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Purpose GPR143 regulates melanosome biogenesis and organelle size in pigment cells. The mechanisms underlying receptor function remain unclear. G protein-coupled receptors (GPCRs) are excellent pharmacologic targets; thus, we developed and applied a screening approach to identify potential GPR143 ligands and chemical modulators. Methods GPR143 interacts with β-arrestin; we therefore established a β-arrestin recruitment assay to screen for compounds that modulate activity. Because GPR143 is localized intracellularly, screening with the wild-type receptor would be restricted to agents absorbed by the cell. For the screen we used a mutant receptor, which shows similar basal activity as the wild type but traffics to the plasma membrane. We tested two compound libraries and investigated validated hits for their effects on melanocyte pigmentation. Results GPR143, which showed high constitutive activity in the β-arrestin assay, was inhibited by several compounds. The three validated inhibitors (pimozide, niclosamide, and ethacridine lactate) were assessed for impact on melanocytes. Pigmentation and expression of tyrosinase, a key melanogenic enzyme, were reduced by all compounds. Because GPR143 appears to be constitutively active, these compounds may turn off its activity. Conclusions X-linked ocular albinism type I, characterized by developmental eye defects, results from GPR143 mutations. Identifying pharmacologic agents that modulate GPR143 activity will contribute significantly to our understanding of its function and provide novel tools with which to study GPCRs in melanocytes and retinal pigment epithelium. Pimozide, one of three GPR143 inhibitors identified in this study, maybe be a good lead structure for development of more potent compounds and provide a platform for design of novel therapeutic agents.

Purpose To identify the causes of autosomal dominant retinitis pigmentosa (adRP) in a cohort of families without mutations in known adRP genes and consequently to characterize a novel dominant-acting missense mutation in SAG. Methods Patients underwent ophthalmologic testing and were screened for mutations using targeted-capture and whole-exome next-generation sequencing. Confirmation and additional screening were done by Sanger sequencing. Haplotypes segregating with the mutation were determined using short tandem repeat and single nucleotide variant polymorphisms. Genealogies were established by interviews of family members. Results Eight families in a cohort of 300 adRP families, and four additional families, were found to have a novel heterozygous mutation in the SAG gene, c.440G>T; p.Cys147Phe. Patients exhibited symptoms of retinitis pigmentosa and none showed symptoms characteristic of Oguchi disease. All families are of Hispanic descent and most were ascertained in Texas or California. A single haplotype including the SAG mutation was identified in all families. The mutation dramatically alters a conserved amino acid, is extremely rare in global databases, and was not found in 4000+ exomes from Hispanic controls. Molecular modeling based on the crystal structure of bovine arrestin-1 predicts protein misfolding/instability. Conclusions This is the first dominant-acting mutation identified in SAG, a founder mutation possibly originating in Mexico several centuries ago. The phenotype is clearly adRP and is distinct from the previously reported phenotypes of recessive null mutations, that is, Oguchi disease and recessive RP. The mutation accounts for 3% of the 300 families in the adRP Cohort and 36% of Hispanic families in this cohort.

PURPOSE Previous studies discovered cone phototransduction shutoff occurs normally for Arr1-/- and Arr4-/-; however, it is defective when both visual arrestins are simultaneously not expressed (Arr1-/-Arr4-/-). We investigated the roles of visual arrestins in an all-cone retina (Nrl-/-) since each arrestin has differential effects on visual function, including ARR1 for normal light adaptation, and ARR4 for normal contrast sensitivity and visual acuity. METHODS We examined Nrl-/-, Nrl-/-Arr1-/-, Nrl-/-Arr4-/-, and Nrl-/-Arr1-/-Arr4-/- mice with photopic electroretinography (ERG) to assess light adaptation and retinal responses, immunoblot and immunohistochemical localization analysis to measure retinal expression levels of M- and S-opsin, and optokinetic tracking (OKT) to measure the visual acuity and contrast sensitivity. RESULTS Study results indicated that Nrl-/- and Nrl-/-Arr4-/- mice light adapted normally, while Nrl-/-Arr1-/- and Nrl-/-Arr1-/-Arr4-/- mice did not. Photopic ERG a-wave, b-wave, and flicker amplitudes followed a general pattern in which Nrl-/-Arr4-/- amplitudes were higher than the amplitudes of Nrl-/-, while the amplitudes of Nrl-/-Arr1-/- and Nrl-/-Arr1-/-Arr4-/- were lower. All three visual arrestin knockouts had faster implicit times than Nrl-/- mice. M-opsin expression is lower when ARR1 is not expressed, while S-opsin expression is lower when ARR4 is not expressed. Although M-opsin expression is mislocalized throughout the photoreceptor cells, S-opsin is confined to the outer segments in all genotypes. Contrast sensitivity is decreased when ARR4 is not expressed, while visual acuity was normal except in Nrl-/-Arr1-/-Arr4-/-. CONCLUSIONS Based on the opposite visual phenotypes in an all-cone retina in the Nrl-/-Arr1-/- and Nrl-/-Arr4-/- mice, we conclude that ARR1 and ARR4 perform unique modulatory roles in cone photoreceptors.

