Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

known as top-down proteomics, has delivered encouraging results (Sze et al., 2002; Ge et al., 2002). Just recently the strategy’s main limitation, that it could not be applied to proteins larger than 50 kd, was alleviated (Han et al., 2006), bringing routine proteome characterization within reach. Other modern mass spectrometry techniques have been used to determine the seminative quaternary structure of α- crystallin and could be applied to other protein species as well (Aquilina et al., 2003). A technique known as imaging mass spectrometry can be used to determine the distribution of proteins and protein species in tissues or organs (Crecelius et al., 2005). The combination of these and other technologies with an established proteome should allow the functional analysis of the totality of the protein species and make a true, in-depth understanding of the native situation conceivable in the eye lens and other tissues.

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The vertebrate eye paints upon the retina with bleached photopigments a picture of the luminous world outside.

—W. A. H. Rushton

Our retinas have evolved to provide useful detection of light over an extraordinary range of illumination intensities by using two sets of photoreceptors, rods and cones, which complement each other in sensitivity and in the speed and dynamic range of response. With this system, differentiation of the horizon from the moonless night sky is possible even though each rod photoreceptor of the human retina absorbs a photon on average once every 85 minutes. At the other end of the scale, a white ptarmigan can still be seen on a snowy slope in bright daylight, when the average cone photoreceptor absorbs more than 106 photons per second (Rodieck, 1998).

Despite enormous differences in physiological responses, the chemistry of light detection involved in both rods and cones of all vertebrate species is the same and involves photoisomerization of 11-cis-retinal, or a closely related derivative, to all-trans-retinal. Enzymatic regeneration of 11- cis-retinal takes place in all vertebrates in cells adjacent to the photoreceptor cells in a process originally called the visual cycle and more recently the retinoid cycle. Great advances have been made in the past decade in our understanding of the molecular events responsible for the regeneration of rod visual pigments. Detailed knowledge of a cone visual cycle remains elusive, although the evidence for involvement of Müller cells continues to accumulate.

This chapter considers advances in our understanding of the visual cycle that critically depended on the use of mouse genetics, alteration of the mouse genome, and proteomics. The rich history associated with many visual cycle components is mentioned only in passing, with relevant reviews cited. Throughout this chapter, we use standard conventions for denoting genes and gene products (examples: human gene, RPE65; mouse gene, Rpe65; protein, RPE65).

A variety of techniques have been employed over the years to study molecular aspects of the rod visual cycle. Early investigators made use of the color changes that accompanied photoisomerization of 11-cis-retinal, bound to opsin, to all-trans-retinal (purple to yellow) and reduction of all-trans-

retinal to all-trans-retinol (yellow to colorless). Later, classic enzymology provided valuable information about the various reactions that make up the cycle, primarily relying on bovine tissue extracts. Molecular cloning of cDNAs and ectopic cDNA expression allowed the assignment of enzymatic activities to molecular entities. The sequencing of complete genomes identified orthologues of key enzymes and opened up comparative sequence analysis for mechanistic studies. Assignment of an in vivo activity to a molecular entity was not a trivial matter and required techniques for generating transgenic and knockout mice. For one enzyme of the cycle (lecithin:retinol acyltransferase, LRAT), the enzymatic activity in vitro clearly pointed to an in vivo function, as proven by the phenotype of mice with targeted disruption of the gene. In other cases, disruption of the genes had either little effect (Rdh5, Rdh11, prRDH, Rdh12, Irbp) or a partial effect (Rlbp1) on the rate of rhodopsin regeneration, suggesting functional redundancy at these steps. Analysis of diseasecausing mutations in humans was not always helpful, presumably because of differences between the human and mouse visual systems.

Additional information regarding the visual cycle resulted from the identification and characterization of proteins using mass spectrometry, methodology referred to as proteomics. Indeed, the sensitivity and accuracy of these techniques have revolutionized structural component analysis, and future utilization promises to open up our understanding of the cell biology of retinoid processing.

Genetic analysis of the mouse visual cycle

The rod visual cycle is made up of a mixture of wellestablished and novel reactions occurring in two adjacent cell types. Diffusion of retinoids between the two cell types links the reactions into a cycle. Decades of research have established the reaction sequence in rod photoreceptors and retinal pigmented epithelium (RPE), and many of the enzymes that carry out these reactions in vivo have been identified. Nonetheless, details regarding the mechanisms of the reactions and possible reaction controls are lacking, and detailed information about the cell biology of retinoid processing in RPE is almost nonexistent. Furthermore, the

nature of the diffusion of retinoids between the cell types is poorly understood.

