Feedback between a retinoid-related nuclear receptor and the let-7 microRNAs controls the pace and number of molting cycles in C. elegans.

Animal development requires coordination among cyclic processes, sequential cell fate specifications, and once-a-lifetime morphogenic events, but the underlying timing mechanisms are not well understood. Caenorhabditis elegans undergoes four molts at regular 8 to 10 hour intervals. The pace of the cycle is governed by PERIOD/lin-42 and other as-yet unknown factors. Cessation of the cycle in young adults is controlled by the let-7 family of microRNAs and downstream transcription factors in the heterochronic pathway. Here, we characterize a negative feedback loop between NHR-23, the worm homolog of mammalian retinoid-related orphan receptors (RORs), and the let-7 family of microRNAs that regulates both the frequency and finite number of molts. The molting cycle is decelerated in nhr-23 knockdowns and accelerated in let-7(-) mutants, but timed similarly in let-7(-) nhr-23(-) double mutants and wild-type animals. NHR-23 binds response elements (ROREs) in the let-7 promoter and activates transcription. In turn, let-7 dampens nhr-23 expression across development via a complementary let-7-binding site (LCS) in the nhr-23 3' UTR. The molecular interactions between NHR-23 and let-7 hold true for other let-7 family microRNAs. Either derepression of nhr-23 transcripts by LCS deletion or high gene dosage of nhr-23 leads to protracted behavioral quiescence and extra molts in adults. NHR-23 and let-7 also coregulate scores of genes required for execution of the molts, including lin-42. In addition, ROREs and LCSs isolated from mammalian ROR and let-7 genes function in C. elegans, suggesting conservation of this feedback mechanism. We propose that this feedback loop unites the molting timer and the heterochronic gene regulatory network, possibly by functioning as a cycle counter.

Gut commensals expand vitamin A metabolic capacity of the mammalian host.

Conversion of dietary vitamin A (VA) into retinoic acid (RA) is essential for many biological processes and thus far studied largely in mammalian cells. Using targeted metabolomics, we found that commensal bacteria in the mouse gut lumen produced a high concentration of the active retinoids, all-trans-retinoic acid (atRA) and 13-cis-retinoic acid (13cisRA), as well as the principal circulating retinoid, retinol. Ablation of anerobic bacteria significantly reduced retinol, atRA, and 13cisRA, whereas introducing these bacteria into germ-free mice significantly enhanced retinoids. Remarkably, cecal bacterial supplemented with VA produced active retinoids in vitro, establishing that gut bacteria encode metabolic machinery necessary for multistep conversion of dietary VA into its active forms. Finally, gut bacteria Lactobacillus intestinalis metabolized VA and specifically restored RA levels in the gut of vancomycin-treated mice. Our work establishes vitamin A metabolism as an emergent property of the gut microbiome and lays the groundwork for developing probiotic-based retinoid therapy.

Retinal Pigment Epithelium in Human Donor Eyes Contains Higher Levels of Bisretinoids Including A2E in Periphery than Macula.

Purpose: With age, human retinal pigment epithelium (RPE) accumulates bisretinoid fluorophores that may impact cellular function and contribute to age-related macular degeneration (AMD). Bisretinoids are comprised of a central pyridinium, dihydropyridinium, or cyclohexadiene ring. The pyridinium bisretinoid A2E has been extensively studied, and its quantity in the macula has been questioned. Age-changes and distributions of other bisretinoids are not well characterized. We measured levels of three bisretinoids and oxidized A2E in macula and periphery in human donor eyes of different ages. Methods: Eyes (N = 139 donors, 61 women and 78 men, aged 40-80 years) were dissected into 8 mm diameter macular and temporal periphery punches. Using liquid chromatography - electrospray ionization - mass spectrometry (LC-ESI-MS) and an authentic synthesized standard, we quantified A2E (ng). Using LC-ESI-MS and a 50-eye-extract of A2E, we semiquantified A2E and 3 other compounds (eye extract equivalent units [EEEUs): A2-glycerophosphoethanolamine (A2GPE), dihydropyridine phosphatidyl ethanolamine (A2DHPE), and monofuranA2E (MFA2E). Results: A2E quantities in ng and EEEUs were highly correlated (r = 0.97, P < 0.001). From 262 eyes, 5 to 9-fold higher levels were observed in the peripheral retina than in the macula for all assayed compounds. A2E, A2DHPE, and MFA2E increased with age, whereas A2GPE remained unaffected. No significant right-left or male-female differences were detected. Conclusions: Significantly higher levels were observed in the periphery than in the macula for all assayed compounds signifying biologic differences between these regions. Levels of oxidized A2E parallel native A2E and not the distribution of retinal illuminance. Data will assist with the interpretion of clinical trial outcomes of agents targeting bisretinoid-related pathways.

