The Journal of Neuroscience, July 13, 2011 • 31(28):10403–10411 • 10403 Cellular/Molecular Regeneration of Photopigment Is Enhanced in Mouse Cone Photoreceptors Expressing RPE65 Protein Peter H. Tang,1 Lee Wheless,2 and Rosalie K. Crouch1,3 Departments of 1Neuroscience, 2Medicine–Division of Biostatistics and Epidemiology, and 3Ophthalmology, Medical University of South Carolina, Charleston, South Carolina 29425 As cone photoreceptors mediate vision in bright light, their photopigments are bleached at a rapid rate and require substantial recycling of the chromophore 11-cis-retinal (RAL) for continued function. The retinal pigment epithelium (RPE) supplies 11-cis-RAL to both rod and cone photoreceptors; however, stringent demands imposed by the function of cones in bright light exceed the output from this source. Recent evidence has suggested that cones may be able to satisfy this demand through privileged access to an additional source of chromophore located within the inner retina. In this study, we demonstrate that the protein RPE65, previously identified in RPE as the isomerohydrolaseoftheRPE–retinalvisualcycle,isfoundwithinconesoftherod-dominantmouseretina,andthelevelofRPE65incones is inversely related to the level in the RPE. The light sensitivity of cone ERGs of BALB/c mice, which had an undetectable level of cone RPE65, was enhanced by approximately threefold with administration of exogenous chromophore, indicating that the cones of these animals are chromophore deficient. This enhancement with chromophore administration was not observed in C57BL/6 mice, whose cones contain RPE65. These results demonstrate that RPE65 within cones may be essential for the efficient regeneration of cone pho- topigments under bright-light conditions. Introduction Moiseyev et al., 2005; Redmond et al., 2005). Once 11-cis-RAL Vitamin A aldehyde, 11-cis-retinal (RAL), acts as the chro- is regenerated, it is shuttled to photoreceptors to reform pho- mophore within rod and cone photoreceptors by covalently topigment. The levels of RPE65 in the RPE have been shown to binding opsin G-protein-coupled receptors to form photopig- vary in different strains of wild-type mice (Lyubarsky et al., ment. When photons are absorbed by the photopigment, 11-cis- 2005). RAL is isomerized to the all-trans conformation, triggering the Numerous studies have suggested that the mechanisms by phototransduction cascade for propagating neuronal signals to which rods and cones obtain chromophore may differ (Wang the visual cortex. To replenish photopigments, all-trans RAL is and Kefalov, 2011). Cones function in daylight to mediate recycled back to 11-cis-RAL through a series of enzymatic high-resolution color vision, and their rates of sensitivity re- steps known as the visual cycle. The most well known pathway covery (Perry and McNaughton, 1991) and photopigment re- is a cycling of vitamin A derivatives (retinoids) from photore- generation (Schnapf et al., 1990) are greater than that of rods ceptors through the retinal pigment epithelium (RPE), known (Baylor et al., 1979, 1984), which function in low-light condi- as the RPE–retinal visual cycle (Travis et al., 2007). Within the tions. Concurrently, the rate of photopigment regeneration RPE, the protein RPE65 acts as the isomerohydrolase to con- that is necessary for sustaining cone function in bright light vert all-trans retinoids back to 11-cis-forms (Jin et al., 2005; exceeds the capabilities of the RPE to recycle chromophore (Mata et al., 2002). Received Jan. 11, 2011; revised May 19, 2011; accepted May 30, 2011. A second visual cycle may exist to supplement cones with Author contributions: P.H.T. and R.K.C. designed research; P.H.T. performed research; P.H.T. contributed unpub- chromophore (Muniz et al., 2007). This alternate pathway is lo- lished reagents/analytic tools; P.H.T., L.W., and R.K.C. analyzed data; P.H.T., L.W., and R.K.C. wrote the paper. calized entirely within the retina (intraretinal visual cycle) and The authors declare no competing financial interests. functions in parallel with the RPE–retinal visual cycle. Cones can ThisstudywassupportedinpartbyNationalInstitutesofHealthGrantsR01EY04939(R.K.C.)andC06RR015455 [Medical University of South Carolina (MUSC)], Foundation Fighting Blindness (R.K.C.), an unrestricted grant (De- use vitamin A alcohol, 11-cis-retinol (ROL), as a source of chro- partment of Ophthalmology, MUSC) from Research to Prevent Blindness (RPB), a RPB Senior Scientific Investigator mophore, presumably by oxidizing 11-cis-ROL to 11-cis-RAL Award (R.K.C.), and a RPB Medical Student Research Fellowship (P.H.T.). We thank Debra Thompson (University of through an uncharacterized dehydrogenase activity (Jones et al., Michigan, Ann