Expression of opsin and visual cycle-related enzymes in fetal rat skin keratinocytes and cellular response to blue light.

The mechanism by which the skin, a non-visual tissue, responds to light remains unknown. To date, opsin expression has been demonstrated in keratinocytes, melanocytes, and fibroblasts, all of which are skin-derived cells. In this study, we examined whether the visual cycle, by which opsin activity is maintained, is present in skin keratinocytes. We also identified the wavelengths of light to which opsin in keratinocytes responds and explored their effects on skin keratinocytes. The fetal rat skin keratinocytes used in this study expressed OPN2, 3, and 5 in addition to enzymes involved in the visual cycle, and all-trans-retinal, which is produced by exposure to light, was reconverted to 11-cis-retinal, resulting in opsin activation. Using the production of all-trans-retinal after light exposure as an indicator, we discovered that keratinocytes responded to light at 450 nm. Furthermore, actin alpha cardiac muscle 1 expression in keratinocytes was enhanced and cell migration was suppressed by exposure to light at these wavelengths. These results indicate that keratinocytes express various opsins and have a visual cycle that keeps opsin active. Moreover, keratinocytes were shown to respond to the blue/UV region of the light spectrum, suggesting that opsin plays a role in the light response of the skin.

Long-term exposure to prometryn damages the visual system and changes color preference of female zebrafish (Danio rerio).

Color vision, initiated from the cone photoreceptors, is essential for fish to obtain environmental information. Although the visual impairment of triazine herbicide prometryn has been reported, data on the effect of herbicide such as prometryn on natural color sensitivity of fish is scarce. Here, zebrafish were exposed to prometryn (0, 1, 10, and 100 µg/L) from 2 h post-fertilization to 160 days post-fertilization, to explore the effect and underlying mechanism of prometryn on color perception. The results indicated that 10 and 100 µg/L prometryn shortened the height of red-green cone cells, and down-regulated expression of genes involved in light transduction pathways (arr3a, pde6h) and visual cycle (lrata, rpe65a); meanwhile, 1 µg/L prometryn increased all-trans-retinoic acid levels in zebrafish eyes, and up-regulated the expression of genes involved in retinoid metabolism (rdh10b, aldh1a2, cyp26a1), finally leading to weakened red and green color perception of female zebrafish. This study first clarified how herbicide such as prometryn affected color vision of a freshwater fish after a long-term exposure from both morphological and functional disruption, and its hazard on color vision mediated-ecologically relevant tasks should not be ignored.

RDH12 allows cone photoreceptors to regenerate opsin visual pigments from a chromophore precursor to escape competition with rods.

Capture of a photon by an opsin visual pigment isomerizes its 11-cis-retinaldehyde (11cRAL) chromophore to all-trans-retinaldehyde (atRAL), which subsequently dissociates. To restore light sensitivity, the unliganded apo-opsin combines with another 11cRAL to make a new visual pigment. Two enzyme pathways supply chromophore to photoreceptors. The canonical visual cycle in retinal pigment epithelial cells supplies 11cRAL at low rates. The photic visual cycle in Müller cells supplies cones with 11-cis-retinol (11cROL) chromophore precursor at high rates. Although rods can only use 11cRAL to regenerate rhodopsin, cones can use 11cRAL or 11cROL to regenerate cone visual pigments. We performed a screen in zebrafish retinas and identified ZCRDH as a candidate for the enzyme that converts 11cROL to 11cRAL in cone inner segments. Retinoid analysis of eyes from Zcrdh-mutant zebrafish showed reduced 11cRAL and increased 11cROL levels, suggesting impaired conversion of 11cROL to 11cRAL. By microspectrophotometry, isolated Zcrdh-mutant cones lost the capacity to regenerate visual pigments from 11cROL. ZCRDH therefore possesses all predicted properties of the cone 11cROL dehydrogenase. The human protein most similar to ZCRDH is RDH12. By immunocytochemistry, ZCRDH was abundantly present in cone inner segments, similar to the reported distribution of RDH12. Finally, RDH12 was the only mammalian candidate protein to exhibit 11cROL-oxidase catalytic activity. These observations suggest that RDH12 in mammals is the functional ortholog of ZCRDH, which allows cones, but not rods, to regenerate visual pigments from 11cROL provided by Müller cells. This capacity permits cones to escape competition from rods for visual chromophore in daylight-exposed retinas.