The phototransduction cascade is activated by the absorption of a photon by 11-cis-retinal, the chromophore attached to rod opsin (figure 59.1). The photoisomerization product, all-trans-retinal, dissociates from opsin and is reduced to all-trans-retinol by NADPH, catalyzed by one or more retinol dehydrogenases (RDHs) of the rod outer segment. All-trans-retinol diffuses from the rod cell through the interphotoreceptor matrix and enters the RPE, where LRAT catalyzes its esterification with a long chain fatty acid. Isomerohydrolase (RPE65) simultaneously hydrolyzes the ester bond of all-trans-retinyl ester and isomerizes the double bond at position 11–12 to yield 11-cis-retinol and a fatty acid. Oxidation of 11-cis-retinol to 11-cis-retinal by NAD is catalyzed by one or more cis-specific RDHs (cis- RDHs) and returned to the rod outer segment for conjugation with opsin and regeneration of the visual pigment. Cellular retinol-binding protein type I (CRBPI) and cellular retinaldehyde-binding protein (CRALBP) are present in RPE and facilitate esterification and isomerization/oxidation of retinoids, respectively. Interphotoreceptor retinoid-

binding protein (IRBP) is present in the interphotoreceptor matrix (IPM) and may protect retinoids during diffusion between cells and reduce their toxic effects. A brief description of our current understanding of the reactions of the rod visual cycle follows.

All-TRANS-Retinol Dehydrogenase Following photoisomerization of 11-cis-retinal, all-trans-retinal is released from opsin and reduced to all-trans-retinol by NADPH in the outer segments of photoreceptor cells. This reaction is accompanied by a change in color from yellow (all-trans- retinal) to colorless (all-trans-retinol) and was the first enzymatic visual cycle reaction to be studied in detail (Wald and Hubbard, 1949).

Based on their kinetic properties and substrate specificities, several short chain dehydrogenases/reductases (SDRs) have been proposed to catalyze the reduction of all-trans- retinal in vivo. However, studies of knockout mice have failed to identify a single enzyme whose absence has a major effect on the rate of visual pigment regeneration in vivo. This example points to the importance of mouse genetics in assigning in vivo functions to in vitro activities.

Figure 59.1 Hypothetical schematic of the rod photoreceptor visual cycle. Transcellular diffusion and intracellular enzymatic processing of visual cycle retinoids are shown. A retinal pigmented epithelial (RPE) cell is depicted with apical process extending toward a rod outer segment, with one disc membrane. Reactions and processes are as discussed in the text. Circular insets depict putative roles of components in RPE apical processes. cis-RDH, 11-cis- retinal dehydrogenase; CRALBP, cellular retinaldehyde-binding protein; CRBPI, cellular retinol-binding protein type I; EBP50,

ERM-binding phosphoprotein of 50 kd, also known as NHERF-1 (sodium hydrogen exchanger regulatory factor type 1); IRBP, interphotoreceptor retinoid-binding protein; ISOMERO, isomerohydrolase, recently identified as RPE65; LRAT, lecithin : retinol acyltransferase; PDZ, a scaffold domain protein; RGR, retinal G protein–coupled receptor; RDH, all-trans-retinol dehydrogenase; Rho, rhodopsin; Rho*, activated rhodopsin (metarhodopsin II); RPE65, retinal pigmented epithelial protein of 65 kd. See color plate 68.

A cDNA encoding RetSDR1 was cloned during a search of a retinal cDNA library for SDR homologues (Haeseleer et al., 1998). In situ hybridization and immunocytochemistry revealed the gene to be highly expressed in cone outer segments; however, expression was also noted in cells of the inner retina. The expressed enzyme catalyzed the reduction of all-trans- but not 11-cis-retinal and required NADPH. Several hydroxysteroids were also substrates for the enzyme, and transcripts were noted in several other tissues (Cerignoli et al., 2002). At present, the function of this enzyme in cone outer segments is not known.