Genetic dissection in mice reveals a dynamic crosstalk between the delivery pathways of vitamin A.

Vitamin A is distributed within the body to support chromophore synthesis in the eyes and retinoid signaling in most other tissues. Two pathways exist for the delivery of vitamin A: the extrinsic pathway transports dietary vitamin A in lipoproteins from intestinal enterocytes to tissues, while the intrinsic pathway distributes vitamin A from hepatic stores bound to serum retinol binding protein (RBP). Previously, the intestine-specific homeodomain transcription factor (ISX) and the RBP receptor STRA6 were identified as gatekeepers of these pathways; however, it is not clear how mutations in the corresponding genes affect retinoid homeostasis. Here, we used a genetic dissection approach in mice to examine the contributions of these proteins in select tissues. We observed that ISX deficiency increased utilization of both preformed and provitamin A. We found that increased storage of retinoids in peripheral tissues of ISX-deficient mice was dependent on STRA6 and induced by retinoid signaling. In addition, double-mutant mice exhibited a partial rescue of the Stra6 mutant ocular phenotype. This rescue came at the expense of a massive accumulation of vitamin A in other tissues, demonstrating that vitamin A is randomly distributed when present in excessive amounts. Remarkably, provitamin A supplementation of mutant mice induced the expression of the RBP receptor 2 in the liver and was accompanied by increased hepatic retinyl ester stores. Taken together, these findings indicate dynamic crosstalk between the delivery pathways for this essential nutrient and suggest that hepatic reuptake of vitamin A takes place when excessive amounts circulate in the blood.

Effect of Radiation on the Essential Nutrient Homeostasis and Signaling of Retinoids in a Non-human Primate Model with Minimal Bone Marrow Sparing.

ABSTRACT: High-dose radiation exposure results in hematopoietic (H) and gastrointestinal (GI) acute radiation syndromes (ARS) followed by delayed effects of acute radiation exposure (DEARE), which include damage to lung, heart, and GI. Whereas DEARE includes inflammation and fibrosis in multiple tissues, the molecular mechanisms contributing to inflammation and to the development of fibrosis remain incompletely understood. Reports that radiation dysregulates retinoids and proteins within the retinoid pathway indicate that radiation disrupts essential nutrient homeostasis. An active metabolite of vitamin A, retinoic acid (RA), is a master regulator of cell proliferation, differentiation, and apoptosis roles in inflammatory signaling and the development of fibrosis. As facets of inflammation and fibrosis are regulated by RA, we surveyed radiation-induced changes in retinoids as well as proteins related to and targets of the retinoid pathway in the non-human primate after high dose radiation with minimal bone marrow sparing (12 Gy PBI/BM2.5). Retinoic acid was decreased in plasma as well as in lung, heart, and jejunum over time, indicating a global disruption of RA homeostasis after IR. A number of proteins associated with fibrosis and with RA were significantly altered after radiation. Together these data indicate that a local deficiency of endogenous RA presents a permissive environment for fibrotic transformation.

Retinoids in Cutaneous Squamous Cell Carcinoma.

Animal studies as early as the 1920s suggested that vitamin A deficiency leads to squamous cell metaplasia in numerous epithelial tissues including the skin. However, humans usually die from vitamin A deficiency before cancers have time to develop. A recent long-term cohort study found that high dietary vitamin A reduced the risk of cutaneous squamous cell carcinoma (cSCC). cSCC is a form of nonmelanoma skin cancer that primarily occurs from excess exposure to ultraviolet light B (UVB). These cancers are expensive to treat and can lead to metastasis and death. Oral synthetic retinoids prevent the reoccurrence of cSCC, but side effects limit their use in chemoprevention. Several proteins involved in vitamin A metabolism and signaling are altered in cSCC, which may lead to retinoid resistance. The expression of vitamin A metabolism proteins may also have prognostic value. This article reviews what is known about natural and synthetic retinoids and their metabolism in cSCC.