Dominant role for pigment epithelial CRALBP in supplying visual chromophore to photoreceptors.

Cellular retinaldehyde-binding protein (CRALBP) supports production of 11-cis-retinaldehyde and its delivery to photoreceptors. It is found in the retinal pigment epithelium (RPE) and Müller glia (MG), but the relative functional importance of these two cellular pools is debated. Here, we report RPE- and MG-specific CRALBP knockout (KO) mice and examine their photoreceptor and visual cycle function. Bulk visual chromophore regeneration in RPE-KO mice is 15-fold slower than in controls, accounting for their delayed rod dark adaptation and protection against retinal phototoxicity, whereas MG-KO mice have normal bulk visual chromophore regeneration and retinal light damage susceptibility. Cone pigment regeneration is significantly impaired in RPE-KO mice but mildly affected in MG-KO mice, disclosing an unexpectedly strong reliance of cone photoreceptors on the RPE-based visual cycle. These data reveal a dominant role for RPE-CRALBP in supporting rod and cone function and highlight the importance of RPE cell targeting for CRALBP gene therapies.

Reduced light exposure mitigates streptozotocin-induced vascular changes and gliosis in diabetic retina by an anti-inflammatory effect and increased retinal cholesterol turnover.

Diabetic retinopathy is not cured efficiently and changes of lifestyle measures may delay early retinal injury in diabetes. The aim of our study was to investigate the effects of reduced daily light exposure on retinal vascular changes in streptozotocin (STZ)-induced model of DM with emphasis on inflammation, Aqp4 expression, visual cycle and cholesterol metabolism-related gene expression in rat retina and RPE. Male Wistar rats were divided into the following groups: 1. control; 2. diabetic group (DM) treated with streptozotocin (100 mg/kg); 3. group exposed to light/dark cycle 6/18 h (6/18); 4. diabetic group exposed to light/dark cycle 6/18 h (DM+6/18). Retinal vascular abnormalities were estimated based on lectin staining, while the expression of genes involved in the visual cycle, cholesterol metabolism, and inflammation was determined by qRT-PCR. Reduced light exposure alleviated vasculopathy, gliosis and the expression of IL-1 and TNF-α in the retina with increased perivascular Aqp4 expression. The expression of genes involved in visual cycle and cholesterol metabolism was significantly up-regulated in RPE in DM+6/18 vs. DM group. In the retina only the expression of APOE was significantly higher in DM+6/18 vs. DM group. Reduced light exposure mitigates vascular changes and gliosis in DM via its anti-inflammatory effect, increased retinal cholesterol turnover and perivascular Aqp4 expression.

Retinylidene chromophore hydrolysis from mammalian visual and non-visual opsins.

Rhodopsin (Rho) and cone opsins are essential for detection of light. They respond via photoisomerization, converting their Schiff-base-adducted 11-cis-retinylidene chromophores to the all-trans configuration, eliciting conformational changes to activate opsin signaling. Subsequent Schiff-base hydrolysis releases all-trans-retinal, initiating two important cycles that maintain continuous vision-the Rho photocycle and visual cycle pathway. Schiff-base hydrolysis has been thoroughly studied with photoactivated Rho but not with cone opsins. Using established methodology, we directly measured the formation of Schiff-base between retinal chromophores with mammalian visual and nonvisual opsins of the eye. Next, we determined the rate of light-induced chromophore hydrolysis. We found that retinal hydrolysis from photoactivated cone opsins was markedly faster than from photoactivated Rho. Bovine retinal G protein-coupled receptor (bRGR) displayed rapid hydrolysis of its 11-cis-retinylidene photoproduct to quickly supply 11-cis-retinal and re-bind all-trans-retinal. Hydrolysis within bRGR in native retinal pigment epithelium microsomal membranes was >6-times faster than that of bRGR purified in detergent micelles. N-terminal-targeted antibodies significantly slowed bRGR hydrolysis, while C-terminal antibodies had no effect. Our study highlights the much faster photocycle of cone opsins relative to Rho and the crucial role of RGR in chromophore recycling in daylight. By contrast, in our experimental conditions, bovine peropsin did not form pigment in the presence of all-trans-retinal nor with any mono-cis retinal isomers, leaving uncertain the role of this opsin as a light sensor.