A cDNA-encoding prRDH (RDH8) was cloned during a screen of retina-specific genes (Rattner et al., 2000). Antibodies revealed that the enzyme was expressed in rod and cone photoreceptor outer segments. The expressed enzyme was specific for all-trans-retinal and NADPH, similar to the activity in rod outer segments. prRdh−/− mice developed normally, and their retinal morphology was normal (Maeda et al., 2005). As predicted, the rate of reduction of all-trans- retinal was reduced in prRdh−/− mouse extracts following a flash; however, the rate of rhodopsin regeneration was normal. This result is an indication that the overall rate of the visual cycle is controlled by a step downstream of RDH, presumably the isomerohydrolase step, and that another dehydrogenase remained to be identified.

Mutations in the RDH12 gene in humans resulted in a severe form of retinal degeneration beginning in early childhood (Janecke et al., 2004). However, the enzyme is found in rod inner segments and not in outer segments, and disruption of the Rdh12 gene in mice did not affect the rate of visual pigment regeneration or ERG responses (Kurth et al., 2007). Rdh12−/− mice also exhibited accelerated 11-cis-retinal production and increased susceptibility to light-induced photoreceptor degeneration (Maeda et al., 2006). The cause of the severe form of retinal dystrophy in humans with mutations in this gene remains unexplained but may be related to light damage.

ATP-Binding Cassette A4 The disc membrane of the rod photoreceptor cell is equipped with a transporter of the ATP-binding cassette (ABC)-transporter family that retrieves all-trans-retinal from the luminal side of the disc membrane. Topologically, the compartment within the discs is equivalent to the outside of the cell, removed from the cytosolic site of production of NADPH and presumably from the active site of all-trans-RDH. The transporter, originally known as ABCR and now called ABCA4, utilizes ATP to “flip” all- trans-retinal from the luminal side of the disc to the cytosolic side, where it can be reduced to all-trans-retinol. The actual substrate for the translocation may be all-trans-retinal linked to phosphatidylethanolamine as a Schiff base. Characterization of the phenotype of Abca4−/− mice (Weng et al., 1999) revealed that the rate of rhodopsin regeneration

was normal, indicating that most of the all-trans-retinal must be released on the cytosolic side of the disc membrane. However, Abca4−/− mice accumulated all-trans-retinal and A2E, a novel pyridinium bis-retinoid characterized earlier as a component of lipofuscin. Mutations in the human ABCA4 gene cause autosomal recessive Stargardt disease (Allikmets et al., 1997), a form of macular dystrophy with large accumulations of lipofuscin in RPE. Based on these observations, it seems likely that the main physiological role of ABCR is to retrieve the small (in comparison to the total flux of the visual cycle) amount of all-trans-retinal that is released on the lumenal side of the discs and thus to minimize accumulation of its toxic condensation products.

Lecithin:Retinol Acyltransferase In RPE, retinyl esters are primarily synthesized by LRAT via transfer of a fatty acyl group from the sn-1 position of phosphatidylcholine to the hydroxyl of retinol (Berry et al., 1989; Saari and Bredberg, 1989). Molecular characterization of LRAT from retina revealed a small hydrophobic protein (25.3 kd) (Ruiz et al., 1999) encoded by a single gene in mouse and humans (Ruiz et al., 2001).

Lrat−/− mice were normal in appearance and grossly unremarkable; however, reduced fertility was noted in males. Development of the retina in Lrat−/− mice appeared normal; however, rod outer segments were shorter than normal in 4.5-month-old animals. Rhodopsin and retinoids, except for small amounts of all-trans-retinol, were virtually absent from retinas. ERG responses were grossly diminished, and the scotopic threshold was elevated by 5–6 log units (Batten et al., 2004).

The virtual absence of retinyl esters and 11-cis-retinal in retinas of Lrat−/− mice is consistent with the proposed role of LRAT in esterification of all-trans-retinol and of all-trans- retinyl ester as the substrate for the isomerohydrolase in the visual cycle (Deigner et al., 1989). Thus, in the absence of LRAT, the visual cycle would be blocked at the isomerohydrolase step by the absence of substrate. Other studies ruled out impaired uptake of blood-borne retinoids by RPE because oral gravage of 9-cis-retinyl acetate or succinate esters resulted in formation of isorhodopsin in the retina (Batten et al., 2005).

Isomerohydrolase The search for an isomerase is a fascinating story in which investigators followed two different paths for years, a search for a protein associated with the isomerase activity, and a search for a function for isomerohydrolase (RPE65), before realizing the two paths intersected.

