Rhodopsin, the visual pigment mediating vision under dim light, is composed of the apoprotein opsin and the chromophore ligand 11-cis-retinal. A P23H mutation in the opsin gene is one of the most prevalent causes of the human blinding disease, autosomal dominant retinitis pigmentosa. Although P23H cultured cell and transgenic animal models have been developed, there remains controversy over whether they fully mimic the human phenotype; and the exact mechanism by which this mutation leads to photoreceptor cell degeneration remains unknown. By generating P23H opsin knock-in mice, we found that the P23H protein was inadequately glycosylated with levels 1-10% that of wild type opsin. Moreover, the P23H protein failed to accumulate in rod photoreceptor cell endoplasmic reticulum but instead disrupted rod photoreceptor disks. Genetically engineered P23H mice lacking the chromophore showed accelerated photoreceptor cell degeneration. These results indicate that most synthesized P23H protein is degraded, and its retinal cytotoxicity is enhanced by lack of the 11-cisretinal chromophore during rod outer segment development.Greater understanding of a genetically heterogeneous group of retinal disorders is now possible due to the results of studies that have revealed their causative genes. Many genetic loci can cause such retinopathies (RetNet) (1). Mutations in phototransduction genes, including those in opsin genes (2), constitute one of the major known causes of inherited blinding diseases (3). Among them, retinitis pigmentosa (RP) 2 refers to a group that displays genetic heterogeneity and a range of clinical phenotypes (4). RP manifested predominantly by death of rod photoreceptor cells is a progressive disease characterized by night blindness that progresses to loss of peripheral vision and eventually all useful vision over decades (5). Of more than 100 mutant opsins associated with autosomal dominant RP (adRP), the most frequent mutation is P23H (6), accounting for ϳ10% of human cases (7,8).In vitro studies have shown that the P23H opsin associated with adRP is misfolded and retained in the ER (9 -12). Consequently, this protein is not transported to the cell membrane (12) but instead was degraded by the ubiquitin-proteosome system (13). Co-expression of adRP-linked opsin folding-deficient mutants and wild type (WT) opsin resulted in enhanced proteosome-mediated degradation and steady-state ubiquitination of both mutant and WT opsin in an experimental cell line (14). These results imply that in vivo, a misfolded monomer of P23H opsin can also induce co-aggregation with WT rhodopsin preventing rod outer segment (ROS) formation. This dominant negative effect on ROS formation has been considered as the underlying reason for the adRP inheritance of P23H in humans.The retinal structure in heterozygous transgenic mice and rats expressing the P23H opsin partially mimics that of adRP in humans carrying this mutation (15)(16)(17)(18)(19). Mislocalization of the P23H opsin in the retina also has been reported in transgenic ani...