Updates on Emerging Interventions for Autosomal Recessive ABCA4-Associated Stargardt Disease.

Autosomal recessive Stargardt disease (STGD1) is an inherited retinal degenerative disease associated with a mutated ATP-binding cassette, subfamily A, member 4 (ABCA4) gene. STGD1 is the most common form of juvenile macular degeneration with onset in late childhood to early or middle adulthood and causes progressive, irreversible visual impairment and blindness. No effective treatment is currently available. In the present article, we review the most recent updates in clinical trials targeting the management of STGD1, including gene therapy, small molecule therapy, and stem cell therapy. In gene therapy, dual adeno-associated virus and non-viral vectors have been successful in delivering the human ABCA4 gene in preclinical studies. For pharmaceutical therapies ALK-001, deuterated vitamin A shows promise with preliminary data for phase 2 trial, demonstrating a decreased atrophy growth rate after two years. Stem cell therapy using human pluripotent stem cell-derived retinal pigment epithelium cells demonstrated long-term safety three years after implantation and visual acuity improvements in the first two years after initiation of therapy. Many other treatment options have ongoing investigations and clinical trials. While multiple potential interventions have shown promise in attenuating disease progression, further exploration is necessary to demonstrate treatment safety and efficacy.

Rapid RGR-dependent visual pigment recycling is mediated by the RPE and specialized Müller glia.

In daylight, demand for visual chromophore (11-cis-retinal) exceeds supply by the classical visual cycle. This shortfall is compensated, in part, by the retinal G-protein-coupled receptor (RGR) photoisomerase, which is expressed in both the retinal pigment epithelium (RPE) and in Müller cells. The relative contributions of these two cellular pools of RGR to the maintenance of photoreceptor light responses are not known. Here, we use a cell-specific gene reactivation approach to elucidate the kinetics of RGR-mediated recovery of photoreceptor responses following light exposure. Electroretinographic measurements in mice with RGR expression limited to either cell type reveal that the RPE and a specialized subset of Müller glia contribute both to scotopic and photopic function. We demonstrate that 11-cis-retinal formed through photoisomerization is rapidly hydrolyzed, consistent with its role in a rapid visual pigment regeneration process. Our study shows that RGR provides a pan-retinal sink for all-trans-retinal released under sustained light conditions and supports rapid chromophore regeneration through the photic visual cycle.

Human nutritional relevance and suggested nutritional guidelines for vitamin A5/X and provitamin A5/X.

In the last century, vitamin A was identified that included the nutritional relevant vitamin A1 / provitamin A1, as well as the vitamin A2 pathway concept. Globally, nutritional guidelines have focused on vitamin A1 with simplified recommendations and calculations based solely on vitamin A. The vitamin A / provitamin A terminology described vitamin A with respect to acting as a precursor of 11-cis-retinal, the chromophore of the visual pigment, as well as retinoic acid(s), being ligand(s) of the nuclear hormone receptors retinoic acid receptors (RARs) α, ß and γ. All-trans-retinoic acid was conclusively shown to be the endogenous RAR ligand, while the concept of its isomer 9-cis-retinoic acid, being "the" endogenous ligand of the retinoid-X receptors (RXRs), remained inconclusive. Recently, 9-cis-13,14-dihydroretinoic acid was conclusively reported as an endogenous RXR ligand, and a direct nutritional precursor was postulated in 2018 and further confirmed by Rühl, Krezel and de Lera in 2021. This was further termed vitamin A5/X / provitamin A5/X. In this review, a new vitamin A5/X / provitamin A5/X concept is conceptualized in parallel to the vitamin A(1) / provitamin A(1) concept for daily dietary intake and towards dietary guidelines, with a focus on the existing national and international regulations for the physiological and nutritional relevance of vitamin A5/X. The aim of this review is to summarize available evidence and to emphasize gaps of knowledge regarding vitamin A5/X, based on new and older studies and proposed future directions as well as to stimulate and propose adapted nutritional regulations.