The isomerase activity, which regenerates the 11-cis- retinoid configuration, is the signature enzyme of the visual cycle. Although many investigators assumed that all-trans- retinal would be isomerized directly to 11-cis-retinal, Rando’s

laboratory determined that the isomerization took place at the oxidation level of all-trans-retinol and discovered an enzymatic activity that converted all-trans-retinyl esters to 11-cis-retinol and a free fatty acid, a reaction termed an isomerohydrolase (Bernstein and Rando, 1986; Bernstein et al., 1987). Hydrolysis of the ester bond was proposed to provide the energy needed for generation of the hindered 11-cis-retinoid (Deigner et al., 1989). Attempts to purify the enzyme or to isolate a cDNA encoding the enzyme were unsuccessful for nearly two decades following the discovery of its activity.

Meanwhile, attempts to identify a retinol-binding protein (RBP) receptor led to the characterization of a major protein of RPE microsomes called p63 (Båvik et al., 1991). Subsequent studies demonstrated that its localization was not consistent with its proposed function. Other investigators, interested in proteins exclusively expressed by RPE cells, characterized a major protein of RPE microsomes, RPE65, which was identical to p63 (Hamel et al., 1993). Rpe65 −/− mice were unable to synthesize 11-cis-retinoids and accumulated large amounts of all-trans-retinyl esters in their RPE (Redmond et al., 1998). Both rod and cone functions were affected, indicating that RPE65 was involved in rod and cone visual cycles (Seeliger et al., 2001). Although this phenotype would be expected if RPE65 were the isomerohydrolase, attempts to provide direct evidence for this contention were unsuccessful until 2005, when three laboratories, using different approaches, demonstrated that RPE65 was responsible for isomerohydrolase activity (Jin et al., 2005; Redmond et al., 2005; Moiseyev et al., 2006).

Differences in susceptibility to light damage observed in several strains of mice were traced to a L450M sequence variation in the Rpe65 gene (Danciger et al., 2000). Subsequent studies revealed that this sequence variation also correlated with differences in the rate of rhodopsin regeneration. Strains of mice with L450 regenerated their visual pigment approximately four times faster and were more susceptible to light damage than strains of mice with M450 (Wenzel et al., 2001). The RPE65 content of M450 strains of mice was significantly lower than that of L450 strains, suggesting that the sequence variation affected the stability of RPE65. The rate of the isomerase reaction is the slow step in the mouse visual cycle (see discussion of retinoid flow). Thus, a decrease in the amount of RPE65 would be expected to reduce the flux of retinoid through this reaction, allow retinoid to accumulate as relatively innocuous all-trans-retinyl esters, and reduce the amount of visual pigment available for photoisomerization. (See also the discussion of CRALBP and light damage.)

The isomerohydrolase reaction also requires a lipidbinding protein for activity. Apo-cellular retinaldehydebinding protein (CRALBP) performs this function most efficiently in vitro (Winston and Rando, 1998), but other proteins are effective at much higher concentrations. Pre-

sumably, the retinoid-binding protein relieves inhibition of the enzyme by binding the product of the reaction, 11-cis- retinol (Winston and Rando, 1998). This explanation is in keeping with the visual phenotype of Rlbp1−/− (Cralbp−/−) mice, which show a delay in the visual cycle at the isomerohydrolase step (Saari et al., 2001). However, the requirement for CRALBP is not absolute because the visual cycle functions in its absence, although very slowly (see later discussion of CRALBP).

Detailed examination of RPE65 structure has provided considerable insight into molecular aspects of the catalytic activity (Moiseyev et al., 2006). RPE65 is a member of the carotenoid-cleavage oxygenase family of enzymes, which includes β-carotene 15,15′-monooxygenase (carotene cleavage enzyme). These enzymes coordinate an active site Fe2+ via four histidines, which are conserved in RPE65 and other members of the family. The crystal structure of an apocarotenoid 15,15′-oxygenase suggests that all-trans-apo- carotenoids are transiently isomerized during catalysis of cleavage (Kloer et al., 2005). Mutation of the corresponding histidine residues in RPE65 abolished the isomerohydolase activity of the protein (Redmond et al., 2005). The addition of chelating agents to native RPE65 enzyme reduced the activity of the enzyme and the addition of Fe(II) restored the activity. Finally, native RPE65 bound Fe(II) with a nearly 1 to 1 stoichiometry. These studies suggest, in an evolutionary sense, that an ancestral Fe-binding protein gave rise to a branch with isomerase and carotenoid cleavage activity (e.g., carotene cleavage enzyme) and a branch with just isomerase activity (e.g., RPE65) and demonstrate a role for iron in the regeneration of visual pigments.

