Lecithin-retinol acyltransferase (LRAT), an enzyme present mainly in the retinal pigmented epithelial cells and liver, converts all-trans-retinol into all-trans-retinyl esters. In the retinal pigmented epithelium, LRAT plays a key role in the retinoid cycle, a two-cell recycling system that replenishes the 11-cis-retinal chromophore of rhodopsin and cone pigments. We disrupted mouse Lrat gene expression by targeted recombination and generated a homozygous Lrat knock-out (Lrat؊/؊) mouse. Despite the expression of LRAT in multiple tissues, the Lrat؊/؊ mouse develops normally. The histological analysis and electron microscopy of the retina for 6 -8-week-old Lrat؊/؊ mice revealed that the rod outer segments are ϳ35% shorter than those of Lrat؉/؉ mice, whereas other neuronal layers appear normal. Lrat؊/؊ mice have trace levels of all-trans-retinyl esters in the liver, lung, eye, and blood, whereas the circulating all-trans-retinol is reduced only slightly. Scotopic and photopic electroretinograms as well as pupillary constriction analyses revealed that rod and cone visual functions are severely attenuated at an early age. We conclude that Lrat؊/؊ mice may serve as an animal model with early onset severe retinal dystrophy and severe retinyl ester deprivation.Lecithin-retinol acyltransferase (LRAT) 1 converts all-transretinol (vitamin A) to all-trans-retinyl esters in several tissues, including the liver, lung, pancreas, intestine, testis, and the retinal pigmented epithelium (RPE) (1-5). LRAT activity in the RPE has been studied for more than 60 years (6), but the enzyme was only recently identified on the molecular level as a 25-kDa integral membrane protein (7). All-trans-retinyl esters are intermediate compounds in a metabolic pathway ("visual cycle" or "retinoid cycle") that recycles 11-cis-retinal, the chromophore of rhodopsin and cone pigments (for review, see Refs. 8 -10). In this cycle, all-trans-retinal dissociates from rhodopsin and cone pigments after photobleaching. In the photoreceptors, all-trans-retinal is reduced to all-trans-retinol and subsequently exported to the adjacent RPE. In the RPE, alltrans-retinol is esterified by LRAT and stored. All-trans-retinyl esters have been suggested to be the substrate for a putative isomerohydrolase in the RPE (11) and for a retinyl ester hydrolase that produces all-trans-retinol, a substrate for the putative isomerase (for review, see Ref. 12). Ultimately, 11-cisretinol is produced, oxidized to 11-cis-retinal, and exported to the photoreceptors. In the rod and cone photoreceptor outer segments, 11-cis-retinal recombines with opsins to form rhodopsin and cone pigments (for review, see Ref. 8).Human LRAT cDNA was cloned from a retinal-RPE cDNA library (7) and rodent Lrat cDNA from liver and other tissues (13-15). Lrat mRNA was shown to be a 5.0-kb species expressed in the RPE, and the multiple transcripts based on differential polyadenylation were detected in several other tissues known for the highest LRAT activity (13). The human LRAT polypeptide consisted of 230 ...
Visual sensation in vertebrates is triggered when light strikes retinal photoreceptor cells causing photoisomerization of the rhodopsin chromophore 11-cis-retinal to all-trans-retinal. The regeneration of preillumination conditions of the photoreceptor cells requires formation of 11-cis-retinal in the adjacent retinal pigment epithelium (RPE). Using the intrinsic fluorescence of all-trans-retinyl esters, noninvasive two-photon microscopy revealed previously uncharacterized structures (6.9 ± 1.1 μm in length and 0.8 ± 0.2 μm in diameter) distinct from other cellular organelles, termed the retinyl ester storage particles (RESTs), or retinosomes. These structures form autonomous all-trans-retinyl ester-rich intracellular compartments distinct from other organelles and colocalize with adipose differentiation-related protein. As demonstrated by in vivo experiments using wild-type mice, the RESTs participate in 11-cis-retinal formation. RESTs accumulate in Rpe65 −/− mice incapable of carrying out the enzymatic isomerization, and correspondingly, are absent in the eyes of Lrat −/− mice deficient in retinyl ester synthesis. These results indicate that RESTs located close to the RPE plasma membrane are essential components in 11-cis-retinal production.
BackgroundLeber congenital amaurosis (LCA), a heterogeneous early-onset retinal dystrophy, accounts for ~15% of inherited congenital blindness. One cause of LCA is loss of the enzyme lecithin:retinol acyl transferase (LRAT), which is required for regeneration of the visual photopigment in the retina.Methods and FindingsAn animal model of LCA, the Lrat −/− mouse, recapitulates clinical features of the human disease. Here, we report that two interventions—intraocular gene therapy and oral pharmacologic treatment with novel retinoid compounds—each restore retinal function to Lrat −/− mice. Gene therapy using intraocular injection of recombinant adeno-associated virus carrying the Lrat gene successfully restored electroretinographic responses to ~50% of wild-type levels (p < 0.05 versus wild-type and knockout controls), and pupillary light responses (PLRs) of Lrat −/− mice increased ~2.5 log units (p < 0.05). Pharmacological intervention with orally administered pro-drugs 9-cis-retinyl acetate and 9-cis-retinyl succinate (which chemically bypass the LRAT-catalyzed step in chromophore regeneration) also caused long-lasting restoration of retinal function in LRAT-deficient mice and increased ERG response from ~5% of wild-type levels in Lrat −/− mice to ~50% of wild-type levels in treated Lrat −/− mice (p < 0.05 versus wild-type and knockout controls). The interventions produced markedly increased levels of visual pigment from undetectable levels to 600 pmoles per eye in retinoid treated mice, and ~1,000-fold improvements in PLR and electroretinogram sensitivity. The techniques were complementary when combined.ConclusionIntraocular gene therapy and pharmacologic bypass provide highly effective and complementary means for restoring retinal function in this animal model of human hereditary blindness. These complementary methods offer hope of developing treatment to restore vision in humans with certain forms of hereditary congenital blindness.
Retinoid cycle describes a set of chemical transformations that occur in the photoreceptor and retinal pigment epithelial cells. The hydrophobic and labile nature of the retinoid substrates and the two-cell chromophore utilization-regeneration system imposes significant constraints on the experimental biochemical approaches employed to understand this process. A brief description of the recent developments in the investigation of the retinoid cycle is the current topic, which includes a review of novel results and techniques pertaining to the retinoid cycle. The chemistry of the all-trans-retinol to 11-cis-retinol isomerization is also discussed.
The embryonic chicken iris constricts to light ex vivo, but with characteristics atypical of visual phototransduction. The chick iris was most sensitive to short-wavelength light, demonstrating an action spectrum consistent with cryptochrome rather than with opsin pigments. Pupillary responses did not attenuate after saturating light exposure, but showed paradoxical potentiation. Iris photosensitivity was not affected by retinoid depletion or inhibitors of visual phototransduction. Knockdown of cryptochrome expression, but not of melanopsin expression, decreased iris photosensitivity. These data characterize a non-opsin photoreception mechanism in a vertebrate eye and suggest a conserved photoreceptive role for cryptochromes in vertebrates.
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