Environmental Light Has an Essential Effect on the Disease Expression in a Dominant RPE65 Mutation.

The retina pigmented epithelium 65 kDa protein (RPE65) is an essential enzyme in the visual cycle that regenerates the 11-cis-retinal chromophore obligatory for vision. Mutations in RPE65 are associated with blinding diseases. D477G (C.1430G > A) is the only known RPE65 variant to cause autosomal dominant retinitis pigmentosa (adRP). Previously, we reported that the heterozygous D477G knock-in (WT/KI) mice exposed to dim light intensity demonstrated delayed chromophore regeneration rates and slowed recovery of photoreceptor sensitivity following photobleaching. However, visual function and retinal architecture were indistinguishable from the wild-type (WT) mice. In this study, when maintained under the physiological day-light intensity (2 K lux), the WT/KI heterozygous mice displayed retina degeneration and reduced electroretinography (ERG) amplitude, recapitulating that observed in human patients. Our findings indicated the importance of the light environment in the mechanism of RPE65 D477G pathogenicity.

The Amphipathic Helix in Visual Cycle Proteins: A Review.

The visual cycle is a complex biological process that involves the sequential action of proteins in the retinal pigment epithelial (RPE) cells and photoreceptors to modify and shuttle visual retinoids. A majority of the visual cycle proteins are membrane proteins, either integral or peripheral membrane proteins. Despite significant progress in understanding their physiological function, very limited structural information is available for the visual cycle proteins. Moreover, the mechanism of membrane interaction is not yet clear in all cases. Here, we demonstrate the presence of an amphipathic helix in selected RPE visual cycle proteins, using in silico tools, and highlight their role in membrane association and function.

Congenital cataracts affect the retinal visual cycle and mitochondrial function: A multi-omics study of GJA8 knockout rabbits.

Congenital cataracts are a threat to visual development in children, and the visual impairment persists after surgical treatment; however, the mechanisms involved remain unclear. Previous clinical studies have identified the effect of congenital cataracts on retinal morphology and function. To further understand the molecular mechanisms by which congenital cataracts affect retinal development, we analyzed retina samples from 7-week-old GJA8-knockout rabbits with congenital cataracts and controls by four-dimensional label-free quantification proteomics and untargeted metabolomics. Bioinformatics analysis of proteomic data showed that retinol metabolism, oxidative phosphorylation, and fatty acid degradation pathways were downregulated in the retinas of rabbits with congenital cataracts, indicating that their visual cycle and mitochondrial function were affected. Additional validation of differentially abundant proteins related to the visual cycle and mitochondrial function was performed using Parallel reaction monitoring and western blot experiments. Untargeted metabolome analysis showed significant upregulation of the antioxidant glutathione and ascorbic acid in the retinas of rabbits with congenital cataracts, indicating that their oxidative stress balance was not dysregulated. SIGNIFICANCE: Congenital cataracts in children can alter retinal structure and function, yet the mechanisms are uncertain. Here is the first study to use proteomics and metabolomics approaches to investigate the effects of congenital cataracts on retinal development in the early postnatal period. Our findings suggest that congenital cataracts have an impact on the retinal visual cycle and mitochondrial function. These findings give insight on the molecular pathways behind congenital cataract-induced visual function impairment in the early postnatal period.