Retinal G Protein–Coupled Receptor Retinal G pro- tein–coupled receptor (RGR) was first detected during a screen for RPE-specific proteins (Jiang et al., 1993). It was later found to be present in Müller cells also. The importance of RGR for retinal function and health was emphasized by the observation that mutations in the RGR gene in humans resulted in degeneration of the retina (Morimura et al., 1999). Sequence analysis revealed it to be a member of the G protein–coupled receptor family and to bind all-trans- retinal. Illumination of an RGR·all-trans-retinal complex resulted in photoisomerization of the retinoid to 11-cis- retinal, similar to the photoisomerization of all-trans-retinal bound to squid retinochrome. This striking result immediately suggested that a function of the protein might be to provide 11-cis-retinal during illumination via a photoisomerization reaction. Initially, characterization of Rgr−/− mice provided conflicting results regarding the effect of the absence of RGR on the rate of regeneration in the dark. More recently, careful attention to the sequence variation of the Rpe65 gene at position 450 (see discussion of RPE65) in control and experimental groups of animals clearly revealed that the rate

of regeneration of rhodopsin was three times slower in Rgr−/− mice (Wenzel et al., 2005). Surprisingly, the absence of RGR in mice affected regeneration both during light exposure and in the dark, indicating that RGR was not a photoisomerase. Molecular details about the role of RGR remain to be determined.

11-CIS-Retinol Dehydrogenase In RPE, 11-cis-retinol, produced by the isomerohydrolase, is oxidized to 11-cis- retinal by an SDR. As with the SDRs in the outer segment, there is functional redundancy at this step. Two SDRs have been identified in extracts of RPE. Surprisingly, neither enzyme alone or in combination accounts completely for 11-cis-retinol oxidizing activity in vivo in the mouse.

Shortly after its discovery, RPE65, then known as p63, was noted to interact with another protein in RPE microsomes (Simon et al., 1995). Cloning and studies of the expressed protein revealed it to be a member of the SDR family of proteins (RDH5) and to catalyze the oxidation of 11-cis-retinol by NAD. Humans with mutations in the RDH5 gene have a condition called fundus albipunctatus, which is a slowly progressing form of retinitis pigmentosa characterized by delayed dark adaptation and, in some cases, lateonset cone dystrophy (Yamamoto et al., 1999; Nakamura et al., 2000). Thus, it was a surprise when the rate of rhodopsin regeneration was found to be normal in Rdh5 −/− mice (Driessen et al., 2000). The only abnormality noted in these animals was an accumulation of 13-cis-retinyl esters in RPE. These results suggested that another SDR catalyzed the oxidation of 11-cis-retinol in mice.

RDH11 is expressed in prostate and also in RPE cells of the retina (Haeseleer et al., 2002). Rdh11−/− mice showed a phenotype similar to that of Rdh5 −/− mice (Kim et al., 2005). The phenotype of Rdh5 −/−/Rdh11−/− (double knockout) mice was similar to that of Rdh5 −/− mice but also displayed an abnormality in cone function. However, the flow of retinoids in the visual cycle was normal when moderate amounts of visual pigment were bleached (Kim et al., 2005), suggesting the existence of yet additional 11-cis-RDHs. No disease-causing mutations in the RDH11 gene in humans have been reported. RDH10 is expressed in RPE and Müller cells but appears to be specific for all-trans-retinols (B. X. Wu et al., 2004).

Several lines of evidence suggest that oxidation of 11-cis- retinol by cis-RDHs of RPE microsomes is facilitated by CRALBP. When bound to CRALBP, the aldehyde group of 11-cis-retinal is sequestered from water-soluble carbonyl reagents yet is readily reduced by NADH and RPE microsomes (Saari and Bredberg, 1982). 11-cis-retinol bound to CRALBP is oxidized to 11-cis-retinal by the cis-retinol dehydrogenase(s) of RPE microsomes more rapidly than free retinol (Saari et al., 1994) and has a lower Km (higher affinity) than free retinol for purified recombinant RDH5 (Golovleva et al., 2003).