Proteomic Evaluation of the Natural History of the Acute Radiation Syndrome of the Gastrointestinal Tract in a Non-human Primate Model of Partial-body Irradiation with Minimal Bone Marrow Sparing Includes Dysregulation of the Retinoid Pathway.

Exposure to ionizing radiation results in injuries of the hematopoietic, gastrointestinal, and respiratory systems, which are the leading causes responsible for morbidity and mortality. Gastrointestinal injury occurs as an acute radiation syndrome. To help inform on the natural history of the radiation-induced injury of the partial body irradiation model, we quantitatively profiled the proteome of jejunum from non-human primates following 12 Gy partial body irradiation with 2.5% bone marrow sparing over a time period of 3 wk. Jejunum was analyzed by liquid chromatography-tandem mass spectrometry, and pathway and gene ontology analysis were performed. A total of 3,245 unique proteins were quantified out of more than 3,700 proteins identified in this study. Also a total of 289 proteins of the quantified proteins showed significant and consistent responses across at least three time points post-irradiation, of which 263 proteins showed strong upregulations while 26 proteins showed downregulations. Bioinformatic analysis suggests significant pathway and upstream regulator perturbations post-high dose irradiation and shed light on underlying mechanisms of radiation damage. Canonical pathways altered by radiation included GP6 signaling pathway, acute phase response signaling, LXR/RXR activation, and intrinsic prothrombin activation pathway. Additionally, we observed dysregulation of proteins of the retinoid pathway and retinoic acid, an active metabolite of vitamin A, as quantified by liquid chromatography-tandem mass spectrometry. Correlation of changes in protein abundance with a well-characterized histological endpoint, corrected crypt number, was used to evaluate biomarker potential. These data further define the natural history of the gastrointestinal acute radiation syndrome in a non-human primate model of partial body irradiation with minimal bone marrow sparing.

Probiotic modulation of perfluorobutanesulfonate toxicity in zebrafish: Disturbances in retinoid metabolism and visual physiology.

Perfluorobutanesulfonate (PFBS), an aquatic pollutant of emerging concern, is found to disturb gut microbiota, retinoid metabolism and visual signaling in teleosts, while probiotic supplementation can shape gut microbial community to improve retinoid absorption. However, it remains unknown whether probiotic bacteria can modulate the toxicities of PFBS on retinoid metabolism and visual physiology. In the present study, adult zebrafish were exposed for 28 days to 0, 10 and 100 µg/L PFBS, with or without dietary administration of probiotic Lactobacillus rhamnosus. Interaction between PFBS and probiotic was examined regarding retinoid dynamics (intestine, liver and eye) and visual stimuli transmission. PFBS single exposures remarkably inhibited the absorption of retinyl ester in female intestines, which were, however, restored by probiotic to normal status. Although coexposure scenarios markedly increased the hepatic storage of retinyl ester in females, mobilization of retinol was reduced in livers by single or combined exposures regardless of sex. In the eyes, transport and catalytic conversion of retinol to retinal and retinoic acid were interrupted by PFBS alone, which were efficiently antagonized by probiotic presumably through an indirect action. In response to the availability of retinal chromophore, transcriptions of opsins and arrestin genes were altered adaptively to control visual perception and termination. Neurotransmission across retina circuitry was changed accordingly, centering on epinephrine and norepinephrine. In summary, the present study found the efficient modulation of probiotic on retinoid metabolic disorders of PFBS pollution, which subsequently impacted visual signaling. A future work is warranted to provide mechanistic clues in retinoid interaction.

Systemic administration of the di-apocarotenoid norbixin (BIO201) is neuroprotective, preserves photoreceptor function and inhibits A2E and lipofuscin accumulation in animal models of age-related macular degeneration and Stargardt disease.