Analysis of Retinol Binding Protein 4 and ABCA4 Gene Variation in Non-Neovascular Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) may be associated with ABCA4 variants and is characterized by the accumulation of visual cycle-byproduct lipofuscin. Reducing retinol-binding protein 4 (RBP4), a retinol transporter protein, may reduce lipofuscin production. This study aims to assess the associations between plasma RBP4, the ABCA4 variation, and AMD severity. Sixty-seven participants were grouped into healthy/mild AMD (n = 32) and severe AMD (n = 35) groups. The latter group was older than the former group and had higher levels of RBP4 (36.8 ± 8.3 vs. 30.4 ± 7.0 µg/mL, p = 0.0012). The ten participants with six ABCA4 linked-variants had higher RBP4 than those without (37.8 ± 7.7 vs. 32.4 ± 7.9 µg/mL; p = 0.026), and eight of them had severe AMD. Univariate analyses showed that severe AMD was related to older age (OR, 1.26; 95% CI, 1.13-1.40; p < 0.0001) and to higher RBP4 levels (OR, 1.12; 95% CI, 1.04-1.20; p = 0.003), whereas the linked ABCA4 variants had no associations. After adjustment, however, only age remained significantly associated with severe AMD. This pilot study shows a trend of higher plasma RBP4 levels in severe AMD or the ABCA4-linked variants, and further age-matched studies are warranted.

Chromophore supply modulates cone function and survival in retinitis pigmentosa mouse models.

Retinitis pigmentosa (RP) is an ocular disease characterized by the loss of night vision, followed by the loss of daylight vision. Daylight vision is initiated in the retina by cone photoreceptors, which are gradually lost in RP, often as bystanders in a disease process that initiates in their neighboring rod photoreceptors. Using physiological assays, we investigated the timing of cone electroretinogram (ERG) decline in RP mouse models. A correlation between the time of loss of the cone ERG and the loss of rods was found. To investigate a potential role of the visual chromophore supply in this loss, mouse mutants with alterations in the regeneration of the retinal chromophore, 11-cis retinal, were examined. Reducing chromophore supply via mutations in Rlbp1 or Rpe65 resulted in greater cone function and survival in a RP mouse model. Conversely, overexpression of Rpe65 and Lrat, genes that can drive the regeneration of the chromophore, led to greater cone degeneration. These data suggest that abnormally high chromophore supply to cones upon the loss of rods is toxic to cones, and that a potential therapy in at least some forms of RP is to slow the turnover and/or reduce the level of visual chromophore in the retina.

Retinal-phospholipid Schiff-base conjugates and their interaction with ABCA4, the ABC transporter associated with Stargardt disease.

N-retinylidene-phosphatidylethanolamine (N-Ret-PE), the Schiff-base conjugate formed through the reversible reaction of retinal (Vitamin A-aldehyde) and phosphatidylethanolamine, plays a crucial role in the visual cycle and visual pigment photoregeneration. However, N-Ret-PE can react with another molecule of retinal to form toxic di-retinoids if not removed from photoreceptors through its transport across photoreceptor membranes by the ATP-binding-cassette transporter ABCA4. Loss-of-function mutations in ABCA4 are known to cause Stargardt disease (STGD1), an inherited retinal degenerative disease associated with the accumulation of fluorescent di-retinoids and severe loss in vision. A larger assessment of retinal-phospholipid Schiff-base conjugates in photoreceptors is needed, along with further investigation of ABCA4 residues important for N-Ret-PE binding. In this study we show that N-Ret-PE formation is dependent on pH and phospholipid content. When retinal is added to liposomes or photoreceptor membranes, 40 to 60% is converted to N-Ret-PE at physiological pH. Phosphatidylserine and taurine also react with retinal to form N-retinylidene-phosphatidylserine and N-retinylidene-taurine, respectively, but at significantly lower levels. N-retinylidene-phosphatidylserine is not a substrate for ABCA4 and reacts poorly with retinal to form di-retinoids. Additionally, amino acid residues within the binding pocket of ABCA4 that contribute to its interaction with N-Ret-PE were identified and characterized using site-directed mutagenesis together with functional and binding assays. Substitution of arginine residues and hydrophobic residues with alanine or residues implicated in STGD1 significantly reduced or eliminated substrate-activated ATPase activity and substrate binding. Collectively, this study provides important insight into conditions which affect retinal-phospholipid Schiff-base formation and mechanisms underlying the pathogenesis of STGD1.