The visual (retinoid) cycle is a fundamental metabolic process in vertebrate retina responsible for production of 11-cis-retinal, the chromophore of rhodopsin and cone pigments. 11-cis-Retinal is bound to opsins, forming visual pigments, and when the resulting visual chromophore 11-cis-retinylidene is photoisomerized to all-trans-retinylidene, all-trans-retinal is released from these receptors. Toxic byproducts of the visual cycle formed from all-trans-retinal often are associated with lipofuscin deposits in the retinal pigmented epithelium (RPE), but it is not clear whether aberrant reactions of the visual cycle participate in RPE atrophy, leading to a rapid onset of retinopathy. Here we report that mice lacking both the ATP-binding cassette transporter 4 (Abca4) and enzyme retinol dehydrogenase 8 (Rdh8), proteins critical for all-trans-retinal clearance from photoreceptors, developed severe RPE/photoreceptor dystrophy at an early age. This phenotype includes lipofuscin, drusen, and basal laminar deposits, Bruch's membrane thickening, and choroidal neovascularization. Importantly, the severity of visual dysfunction and retinopathy was exacerbated by light but attenuated by treatment with retinylamine, a visual cycle inhibitor that slows the flow of all-trans-retinal through the visual cycle. These findings provide direct evidence that aberrant production of toxic condensation byproducts of the visual cycle in mice can lead to rapid, progressive retinal degeneration.What discriminates the eye from other organs is its light sensitivity and associated metabolic transformations that restore the light-sensitive chromophore (1). It is unclear if aberrations in the visual cycle and byproduct accumulation could be an underlying cause of retinopathy or merely a nonspecific nonpathogenic reflection of impaired metabolism. To distinguish between these two possibilities, we disrupted two genes critical for clearance of light-generated all-transretinal from rhodopsin and cone visual pigments (2, 3). Both the photoreceptor-specific ATP-binding cassette transporter (ABCA4) (4) and all-trans-retinol dehydrogenases (RDHs) 2 are involved in removal of all-trans-retinal from photoreceptors (5) (Scheme 1).ABCA4, also known as ABCR or the rim protein, localizes to the rim of photoreceptor discs and transfers all-trans-retinal from the inside to the outside of disc membranes once alltrans-retinal is released from visual pigments (4). Diretinoidpyridinium-ethanolamine (A2E) (6, 7) and retinal dimer (RALdi) conjugates (8) are the major fluorophores of lipofuscins produced from all-trans-retinal. Even in the presence of a functional transporter, both A2E and RALdi can accumulate as a consequence of aging (9) and produce toxic effects on RPE cells (10, 11). Patients affected by age-related macular degeneration (AMD), Stargardt disease with a disabled ABCA4 gene, or other retinal diseases associated with lipofuscin accumulation develop retinal degeneration. ABCA4 mutations also are linked with a high risk of AMD (12). However, no such ...

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 ...

CaBP1-8 are neuronal Ca 2+ -binding proteins with similarity to calmodulin (CaM). Here we show that CaBP4 is specifically expressed in photoreceptors, where it is localized to synaptic terminals. The outer plexiform layer, which contains the photoreceptor synapses with secondary neurons, was thinner in the Cabp4 −/− mice than in control mice. Cabp4 −/− retinas also had ectopic synapses originating from rod bipolar and horizontal cells that extended into the outer nuclear layer. Responses of Cabp4 −/− rod bipolars were reduced in sensitivity about 100-fold. Electroretinograms (ERGs) indicated a reduction in cone and rod synaptic function. The phenotype of Cabp4 −/− mice shares similarities with that of incomplete congenital stationary night blindness (CSNB2) patients. CaBP4 directly associated with the C-terminal domain of the Ca v 1.4 α 1 -subunit and shifted the activation of Ca v 1.4 to hyperpolarized voltages in transfected cells. These observations indicate that CaBP4 is important for normal synaptic function, probably through regulation of Ca 2+ influx and neurotransmitter release in photoreceptor synaptic terminals.L-type Ca 2+ channels are involved in neuronal differentiation and outgrowth and in synaptic plasticity 1,2 . At many ribbon synapses, Ca 2+ influx through L-type Ca 2+ channels triggers neurotransmitter release 3-5 . The α 1 -subunit of the L-type Ca v 1.4 channel (Ca v 1.4α1) is specific to photoreceptors and is present at highest density in the synaptic terminals 5,6 . Compared with other L-type Ca 2+ channels, Ca v 1.4 channels are activated at relatively more negative voltages and show slow inactivation 7-9 , important properties for the ability of photoreceptors to sustain continual glutamate release in the dark 4,10 . Null mutations in Ca v 1.4α1 are responsible for an X-linked disorder, CSNB2 (refs. 11 ,12 ). ERGs of these patients indicate that a deficit may occur in transmission of signals from rod photoreceptors to bipolar cells. In mice, deletion of the β 2 -subunit, another component of the photoreceptor L-type channel, alters the expression of Ca v 1.4 and produces a phenotype similar to that seen in CSNB2 patients 13 .CaBPs, a subfamily of calmodulin (CaM)-like neuronal Ca 2+ -binding proteins 14 , modulate voltage-dependent Ca 2+ channels (VDCCs) and inositol triphosphate receptors 15-17 . Here we show that CaBP4, which has only been partially characterized in silico 14 , is found specifically