Atrophic A\age-related macular degeneration (AMD) and Stargardt disease (STGD) are major blinding diseases affecting millions of patients worldwide, but no treatment is available. In dry AMD and STGD oxidative stress and subretinal accumulation of N-retinylidene-N-retinylethanolamine (A2E), a toxic by-product of the visual cycle, causes retinal pigment epithelium (RPE) and photoreceptor degeneration leading to visual impairment. Acute and chronic retinal degeneration following blue light damage (BLD) in BALB/c mice and aging of Abca4-/- Rdh8-/- mice, respectively, reproduce features of AMD and STGD. Efficacy of systemic administrations of 9'-cis-norbixin (norbixin), a natural di-apocarotenoid, prepared from Bixa orellana seeds with anti-oxidative properties, was evaluated during BLD in BALB/c mice, and in Abca4-/- Rdh8-/- mice of different ages, following three experimental designs: "preventive", "early curative" and "late curative" supplementations. Norbixin injected intraperitoneally in BALB/c mice, maintained scotopic and photopic electroretinogram amplitude and was neuroprotective. Norbixin chronic oral administration for 6 months in Abca4-/- Rdh8-/- mice following the "early curative" supplementation showed optimal neuroprotection and maintenance of photoreceptor function and reduced ocular A2E accumulation. Thus, norbixin appears promising as a systemic drug candidate for both AMD and STGD treatment.

Toxic effects of A2E in human ARPE-19 cells were prevented by resveratrol: a potential nutritional bioactive for age-related macular degeneration treatment.

Age-related macular degeneration (AMD) is a late-onset retinal disease and the leading cause of central vision loss in the elderly. Degeneration of retinal pigment epithelial cells (RPE) is a crucial contributing factor responsible for the onset and progression of AMD. The toxic fluorophore N-retinyl-N-retinylidene ethanolamine (A2E), a major lipofuscin component, accumulates in RPE cells with age. Phytochemicals with antioxidant properties may have a potential role in both the prevention and treatment of this age-related ocular disease. Particularly, there is an increased interest in the therapeutic effects of resveratrol (RSV), a naturally occurring polyphenol (3,4',5-trihydroxystilbene). However, the underlying mechanism of the RSV antioxidative effect in ocular diseases has not been well explored. We hypothesized that this bioactive compound may have beneficial effects for AMD. To this end, to investigate the potential profits of RSV against A2E-provoked oxidative damage, we used human RPE cell line (ARPE-19). RSV (25 µM) attenuates the cytotoxicity and the typical morphological characteristics of apoptosis observed in 25 µM A2E-laden cells. RSV pretreatment strengthened cell monolayer integrity through the preservation of the transepithelial electrical resistance and reduced the fluorescein isothiocyanate (FITC)-dextran diffusion rate as well as cytoskeleton architecture. In addition, RSV exhorts protective effects against A2E-induced modifications in the intracellular redox balance. Finally, RSV also prevented A2E-induced mitochondrial network fragmentation. These findings reinforce the idea that RSV represents an attractive bioactive for therapeutic intervention against ocular diseases associated with oxidative stress such as AMD.

Cellular and molecular effects of Baccharis dracunculifolia D.C. and Plectranthus barbatus Andrews medicinal plant extracts on retinoid metabolism in the human hepatic stellate cell LX-2.

BACKGROUND: Chronic hepatic diseases are serious problems worldwide, which may lead to the development of fibrosis and eventually cirrhosis. Despite the significant number of people affected by hepatic fibrosis, no effective treatment is available. In the liver, hepatic stellate cells are the major fibrogenic cell type that play a relevant function in chronic liver diseases. Thus, the characterization of components that control the fibrogenesis in the hepatic stellate cells is relevant in supporting the development of innovative therapies to treat and/or control liver fibrosis. The present study investigated the effects of Baccharis dracunculifolia D.C. and Plectranthus barbatus Andrews medicinal plant extracts in LX-2 transdifferentiation. METHODS: LX-2 is a human immortalized hepatic stellate cell that can transdifferentiate in vitro from a quiescent-like phenotype to a more proliferative and activated behavior, and it provides a useful platform to assess antifibrotic drugs. Then, the antifibrotic effects of hydroalcoholic extracts of Baccharis dracunculifolia and Plectranthus barbatus medicinal plants on LX-2 were evaluated. RESULTS: The results in our cellular analyses, under the investigated concentrations of the plant extracts, indicate no deleterious effects on LX-2 metabolism, such as toxicity, genotoxicity, or apoptosis. Moreover, the extracts induced changes in actin filament distribution of activated LX-2, despite not affecting the cellular markers of transdifferentiation. Consistent effects in cellular retinoid metabolism were observed, supporting the presumed activity of the plant extracts in hepatic lipids metabolism, which corroborated the traditional knowledge about their uses for liver dysfunction. CONCLUSION: The combined results suggested a potential hepatoprotective effect of the investigated plant extracts reinforcing their safe use as coadjuvants in treating imbalanced liver lipid metabolism.

