Noninvasive two-photon imaging reveals retinyl ester storage structures in the eye

Journal ArticleVisual 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 microm in length and 0.8 +/- 0.2 microm 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

Two types of arrestins expressed in medaka rod photoreceptors

AbstractSimilar to visual arrestins of other vertebrates, two subtypes of medaka visual arrestins, KfhArr-R1 and KfhArr-C, are selectively expressed in rods and cones, respectively [Hisatomi et al. (1997) FEBS Lett. 411, 12–18]. We isolated a cDNA encoding the third arrestin, KfhArr-R2, from a medaka retinal cDNA library. Phylogenetic analysis of arrestin sequences suggests that KfhArr-R2 is classified into the rod arrestin subtype. In situ hybridization indicated that KfhArr-R2 mRNA is localized in most of the rod photoreceptors, suggesting that both KfhArr-R1 and -R2 are co-expressed in rods. Antisera against KfhArr-R2 recognized outer segments, but anti-KfhArr-R1 antisera reacted with cell bodies and synaptic termini in light-adapted rods. It seems likely that KfhArr-R1 and -R2 play different roles in rod photoreceptors

Noninvasive two-photon imaging reveals retinyl ester storage structures in the eye

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

Pharmacological and rAAV Gene Therapy Rescue of Visual Functions in a Blind Mouse Model of Leber Congenital Amaurosis

BACKGROUND: Leber 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 FINDINGS: An 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. CONCLUSION: Intraocular 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

Drug Discovery Strategies for Inherited Retinal Degenerations

Inherited retinal degeneration is a group of blinding disorders afflicting more than 1 in 4000 worldwide. These disorders frequently cause the death of photoreceptor cells or retinal ganglion cells. In a subset of these disorders, photoreceptor cell death is a secondary consequence of retinal pigment epithelial cell dysfunction or degeneration. This manuscript reviews current efforts in identifying targets and developing small molecule-based therapies for these devastating neuronal degenerations, for which no cures exist. Photoreceptors and retinal ganglion cells are metabolically demanding owing to their unique structures and functional properties. Modulations of metabolic pathways, which are disrupted in most inherited retinal degenerations, serve as promising therapeutic strategies. In monogenic disorders, great insights were previously obtained regarding targets associated with the defective pathways, including phototransduction, visual cycle, and mitophagy. In addition to these target-based drug discoveries, we will discuss how phenotypic screening can be harnessed to discover beneficial molecules without prior knowledge of their mechanisms of action. Because of major anatomical and biological differences, it has frequently been challenging to model human inherited retinal degeneration conditions using small animals such as rodents. Recent advances in stem cell-based techniques are opening new avenues to obtain pure populations of human retinal ganglion cells and retinal organoids with photoreceptor cells. We will discuss concurrent ideas of utilizing stem-cell-based disease models for drug discovery and preclinical development

K (2001) Gene transfer mediated by recombinant baculovirus into mouse eye. Invest Ophthalmol Vis Sci 42: 3294–3300

PURPOSE. To determine the efficiency of baculoviruses (BVs) to transfer recombinant genes in vivo into murine ocular tissues. METHODS. Recombinant (r)BVs carrying fluorescent protein (FP) cDNA under the control of cytomegalovirus (CMV) immediate early promoter were constructed. Initially, cultured HEK293 and ARPE19 cells were infected with these rBVs and analyzed for efficiency and stability of transgene expression. The rBV-CMV green (G)FP was also injected into the intravitreal and subretinal space of mouse eye. Mice were periodically analyzed to determine the efficiency and stability of expression by histologic examination under fluorescence microscopy. The effect of rBV-CMV-GFP on the physiology of the retina was analyzed by electroretinography. RESULTS. cDNAs encoding fluorescent proteins were efficiently transduced in HEK293 and ARPE19 cells in vitro. GFP expression in vivo was observed exclusively in retinal pigment epithelial (RPE) cells after subretinal injections. Intravitreal injections of rBV resulted in GFP expression in the corneal endothelium, lens, RPE, and retina. GFP expression was observed for up to 14 days after injection. The infiltration of macrophages, observed 2 days after injection in the area of GFP transduction, had dissipated by day 8 after injection. No alteration in ERG responses was observed 6 weeks after injection of rBV-CMV-GFP. CONCLUSIONS. BV efficiently transduces cultured RPE cells and many cell types in vivo in the eye, including endothelial, epithelial, and neuronal cells. BV may be a useful vector for transferring genes in cultured cells and in vivo into ocular tissue. (Invest Ophthalmol Vis Sci. 2001;42:3294 -3300