Retinol-Binding Protein and the RBP Receptor RBP is a small protein (21 kd) secreted into the blood conjugated with all-trans-retinol, where it circulates associated with transthyretin. RBP is synthesized primarily in the liver and in lesser amounts in several tissues, including adipose tissue and RPE. In Rbp−/− mice, which are unable to mobilize hepatic vitamin A but develop normally, dietary vitamin A reaches peripheral tissues as retinyl esters carried by plasma lipoproteins. Retinal function is markedly diminished for the first few months after birth (Quadro et al., 1999), but Rbp−/− mice attain full visual function thereafter if maintained on a vitamin A-sufficient diet. Thus, it appears that for developmental purposes, most tissues are able to obtain vitamin A via circulating dietary retinyl esters, but the eye requires an RBP-dependent mechanism for the amount of vitamin A required for vision (Vogel et al., 2002).

Uptake of vitamin A from RBP was suggested to involve a receptor-mediated process in the 1970s (Bok and Heller, 1976), and decades later an RBP-receptor, STRA6, was identified (Kawaguchi et al., 2007). STRA6 was originally discovered as a gene responsive to retinoic acid. Homozygous mutations in STRA6 in humans result in severe developmental malformations and mental retardation (Passuto et al., 2007). Based on the patterns of STRA6 expression in embryo and adult, it appears that STRA6 may play an important role in delivering vitamin A to many tissues in the embryo and a more restricted role in the adult, where it is primarily expressed in tissues making up blood-brain or blood-organ barriers (e.g., RPE, choroid plexus).

affinity for all-trans-retinol and all-trans-retinal (Napoli, 1999). CRBPI is expressed in many tissues, including RPE and Müller cells of the eye (Bok et al., 1984; Saari et al., 1984). Evidence obtained in vitro suggested that the protein could be involved in promoting esterification and oxidation of retinol and hydrolysis of retinyl esters (Herr and Ong, 1992; Napoli, 1999). However, its physiological role in vivo was not clear until analysis of CrbpI −/− mice. Normal development, reproduction, and behavior of these animals ruled out a major role for CRBPI in retinoic acid production because retinoic acid signaling is required for all these processes. A reduced content and six times faster turnover of hepatic retinyl esters was in keeping with a role for the protein in delivery of all-trans-retinol for esterification by LRAT (Ghyselinck et al., 1999). Stores of retinyl esters in RPE were also reduced relative to wild type, and all-trans-retinol accumulated transiently during recovery from a flash (Saari et al., 2002). Overall, the results were in accord with a role for CRBPI in delivery of all-trans-retinol to LRAT for esterification in RPE and liver.

Interphotoreceptor Retinoid-Binding Protein Interphotoreceptor retinoid-binding protein (IRBP) is a watersoluble protein synthesized by photoreceptor cells and secreted into the interphotoreceptor matrix (IPM) (reviewed in Gonzalez-Fernandez, 2003). Localization of the protein in the extracellular compartment between photoreceptor and RPE cells (Bunt-Milam and Saari, 1983) and its ability to load with all-trans-retinol following bleaches of visual pigment suggested that the protein would be involved in transcellular diffusion of retinoids (reviewed in Pepperberg et al., 1993).

The retinas of Irbp−/− mice showed a loss of photoreceptor nuclei and changes in the structural integrity of photoreceptor outer segments from P11 to P30. However, the rate of progression of the condition was quite slow, and was unaffected by raising the animals in the dark (Ripps et al., 2000). Even after 6 months, the number of photoreceptor nuclei was reduced to only about 50% of the number in wild-type mice (Liou et al., 1998; Ripps et al., 2000). Surprisingly, the rate of visual pigment regeneration was unaffected (Palczewski et al., 1999) or was even modestly faster (Ripps et al., 2000) in the absence of IRBP. Attempts to detect other retinoid-binding proteins in IPM that might have substituted for IRBP have been unsuccessful.

IRBP appears to be necessary for release of 11-cis-retinal from cultured RPE cells (Carlson and Bok, 1992). One explanation for this finding is that a receptor mechanism is involved. However, the rate of visual pigment regeneration in Irbp−/− mice was nomal, and molecular information regarding this receptor has not yet appeared in the literature.