An Overview of Carotenoids, Apocarotenoids, and Vitamin A in Agro-Food, Nutrition, Health, and Disease.

Carotenoids are fascinating compounds that can be converted into many others, including retinoids that also play key roles in many processes. Although carotenoids are largely known in the context of food science, nutrition, and health as natural colorants and precursors of vitamin A (VA), evidence has accumulated that even those that cannot be converted to VA may be involved in health-promoting biological actions. It is not surprising that carotenoids (most notably lutein) are among the bioactives for which the need to establish recommended dietary intakes have been recently discussed. In this review, the importance of carotenoids (including apocarotenoids) and key derivatives (retinoids with VA activity) in agro-food with relevance to health is summarized. Furthermore, the European Network to Advance Carotenoid Research and Applications in Agro-Food and Health (EUROCAROTEN) is introduced. EUROCAROTEN originated from the Ibero-American Network for the Study of Carotenoids as Functional Food Ingredients (IBERCAROT).

Abnormal Cannabidiol Modulates Vitamin A Metabolism by Acting as a Competitive Inhibitor of CRBP1.

Cellular retinol-binding proteins (CRBPs) facilitate the uptake and intracellular transport of vitamin A. They integrate retinoid metabolism, playing an important role in regulating the synthesis of bioactive vitamin A metabolites. Thus, CRBPs constitute potential pharmacological targets to modulate cellular retinoid status that in turn may have applications in the treatment of certain immunological, metabolic, and ocular disorders. Here we identify abnormal cannabidiol (abn-CBD) as a nonretinoid inhibitor of cellular retinol-binding protein 1 (CRBP1). X-ray crystal structures of CRBP1 in complex with abn-CBD and its derivatives revealed a distinctive mode of protein-ligand interaction and provided a molecular basis for the high affinity and selectivity of this compound. We demonstrated that abn-CBD modulates the flux of retinoids via the retinoid cycle in vivo. Furthermore, the biological activity of abn-CBD was evidenced by its ability to protect against light-induced retinal damage in Balb/cJ mice. Altogether, our findings indicate that targeting selected CRBPs with a small-molecule inhibitor can potentially lead to the development of new therapeutic agents to counteract diseases with etiologies involving imbalance in retinoid metabolism or signaling.

Fingerprint Stimulated Raman Scattering Imaging Reveals Retinoid Coupling Lipid Metabolism and Survival.

Retinoids play critical roles in development, immunity, and lipid metabolism, and their deficiency leads to various human disorders. Yet, tools for sensing retinoids in vivo are lacking, which limits the understanding of retinoid distribution, dynamics and functions in living organisms. Here, using hyperspectral stimulated Raman scattering microscopy, we discover a previously unknown cytoplasmic store of retinoids in Caenorahbditis elegans. Following the temporal dynamics of retinoids, we reveal that their levels are positively correlated with fat storage, and their supplementation slows down fat loss during starvation. We also discover that retinoids promote fat unsaturation in response to high-glucose stress, and improve organism survival. Together, our studies report a new method for tracking the spatiotemporal dynamics of retinoids in living organisms, and suggest the crucial roles of retinoids in maintaining metabolic homeostasis and enhancing organism fitness upon developmental and dietary stresses.

Omega-3 Fatty Acids Supplementation: Therapeutic Potential in a Mouse Model of Stargardt Disease.