Genetic hyperactivation of Nrf2 causes larval lethality in Keap1a and Keap1b-double-knockout zebrafish.

The Keap1-Nrf2 pathway is an evolutionarily conserved mechanism that protects cells from oxidative stress and electrophiles. Keap1 is a repressor of Nrf2 in normal cellular conditions but also a stress sensor for Nrf2 activation. Interestingly, fish and amphibians have two Keap1s (Keap1a and Keap1b), of which Keap1b is the ortholog of mammalian Keap1. Keap1a, on the other hand, is a gene found only in fish and amphibians, having been lost during the evolution to amniotes. We have previously shown that keap1b-knockout zebrafish have increased Nrf2 activity and reduced response to certain Nrf2-activating compounds but that they grow normally to adulthood. This may be because the remaining keap1a suppresses the hyperactivation of Nrf2, which is responsible for the post-natal lethality of Keap1-knockout mice. In this study, we analyzed keap1a;keap1b-double-knockout zebrafish to test this hypothesis. We found that keap1a;keap1b-double-knockout zebrafish, like Keap1-knockout mice, showed eating defects and were lethal within a week of hatching. Genetic introduction of the Nrf2 mutation rescued both the eating defects and the larval lethality, indicating that Nrf2 hyperactivation is the cause. However, unlike Keap1-knockout mice, keap1a;keap1b-double-knockout zebrafish showed no physical blockage of the food pathway; moreover, the cause of death was not directly related to eating defects. RNA-sequencing analysis revealed that keap1a;keap1b-double-knockout larvae showed extraordinarily high expression of known Nrf2-target genes as well as decreased expression of visual cycle genes. Finally, trigonelline or brusatol partially rescued the lethality of keap1a;keap1b-double-knockout larvae, suggesting that they can serve as an in vivo evaluation system for Nrf2-inhibiting compounds.

The downregulation of HSP90-controlled CRALBP expression is associated with age-related vision attenuation.

The dysfunction of CRALBP, a key regulator of the visual cycle, is associated with retinitis punctata albescens characterized by night vision loss and retinal degeneration. In this paper, we find that the expression of CRALBP is regulated by heat shock protein 90 (HSP90). Inhibition of HSP90α or HSP90ß expression by using the CRISPR-Cas9 technology downregulates CRALBP's mRNA and protein expression in ARPE-19 cells by triggering the degradation of transcription factor SP1 in the ubiquitin-proteasome pathway. SP1 can bind to CRALBP's promoter, and inhibition of SP1 by its inhibitor plicamycin or siRNA downregulates CRALBP's mRNA expression. In the zebrafish, inhibition of HSP90 by the intraperitoneal injection of IPI504 reduces the thickness of the retinal outer nuclear layer and Rlbp1b mRNA expression. Interestingly, the expression of HSP90, SP1, and CRALBP is correlatedly downregulated in the senescent ARPE-19 and Pig primary RPE cells in vitro and in the aged zebrafish and mouse retinal tissues in vivo. The aged mice exhibit the low night adaption activity. Taken together, these data indicate that the HSP90-SP1 is a novel regulatory axis of CRALBP transcriptional expression in RPE cells. The age-mediated downregulation of the HSP90-SP1-CRALBP axis is a potential etiology for the night vision reduction in senior people.

Glial cells expressing visual cycle genes are vital for photoreceptor survival in the zebrafish pineal gland.