The role of IRBP in visual physiology remains enigmatic. The small enhancement of the rate of rhodopsin regeneration in the absence of IRBP and the slow progression of the associated retinal degeneration are consistent with a role for the protein in buffering the concentration of free (unbound) all-trans-retinal or 11-cis-retinal in the IPM during retinoid diffusion between RPE and photoreceptor cells. However, the absence of protection from retinal degeneration in darkreared animals does not fully accommodate such a function. IRBP binds several hydrophobic substances in addition to retinoids, including docosahexaenoic acid and other fatty acids (Bazan et al., 1985), and may be involved in intercellular diffusion of these substances, including scavenging of oxidatively damaged retinoids and fatty acids.

Cellular Retinaldehyde-Binding Protein CRALBP is a water-soluble protein with high affinity for 11-cis-retinol or 11-cis-retinal, found in abundance in RPE and Müller cells (Saari and Crabb, 2005). These features and the protein’s pronounced effects on enzymatic reactions of the visual cycle in vitro (Saari et al., 1994; Golovleva et al., 2003; Winston and Rando, 1998), suggested that it was involved in the regeneration of visual pigments.

No differences were detected in the gross retinal morphologies of control and Rlbp1−/− mice raised in the dark (Saari et al., 2001). However, the rate of visual pigment regeneration following flash illumination was slower by about 15 times in the Rlbp1−/− animals. PCR analysis of the Rpe65 gene indicated that differences in the rate of regeneration of control and knockout mice could not be accounted for by the L450M sequence variation in the Rpe65 gene (see section on RPE65). Analysis of visual cycle retinoids revealed that retinyl ester accumulated during the delay, suggesting that the isomerohydrolase step in the visual cycle was affected by the absence of CRALBP. Dark adaptation was delayed in both cone and rod visual pathways in Rlbp1−/− animals as measured by ERG. These results are consistent with studies in humans with mutations in the RLBP1 gene demonstrating dramatic delays in both rod and cone branches of dark adaptation curves (Bursedt et al., 2001).

The results of these studies for the rod system are most readily understood if apo-CRALBP accepts 11-cis-retinol from the isomerohydrolase, a role for the protein that is well established in vitro (Winston and Rando, 1998). However, it is important to note that the absence of CRALBP in mice resulted only in a delay in visual pigment regeneration, not a complete block. Some other molecule may be present or induced in the knockout that can fulfill the role of CRALBP, if less efficiently.

Albino Rlbp1−/− mice were protected from light damage (Saari et al., 2001). Other laboratories also observed that delays in the visual cycle resulted in protection from light damage (Sieving et al., 2001; Grimm et al., 2000). Retinoid analysis of Rlbp1−/− mice established that a diminished regeneration rate caused photoreceptor retinoids to accumulate as the relatively innocuous retinyl esters in RPE during constant illumination (Garwin and Saari, 2008). This and the resulting diminished amount of potentially toxic all-trans- retinaldehyde in flux are likely to account for this protective effect. It is interesting to note that pharmacological inhibition of the visual cycle has been proposed as a potential means of delaying the onset of some inherited retinal diseases (reviewed in Travis et al., 2006).

Retinoid Flow in the Visual Cycle The kinetics of appearance and disappearance of visual cycle retinoids during recovery from a flash or from constant illumination has been followed over the years using methods with increasing resolving power and sensitivity. Initial studies reported the flow of retinoids between neural retina and RPE long before sophisticated methods of retinoid separation were available (Dowling, 1960). More recent studies employing high-performance liquid chromatography (HPLC) analysis have identified slow steps based on the accumulation and decay of visual cycle intermediates. Early HPLC studies (Saari et al., 1998) were done in mice of mixed

genetic backgrounds, before the effect of Rpe65 sequence variations on regeneration rates was understood (Wenzel et al., 2001). Nonetheless, these and other studies clearly demonstrated the accumulation and slow decay of all-trans- retinal during recovery in the dark following a flash (Dowling, 1960; Saari et al., 1998; Palczewski et al., 1999; Qtaishat et al., 1999). Later studies with inbred strains of mice corroborated the slow decay of all-trans-retinal and also noted the accumulation and slow decay of all-trans-retinyl esters during recovery in the dark (Saari et al., 2001). Thus, there appeared to be two slow steps in the flow of retinoids leading to regeneration of rhodopsin following a flash, one related to the reduction of all-trans-retinal and the other related to the isomerohydrolase step. Based on the apparent decay rates for the two intermediates, processing of all-trans- retinyl ester, presumably by isomerohydrolase, appeared to be the slowest step. A similar conclusion was reached via mathematical analysis of flash-recovery data (Lamb and Pugh, 2004).