Purpose: To evaluate the therapeutic effects of omega-3 (ω3) fatty acids on retinal degeneration in the ABCA4-/- model of Stargardt disease when the blood level of arachidonic acid (AA)/eicosapentaenoic acid (EPA) ratio is between 1 and 1.5. Methods: Eight-month-old mice were allocated to three groups: wild type (129S1), ABCA4-/- untreated, and ABCA4-/- ω3 treated. ω3 treatment lasted 3 months and comprised daily gavage administration of EPA and docosahexaenoic acid (DHA). Blood and retinal fatty acid analysis was performed using gas chromatography to adjust the blood AA/EPA ∼1 to 1.5. Eyecups were histologically examined using transmission electron microscopy and confocal microscopy to evaluate lipofuscin granules and the photoreceptor layer. Retinal N-retinylidene-N-retinylethanolamine (A2E), a major component of retinal pigment epithelium lipofuscin, was quantified using liquid chromatography and tandem mass spectrometry, in addition to retinal proteomic analysis to determine changes in inflammatory proteins. Results: EPA levels increased and AA levels decreased in the blood and retinas of the treatment group. Significantly less A2E and lipofuscin granules were observed in the treatment group. The thickness of the outer nuclear layer was significantly greater in the treatment group (75.66 ± 4.80 µm) than in the wild-type (61.40 ± 1.84 µm) or untreated ABCA4-/- (56.50 ± 3.24 µm) groups. Proteomic analysis indicated lower levels of complement component 3 (C3) in the treatment group, indicative of lower complement-induced inflammatory response. Conclusions: Three months of ω3 supplementation (AA/EPA ∼1-1.5) reduces A2E levels, lipofuscin granules, and C3 levels in the ABCA4-/- mouse model of Stargardt disease, consistent with slowing of the disease.

ß-apo-10'-carotenoids support normal embryonic development during vitamin A deficiency.

Vitamin A deficiency is still a public health concern affecting millions of pregnant women and children. Retinoic acid, the active form of vitamin A, is critical for proper mammalian embryonic development. Embryos can generate retinoic acid from maternal circulating ß-carotene upon oxidation of retinaldehyde produced via the symmetric cleavage enzyme ß-carotene 15,15'-oxygenase (BCO1). Another cleavage enzyme, ß-carotene 9',10'-oxygenase (BCO2), asymmetrically cleaves ß-carotene in adult tissues to prevent its mitochondrial toxicity, generating ß-apo-10'-carotenal, which can be converted to retinoids (vitamin A and its metabolites) by BCO1. However, the role of BCO2 during mammalian embryogenesis is unknown. We found that mice lacking BCO2 on a vitamin A deficiency-susceptible genetic background (Rbp4-/-) generated severely malformed vitamin A-deficient embryos. Maternal ß-carotene supplementation impaired fertility and did not restore normal embryonic development in the Bco2-/-Rbp4-/- mice, despite the expression of BCO1. These data demonstrate that BCO2 prevents ß-carotene toxicity during embryogenesis under severe vitamin A deficiency. In contrast, ß-apo-10'-carotenal dose-dependently restored normal embryonic development in Bco2-/-Rbp4-/- but not Bco1-/-Bco2-/-Rbp4-/- mice, suggesting that ß-apo-10'-carotenal facilitates embryogenesis as a substrate for BCO1-catalyzed retinoid formation. These findings provide a proof of principle for the important role of BCO2 in embryonic development and invite consideration of ß-apo-10'-carotenal as a nutritional supplement to sustain normal embryonic development in vitamin A-deprived pregnant women.

Modeling Retinal Diseases Using Genetic Approaches in Mice.

Genetic mouse models mimicking human diseases have been developed and utilized for retinal research in various topics, involving anatomy, physiology, biochemistry, and pathology. The main reasons why mouse models are important for retinal research include that rodents share a key retinal homology with humans and that genetic manipulation is relatively easily applicable for mice. Here, we describe genetic mouse models, which are categorized with functions in the retina and relationship with human diseases.

Quercetin-3-O-α-l-arabinopyranoside protects against retinal cell death via blue light-induced damage in human RPE cells and Balb-c mice.

Age-related macular degeneration (AMD) is among the increasing number of diseases causing irreversible blindness in the elderly. Dry AMD is characterized by the accumulation of lipofuscin in retinal pigment epithelium (RPE) cells. N-Retinylidene-N-retinylethanolamine (A2E), a component of lipofuscin, is oxidized to oxo-A2E under blue light illumination, leading to retinal cell death. The aim of this study was to investigate the protective effect and mechanism of quercetin-3-O-α-l-arabinopyranoside (QA) against blue light (BL)-induced damage in both RPE cells and mice models. Treatment by QA inhibited A2E uptake in RPE cells, as determined by a decrease in fluorescence intensity. QA also protected A2E-laden RPE cells against BL-induced apoptosis. QA inhibited C3 complement activation and poly (ADP-ribose) polymerase (PARP) cleavage, as determined by western blotting. QA showed an inhibitory effect on AP1 and NF-kB activity as estimated in a reporter gene assay. In addition, QA activated the gene expression of aryl hydrocarbon receptor target genes (CYP1A1, CYP1B1) in TCDD-treated RPE cells. In the mice model, oral administration of QA protected against retinal degeneration induced by BL exposure as determined by histological analyses (thickness of retinal layers and immunostaining for caspase-3). In addition, QA inhibited apoptosis and inflammation via inhibition of NF-kB p65 translocation, C3 activation, and PARP cleavage. Collectively, these results revealed the protective mechanism of QA against BL-induced retinal damage both in vitro and in vivo.