Photoreceptors in the vertebrate eye are dependent on the retinal pigmented epithelium for a variety of functions including retinal re-isomerization and waste disposal. The light-sensitive pineal gland of fish, birds, and amphibians is evolutionarily related to the eye but lacks a pigmented epithelium. Thus, it is unclear how these functions are performed. Here, we ask whether a subpopulation of zebrafish pineal cells, which express glial markers and visual cycle genes, is involved in maintaining photoreceptors. Selective ablation of these cells leads to a loss of pineal photoreceptors. Moreover, these cells internalize exorhodopsin that is secreted by pineal rod-like photoreceptors, and in turn release CD63-positive extracellular vesicles (EVs) that are taken up by pdgfrb-positive phagocytic cells in the forebrain meninges. These results identify a subpopulation of glial cells that is critical for pineal photoreceptor survival and indicate the existence of cells in the forebrain meninges that receive EVs released by these pineal cells and potentially function in waste disposal.

Ultrafast spectra and kinetics of human green-cone visual pigment at room temperature.

Rhodopsin is the pigment that enables night vision, whereas cone opsins are the pigments responsible for color vision in bright-light conditions. Despite their importance for vision, cone opsins are poorly characterized at the molecular level compared to rhodopsin. Spectra and kinetics of the intermediate states of human green-cone visual pigment (mid-wavelength sensitive, or MWS opsin) were measured and compared with the intermediates and kinetics of bovine rhodopsin. All the major intermediates of the MWS opsin were recorded in the picosecond to millisecond time range. Several intermediates in MWS opsin appear to have characteristics similar to the intermediates of bovine rhodopsin; however, there are some marked differences. One of the most striking differences is in their kinetics, where the kinetics of the MWS opsin intermediates are slower compared to those of the bovine rhodopsin intermediates.

Iron overload and chelation modulates bisretinoid levels in the retina.

Aim: Iron dysregulation in conjunction with other disease processes may exacerbate retinal degeneration. We employed models of iron overload and iron chelation to explore the interactions between iron-catalyzed oxidation and photoreactive bisretinoid lipofuscin. Methods: The mice were injected intravitreally with ferric ammonium citrate (FAC) or were treated using the iron chelator deferiprone (DFP) from birth to 2 months of age. Short-wavelength fundus autofluorescence (SW-AF) and spectral-domain optical coherence tomography (SD-OCT) scans were acquired. The bisretinoid levels were quantified using ultra performance liquid chromatography (UPLC) and in vivo through quantitative fundus autofluorescence (qAF). In histologic sections, the photoreceptor cell viability was assessed by measuring the thickness of the outer nuclear layer (ONL). Results: The levels of bisretinoids, all-trans-retinal dimers, and A2PE were significantly increased in the FAC-injected eyes of C57BL/6J mice. Seven days after FAC injection, hyperautofluorescent foci were visible in fundus autofluorescence (488 nm) images, and in SD-OCT scans, aberrant hyperreflectivity was present in the outer retina and ONL thinning was observed. In FAC-injected Abca4-/- mice with pronounced RPE bisretinoid lipofuscin accumulation, the hyperautofluorescent puncta were more abundant than in the wild-type mice, and the extent of ONL thinning was greater. Conversely, the intravitreal injection of FAC in Mertk-/- mice led to a more modest increase in A2PE after 2 days. In contrast to the effect of iron accumulation, chelation with DFP resulted in significantly increased levels of A2E and A2-GPE and qAF due to the reduced iron-catalyzed oxidation of bisretinoids. In Mertk-/- mice, the A2E level was significantly lower and the ONL area was smaller than in DFP-treated mice. DFP chelation did not impair the visual cycle in BALB/cJ mice. Conclusion: Iron accumulation was associated with progressive impairment in photoreceptor cells that was associated with the increased formation of a bisretinoid species known to form in photoreceptor outer segments as a precursor to A2E. Additionally, disease features such as the development of hyperautofluorescence puncta in fundus AF images, hyperreflectivity in the outer retina of SD-OCT scans, and ONL thinning were more pronounced when iron was delivered to Abca4-/- mice with a greater propensity for bisretinoid formation. Higher bisretinoid levels and enhanced qAF are indicative of lesser bisretinoid loss due to oxidation.