Other studies had demonstrated that the rate of visual pigment regeneration characteristic of various strains of mice was proportional to the amount of RPE65 (isomerohydrolase) in their retinas (Grimm et al., 2000). Retinoid analysis of mice during steady illumination provided additional information (Garwin and Saari, 2008). All-trans-retinal rose to a maximum and decayed to low levels during the illumination period, whereas all-trans-retinyl esters accumulated to a plateau and remained elevated until the light was extinguished. These results demonstrate the two slow steps but indicate that decay of all-trans-retinyl esters (isomerohydrolase reaction) is the slower of the two.

Thus, retinoid analyses identified visual cycle intermediates that accumulated during recovery from either flash or steady illumination. Because both reactions that process these intermediates are complicated, we do not know what limits the rates. For instance, the rate of reduction of all-trans- retinal by NADPH could be controlled by several processes, including the intrinsic turnover rate and amount of the enzyme, the rate of production of NADPH, the rate of release of all-trans-retinal from opsin, or an active control process. Similarly, the rate of isomerization and cleavage of all-trans-retinyl ester could be limited by processes including the rate of the isomerohydrolase reaction itself, removal of product by apo-CRALBP, substrate delivery of all-trans- retinyl ester to the enzyme, or active control of the reaction rate.

Cone visual cycle

The cone visual cycle has been more difficult to approach experimentally because of the relatively few cones in most retinas used in the laboratory for biochemical studies. Circumstantial evidence has implicated Müller cells in cone

visual pigment regeneration for decades. Recently, considerable progress has been made in characterizing reactions that may be involved in a cone visual cycle. It will be of great interest to learn whether novel reactions occur in Müller cells. Our understanding of this putative pathway lags behind that of the rod regeneration pathway.

It is not surprising that many aspects of visual pigment regeneration in rod and cone photoreceptor cells are dramatically different. Rod and cone visual pigments were designed to function in totally different illumination environments. In addition, rods outnumber cones in mammalian retinas by about 30 to one, setting up the possibility of competition for 11-cis-retinal. Bleached salamander cone photoreceptors in vitro will regenerate their visual pigment with exogenous 11-cis-retinol, whereas rods require 11-cis-retinal ( Jones et al., 1989). It is possible that this specificity reduces the competition between rods and cones for 11-cis-retinoids. In salamander retina, isolated cones regenerate when 11-cis- retinal is applied to either their inner or outer segments, whereas rods regenerate only when 11-cis-retinal is applied to the outer segments (Jin et al., 1994). This implies that cones could utilize a source of 11-cis-retinoid near their inner segments, possibly Müller cells. Bleached cone photoreceptors in neural retina, separated from RPE, resensitize in the dark, whereas bleached rod photoreceptors require the apposition of RPE (Goldstein and Wolf, 1973), again implying that a source of 11-cis-retinol must exist within cells of the neural retina. Müller cells contain both CRBPI and CRALBP (Saari and Crabb, 2005), and cultured chicken Müller cells synthesize 11-cis-retinol from all-trans-retinol (Das et al., 1992; Muniz et al., 2006). Recently, enzyme activities of a putative cone visual cycle have been identified in retinal extracts of ground squirrels and chickens (Arshavsky, 2002; Mata et al., 2002). Thus, considerable indirect evidence exists for a distinct cone visual cycle in Müller cells. However, much of the evidence comes from studies of amphibian or avian retina, and it is not certain that the same features apply to mammalian retina. The cone visual cycle depicted in figure 59.2 is based on that proposed by Travis and co-workers (Mata et al., 2002).

Proteomic analysis of the visual cycle

Proteomics, within the context of this article, refers to the identification of the proteins within a tissue, organ, organelle, or tissue fraction using mass spectrometric analysis. Few studies have directly addressed questions related to the visual cycle with this approach. However, the information obtained from studies with other primary goals has often provided useful insight regarding visual cycle components.

Analysis of Interacting Components As discussed elsewhere in this chapter, apo-CRALBP is likely to bind 11-cis-