Transgenic Mice Over-Expressing RBP4 Have RBP4-Dependent and Light-Independent Retinal Degeneration.

Purpose: Transgenic mice overexpressing serum retinol-binding protein (RBP4-Tg) develop progressive retinal degeneration, characterized by microglia activation, yet the precise mechanisms underlying retinal degeneration are unclear. Previous studies showed RBP4-Tg mice have normal ocular retinoid levels, suggesting that degeneration is independent of the retinoid visual cycle or light exposure. The present study addresses whether retinal degeneration is light-dependent and RBP4-dependent by testing the effects of dark-rearing and pharmacological lowering of serum RBP4 levels, respectively. Methods: RBP4-Tg mice reared on normal mouse chow in normal cyclic light conditions were directly compared to RBP4-Tg mice exposed to chow supplemented with the RBP4-lowering compound A1120 or dark-rearing conditions. Quantitative retinal histological analysis was conducted to assess retinal degeneration, and electroretinography (ERG) and optokinetic tracking (OKT) tests were performed to assess retinal and visual function. Ocular retinoids and bis-retinoid A2E were quantified. Results: Dark-rearing RBP4-Tg mice effectively reduced ocular bis-retinoid A2E levels, but had no significant effect on retinal degeneration or dysfunction in RBP4-Tg mice, demonstrating that retinal degeneration is light-independent. A1120 treatment lowered serum RBP4 levels similar to wild-type mice, and prevented structural retinal degeneration. However, A1120 treatment did not prevent retinal dysfunction in RBP4-Tg mice. Moreover, RBP4-Tg mice on A1120 diet had significant worsening of OKT response and loss of cone photoreceptors compared to RBP4-Tg mice on normal chow. This may be related to the very significant reduction in retinyl ester levels in the retina of mice on A1120-supplemented diet. Conclusions: Retinal degeneration in RBP4-Tg mice is RBP4-dependent and light-independent.

Impact of LCA-Associated E14L LRAT Mutation on Protein Stability and Retinoid Homeostasis.

Vitamin A (all-trans-retinol) is metabolized to the visual chromophore (11-cis-retinal) in the eyes and to all-trans-retinoic acid, a hormone like compound, in most tissues. A key enzyme in retinoid metabolism is lecithin:retinol acyltransferase (LRAT), which catalyzes the esterification of vitamin A. The importance of LRAT is indicated by pathogenic missense and nonsense mutations, which cause devastating blinding diseases. Retinoid-based chromophore replacement therapy has been proposed as treatment for these types of blindness based on studies in LRAT null mice. Here, we analyzed the structural and biochemical basis for retinal pathology caused by mutations in the human LRAT gene. Most LRAT missense mutations associated with retinal degeneration are localized within the catalytic domain, whereas E14L substitution is localized in an N-terminal α-helix, which has been implicated in interaction with the phospholipid bilayer. To elucidate the biochemical consequences of this mutation, we determined LRAT(E14L)'s enzymatic properties, protein stability, and impact on ocular retinoid metabolism. Bicistronic expression of LRAT(E14L) and enhanced green fluorescence protein revealed instability and accelerated proteosomal degradation of this mutant isoform. Surprisingly, instability of LRAT(E14L) did not abrogate the production of the visual chromophore in a cell-based assay. Instead, expression of LRAT(E14L) led to a rapid increase in cellular levels of retinoic acid upon retinoid supplementation. Thus, our study unveils the potential role of retinoic acid in the pathology of a degenerative retinal disease with important implications for the use of retinoid-based therapeutics in affected patients.