Exposure to bright light can cause visual dysfunction and retinal photoreceptor damage in humans and experimental animals, but the mechanism(s) remain unclear. We investigated whether the retinoid cycle (i.e. the series of biochemical reactions required for vision through continuous generation of 11-cis-retinal and clearance of all-trans-retinal, respectively) might be involved. Previously, we reported that mice lacking two enzymes responsible for clearing all-trans-retinal, namely photoreceptor-specific ABCA4 (ATP-binding cassette transporter 4) and RDH8 (retinol dehydrogenase 8), manifested retinal abnormalities exacerbated by light and associated with accumulation of diretinoid-pyridinium-ethanolamine (A2E), a condensation product of all-trans-retinal and a surrogate marker for toxic retinoids. Now we show that these mice develop an acute, light-induced retinopathy. However, cross-breeding these animals with lecithin:retinol acyltransferase knock-out mice lacking retinoids within the eye produced progeny that did not exhibit such light-induced retinopathy until gavaged with the artificial chromophore, 9-cis-retinal. No significant ocular accumulation of A2E occurred under these conditions. These results indicate that this acute light-induced retinopathy requires the presence of free all-trans-retinal and not, as generally believed, A2E or other retinoid condensation products. Evidence is presented that the mechanism of toxicity may include plasma membrane permeability and mitochondrial poisoning that lead to caspase activation and mitochondria-associated cell death. These findings further understanding of the mechanisms involved in light-induced retinal degeneration.

Retinal guanylate cyclases 1 and 2 (GC1 and GC2) are responsible for synthesis of cyclic GMP in rods and cones, but their individual contributions to phototransduction are unknown. We report here that the deletion of both GC1 and GC2 rendered rod and cone photoreceptors nonfunctional and unstable. In the rod outer segments of GC double knock-out mice, guanylate cyclase-activating proteins 1 and 2, and cyclic GMP phosphodiesterase were undetectable, although rhodopsin and transducin ␣-subunit were mostly unaffected. Outer segment membranes of GC1 ؊/؊ and GC double knock-out cones were destabilized and devoid of cone transducin (␣-and ␥-subunits), cone phosphodiesterase, and G protein-coupled receptor kinase 1, whereas cone pigments were present at reduced levels. Real time reverse transcription-PCR analyses demonstrated normal RNA transcript levels for the downregulated proteins, indicating that down-regulation is posttranslational. We interpret these results to demonstrate an intrinsic requirement of GCs for stability and/or transport of a set of membrane-associated phototransduction proteins.

Cell-based therapies for myelin disorders, such as multiple sclerosis and leukodystrophies, require technologies to generate functional oligodendrocyte progenitor cells. Here we describe direct conversion of mouse embryonic and lung fibroblasts to “induced” oligodendrocyte progenitor cells (iOPCs) using sets of either eight or three defined transcription factors. iOPCs exhibit a bipolar morphology and global gene expression profile consistent with bona fide OPCs. They can be expanded in vitro for at least five passages while retaining the ability to differentiate into multiprocessed oligodendrocytes. When transplanted to hypomyelinated mice, iOPCs are capable of ensheathing host axons and generating compact myelin. Lineage conversion of somatic cells to expandable iOPCs provides a strategy to study the molecular control of oligodendrocyte lineage identity and may facilitate neurological disease modeling and autologous remyelinating therapies.

Background: High levels of all-trans-retinal (atRAL) are associated with photoreceptor degeneration. Results: atRAL promotes NADPH oxidase-mediated overproduction of intracellular reactive oxygen species. Conclusion: A cascade of signaling events is demonstrated to underlie the action of atRAL in photoreceptor degeneration in mice. Significance: Mechanistic elucidation of atRAL-mediated photoreceptor degeneration is essential for understanding the molecular pathogenesis of Stargardt disease and other types of retinal degeneration.