Leber congenital amaurosis (LCA) comprises a group of genetic disorders in which visual loss or dysfunction is present at birth. Patients typically have hyperopia and nystagmus and reduced electroretinograms (ERGs). The extent of visual loss varies from patient to patient but is usually severe. Mutations have been identified in 15 genes in persons with LCA, each of which is a recessive disorder.1,2 Mutations in the RPE65 gene account for approximately 7% of LCA. RPE65 is uniquely expressed in the retinal pigment epithelium (RPE), where the protein, an enzyme, binds and converts all-trans retinyl ester to 11-cis retinol.3–5 Retinol isomerization is an essential enzymatic step required for functional vision to occur in rod and cone photoreceptors.6,7 More than 60 mutations in the RPE65 gene have been documented in LCA patients. Mutations have been reported in each of the 14 exons of the RPE65 gene and its boundaries.8–14 Typically, mutations in the RPE65 gene result in impaired vision from birth and typically progress to legal blindness in the third decade of life.9,11,12,15,16 Mutations in RPE65 do not necessarily result in early loss of photoreceptors. For example, studies of dog retinas with a naturally occurring RPE65 mutation and mouse retinas that are missing the RPE65 gene show structurally intact photoreceptors visible by optical coherence tomography that appear nonfunctional because of the inability of the RPE to generate 11-cis retinal. The sparing of photoreceptors has allowed RPE65 gene replacement therapy to restore this critical retinol isomerase activity to the RPE with the accompanying restoration of visual function.17,18 In this report we describe the pathology and clinical findings in a woman with a homozygous mutation (Ala132Thr) in the RPE65 gene.12 Unlike most persons with RPE65 mutations, this patient retained some vision into her early fifties. To our knowledge this is the first study of adult postmortem donor eyes from a patient with a homozygous recessive mutation in the RPE65 gene.

The expression and activity of phosphoinositide 3-kinase (PI3K) and the formation of PI3K-generated D3 phosphoinositides have been demonstrated in intact retinal rod outer segment (ROS) membranes.1–4 To date, studies have implicated D3 phosphoinositides in a variety of cellular activities such as vesicular trafficking, cytoskeletal reorganization, cell growth, adhesion, and survival,4,5 as well as photoreceptor-specific functions such as modulation of phototransduction,6 disc biogenesis,7 protein translocation,8 synaptic ribbon formation, and glutamate release.9 These studies clearly demonstrate that phosphoinositides generated by PI3K could facilitate intracellular protein trafficking and modulate phototransduction. However, the functional role of PI3K in any of these activities in rod photoreceptors is not fully established. In adult retina, our previous in vivo studies have shown that moderate light exposure activates PI3K/Akt survival signaling pathway in rod photoreceptor outer segments through light-induced tyrosine phosphorylation of the retinal insulin receptor (IR).10,11 Also, in retina, deletion of the IR leads to photoreceptor degeneration due to increased susceptibility to damage from light-induced stress, in part because of the inability to activate the PI3K/Akt survival pathway.12 Receptor activation of PI3K has been shown to protect 661W,13,14 R28,15 and retinal ganglion cells16 from stress-induced cell death. These findings demonstrate the biological significance of the PI3K signaling pathway in neuroprotection of retinal photoreceptor cells. Systemic deletion of p85α regulatory or p110α catalytic subunits of PI3K results in embryonic or neonatal lethality.17,18 To circumvent the problems associated with the global PI3K KO and to investigate functional significance of PI3K in a particular tissue or subset of cells, several studies have used the Cre-lox technology. Conditional deletion of cardiac pik3r1 resulted in attenuated Akt signaling, reduced heart size, and altered cardiac gene expression.19 Conditional deletion of pik3r1 in skeletal muscle resulted in reduced muscle weight and size.20 The functional role of PI3K in the retina or photoreceptor cells is not known, although earlier studies from our laboratory have suggested its involvement in neuroprotection through the IR/PI3K/Akt signaling pathway.21–23 In the present study, using Cre-lox technology, we conditionally deleted the p85α (pik3r1) regulatory subunit of PI3K in rod photoreceptors to evaluate the functional significance of PI3K in these cells.