Arbor, MI) for generously supplying the 8B11 anti-RPE65 antibody, for extensive discussion of the 1989; Ala-Laurila et al., 2009). Rods lack this capability, and thus experiments, for undertaking experiments with our antibodies in her laboratory, and for comments on a previous version of this manuscript. We also thank Vladimir Kefalov (Washington University at St. Louis, St. Louis, MO), 11-cis-ROL is not a viable substrate for these photoreceptors Venkat Ramshesh, Joseph Palatinus, Patrice Goletz, Jie Fan, Luanna Bartholomew, Ryan Parker, Yiannis Koutalos, (Kono et al., 2008). Mu¨ller cells may contribute to the intraretinal Masahiro Kono, and Ba¨rbel Rohrer (Medical University of South Carolina, Charleston, SC) for technical assistance, visual cycle by producing 11-cis-ROL from all-trans ROL (Das et discussions, and manuscript revisions. al., 1992; Wang and Kefalov, 2009; Wang et al., 2009); however, Correspondence should be addressed to Rosalie K. Crouch, Department of Ophthalmology, Medical University of the mechanism is currently not known. South Carolina, 167 Ashley Avenue, Charleston, SC 29425. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.0182-11.2011 There is evidence to support the existence of RPE65 within Copyright © 2011 the authors 0270-6474/11/3110403-09$15.00/0 cones, in addition to the RPE. The detection of Rpe65 mRNA has 10404 • J. Neurosci., July 13, 2011 • 31(28):10403–10411 Tang et al. • RPE65 in Mouse Cone Photoreceptors Figure 1. Localization of RPE65 protein within the cone OS. A, Frozen sections prepared from 4-week-old C57BL/6 (top) and BALB/c (middle) eyecups and 2-week-old Rpe65Ϫ/Ϫ eyecups (bottom) were costained for RPE65 (PETLET antibody), S-opsin, and nuclei (DAPI). B, Analysis of sequential single-plane images acquired as a z-series (1–3) and the merged image (M) from a single-coneOSisdisplayed,showingthedistributionofRPE65.C,C57BL/6eyecupsectioncostainedforRPE65andGS,showingtheabsenceofRPE65intheMu¨llerglia.D,VisualizationofISinC57BL/6 mouse eyecup using propidium iodide (PI) allows for additional analysis of RPE65 localization within the cone, demonstrating that the majority of RPE65 is localized at the base of the cone OS. E, FITC-conjugated PNA was used to counterstain the glycoprotein sheath surrounding C57BL/6 mouse cone OS and IS, and a single-plane image was acquired to show the glycoprotein sheath surrounding RPE65 immunostaining within the OS. ONL, Outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. been reported in isolated salamander cones (Ma et al., 1998), and Materials and Methods immunohistochemistry (IHC) analysis of retinas from a variety Animals. Experimental procedures and animal care protocols adhered to of animal species confirmed the presence of the protein (Znoiko the Association for Research in Vision and Ophthalmology Statement for et al., 2002). These findings have generated debate because inde- the Use of Animals in Ophthalmic and Vision Research and were ap- pendent studies failed to reproduce the results (Seeliger et al., proved by the Institutional Animal Care and Use Committee of the Med- 2001; Hemati et al., 2005). The current study was undertaken to ical University of South Carolina. C57BL/6 mice were obtained from The reevaluate the localization of RPE65 protein within cones of dif- Jackson Laboratory. BALB/c, 129Sv, and albino C57BL/6 (C57BL/6-Tyrc/ ferent mouse strains/lines and to determine whether there is a BrdCrHsd) mice were obtained from Harlan Laboratories. Rpe65Ϫ/Ϫ relationship between the presence of RPE65 within cones and mice were a generous gift from T. Michael Redmond (National Eye In- their function. stitute, Bethesda, MD). All lines of mice used for experiments were F1 Tang et al. • RPE65 in Mouse Cone Photoreceptors J. Neurosci., July 13, 2011 • 31(28):10403–10411 • 10405 Figure 2. Specificity of anti-RPE65 antibodies. Immunostaining of the 8B11 anti-RPE65 antibody was evaluated with 4-week-old C57BL/6 retinas and eyecups. A, The 8B11 antibody failed to detect RPE65 within cones of the flat-mounted retina. B, Evaluation of eyecup sections provided confirmation, with 8B11 immunostaining detected only within the RPE. C, Immunoblot analysis of whole eyecup homogenates prepared from 2-week-old C57BL/6 and Rpe65Ϫ/Ϫ mice with the PETLET antibody showed detection of a 61 kDa major band corresponding to RPE65 protein in the C57BL/6 sample but not in the Rpe65Ϫ/Ϫ sample. The 8B11 antibody also detected RPE65 protein in the C57BL/6 sample but none in the Rpe65Ϫ/Ϫ sample. Blots were stripped and reprobed for ␤-actin, shown as insets. Arrows represent location of RPE65 band; M, marker; DIC, differential interference contrast; ONL, outer nuclear layer. progeny generated from mating the various strains,