultsTranscriptome 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.ConclusionsIdentification 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.