PURPOSE To test the efficiency of locally administrated tresperimus in experimental autoimmune uveoretinitis (EAU). METHODS EAU was induced in Lewis rats by S-antigen (S-Ag) immunization. Three intravitreal injections of tresperimus (prevention or prevention/treatment protocols) were performed at different time points after immunization. The pharmacokinetics of tresperimus was evaluated in the ocular tissues and plasma. The in vitro effect of tresperimus was evaluated on macrophages. EAU was graded clinically and histologically. Blood ocular barrier permeability was evaluated by protein concentration in ocular fluids. Immune response to S-Ag was examined by delayed type hypersensitivity, the expression of inflammatory cytokines in lymph nodes, ocular fluids and serum by multiplex ELISA, and in ocular cells by RT-PCR. RESULTS In vitro, tresperimus significantly reduced the production of inflammatory cytokines by lipopolysaccharide-stimulated macrophages. In vivo, in the treatment protocol, efficient tresperimus levels were measured in the eye but not in the plasma up to 8 days after the last injection. Tresperimus efficiently reduced inflammation, retinal damage, and blood ocular barrier permeability breakdown. It inhibited nitric oxide synthase-2 and nuclear factor κBp65 expression in ocular macrophages. IL-2 and IL-17 were decreased in ocular media, while IL-18 was increased. By contrast, IL-2 and IL-17 levels were not modified in inguinal lymph nodes draining the immunization site. Moreover, cytokine levels in serum and delayed type hypersensitivity to S-Ag were not different in control and treated rats. In the prevention/treatment protocol, ocular immunosuppressive effects were also observed. CONCLUSIONS Locally administered tresperimus appears to be a potential immunosuppressive agent in the management of intraocular inflammation.

Downregulation of the retinal insulin/mTOR pathway in mouse models of retinitis pigmentosa is linked to cone cell death, which can be delayed by systemic administration of insulin. A classic survival kinase linking extracellular trophic/growth factors with intracellular antiapoptotic pathways is phosphoinositide 3-kinase (PI3K), which the authors have shown to protect rod photoreceptors from stress-induced cell death. The role of PI3K in cones was studied by conditional deletion of its p85α regulatory subunit.Mice expressing Cre recombinase in cones were bred to mice with a floxed pi3k gene encoding the p85α regulatory subunit of the PI3K and were back-crossed to ultimately generate offspring with cone-specific p85α knockout (cKO). Cre expression and cone-specific localization were confirmed by Western blot analysis and immunohistochemistry (IHC), respectively. Cone structural integrity was determined by IHC using peanut agglutinin and an M-opsin-specific antibody. Electroretinography (ERG) was used to assess rod and cone photoreceptor function. Retinal structure was examined by light and electron microscopy.An age-related cone degeneration was found in cKO mice, evidenced by a reduction in photopic ERG amplitudes and loss of cone cells. By 12 months of age, approximately 78% of cones had died, and progressive disorganization of synaptic ultrastructure was noted in surviving cone terminals in cKO retinas. Rod viability was unaffected in p85α cKO mice.The present study suggests that PI3K signaling pathway is essential for cone survival in the mouse retina.

Photoreceptor cyclic nucleotide-gated (CNG) channels are localized to the plasma membrane of photoreceptor outer segments and play a pivotal role in phototransduction.1,2 In the dark, cGMP binding activates rod CNG channels, allowing a steady cation current to flow into the outer segments. Light triggers a sequence of enzymatic reactions that leads to hydrolysis of cGMP, causing the channel to close. On channel closure, the inward current ceases and the cell hyperpolarizes, inhibiting synaptic transmission to second-order neurons. An analogous phototransduction scheme exists in cones, which are responsible for color vision and vision in bright illumination when rods are saturated. However, light sensitivity, cGMP sensitivity, Ca2+ permeation, and functional modulation differ profoundly between rod and cone CNG channels.3–5 CNG channels are composed of two structurally related subunit types, the A and B subunits. The rod channel consists of CNGA1 and CNGB1 subunits, whereas the cone channel is composed of CNGA3 and CNGB3 subunits. Heterologous expression studies have shown that the A subunits are responsible for the ion-conducting activity of the channel, whereas the B subunits function as modulators.2,6 Mutations in the rod CNG channel were identified in patients with retinitis pigmentosa,7 whereas mutations in the cone CNG channel have been associated with a variety of cone diseases, including achromatopsia, progressive cone dystrophy, and early-onset macular degeneration.8–11 To date more than 70 mutations have been identified in genes encoding the human CNGA3 and CNGB3 subunits, with mutations in CNGB3 alone found in more than 50% of patients with achromatopsia,9 a retinal disorder that affects approximately 1 in every 33,000 Americans. The condition is associated with color blindness, visual acuity loss, extreme light sensitivity, and nystagmus.8,11,12 The most frequently occurring mutation in CNGB3 is the Thr383fsx mutation, which accounts for more than 80% of all CNGB3 mutant alleles.9,13,14 This frame-shift mutation results in truncation of the pore-forming loop and the C-terminal cytoplasmic domain; as a result, no intact CNGB3 is formed. Thus, a loss-of-function defect is the primary mechanism of action for CNGB3 mutation in humans. Although CNGB3 shares a common topology with CNGA3 and has a pore-forming region, this subunit, when expressed alone, does not form a functional channel, in contrast to CNGA3, which forms a functional homomeric channel.6,15 Heteromeric CNGA3/CNGB3 channels display properties typical of native CNG channels,6,16 suggesting that CNGB3 modulates the channel's physiological properties in vivo. Our previous experimental work showing the interaction between CNGA3 and CNGB3 in the mouse retina17 and cone defects in CNGB3−/− mice18 strongly supports this view. Here we investigate the progression of cone defects caused by CNGB3 deficiency. CNGB3−/− mice displayed an early-onset, slow progression of cone dysfunction and degeneration, similar to the human achromatopsia found in patients with mutations in CNGB3. Furthermore, photoreceptor terminals appeared normal in the CNGB3−/− retina, suggesting that deficits in cone signaling result from disrupted phototransduction and cone loss rather than from synaptic defects. Thus, the CNGB3−/− mouse line will be a valuable model with which to study the functional and structural role of CNGB3 in cones and in retinal pathogenesis associated with achromatopsia.

Purpose To characterize light and circadian control of photoreceptor phagocytosis in a diurnal cone-rich rodent. Methods Diurnal Arvicanthis ansorgei were maintained under standard cyclic light (12 hours 300 lux white light [L]/12 hours dark [D]) and were divided into five groups: 1, maintained in LD; 2, transferred to constant darkness (DD); 3, transferred to constant light (LL, 300 lux); 4, subjected to 6-hour advance in light onset; and 5, subjected to 6-hour delay in light onset. Animals were killed every 3 hours during 24 hours, and their eyes were rapidly enucleated and fixed. Cryosections were stained using specific rod (rhodopsin) and cone (MW cone opsin) antibodies. Immunopositive inclusions within the retinal pigment epithelium layer were quantified for each time point. Results In LD, both rod and cone phagocytosis showed coincident synchronized profiles with sharp peaks 1 to 2 hours after light onset. In groups 2 and 3, phagocytic activity shifted partially to the new light schedules. In DD, the temporal phagocytic profile resembled largely that of LD. On the contrary, LL animals exhibited large alterations in both rod and cone phagocytosis. There was no longer any peak, with both showing relatively uniform profiles. In addition, the number of cone phagosomes was much higher ( approximately 250% increase) compared with LD or DD. Conclusions These data are the first to measure photoreceptor phagocytosis in a diurnal mammal under different lighting conditions and to highlight the disruptive effects of constant light, especially on cone photoreceptor function.

Rhodopsin mutations are common, with more than 100 distinct rhodopsin mutations described in patients who have autosomal dominant retinitis pigmentosa (ADRP).1 Symptoms of RP include loss of sensitivity to dim light, abnormal visual function, and characteristic bone spicule deposits of pigment in the retina. Affected individuals progressively lose visual field and visual acuity, and photoreceptor cell death can ultimately lead to blindness. Although mutations in several photoreceptor-specific and some nonspecific genes cause RP, mutations in rhodopsin are the most prevalent class identified to date, resulting in 30% to 40% of all ADRP.2 A fraction of these rhodopsin mutations alter the C-terminal tail of the protein, such as the point mutations P347L, P347S, P347R, and V345M.3–5 In addition, two frame-shift mutations (fs 341del and fs 341-343del) are predicted to add additional residues to the C terminus,6 whereas Q344ter results in a C-terminal truncation, and an intron splice mutation (N88) is thought to remove the entire C-terminal tail of rhodopsin.7,8 Previously, transgenic mice were generated with a C-terminal truncation S334ter to remove all the putative sites of rhodopsin shutoff by arrestin and phosphorylation, to study the effect on phototransduction.9 With the subsequent identification of several mutations within this region in patients with RP, these mice have become increasingly useful in the study of RP pathogenesis, as well as intracellular sorting of rhodopsin in photoreceptors. Other transgenic mouse models, Q334ter and P347S, have been created with mutations in the C-terminal cytoplasmic domain.10,11 In these animals, extracellular mislocalization of rhodopsin P347S, and intracellular delocalization of Q344ter to inner segment and synaptic terminal membranes strongly suggest a role for the C-terminal tail in opsin sorting and transport. Rhodopsin trafficking has been further investigated in vitro and in vivo with other systems. Evidence supporting the role of the C-terminal residues for proper sorting into post-Golgi vesicles has been obtained with a retinal cell-free system.12 The final five amino acids of the C-terminal (QVAPA in human) were found to be critical for this sorting process. In other studies, the dynein light chain Tctex-1 has been shown to interact directly with the C-terminal tail of rhodopsin and has been hypothesized to mediate transport from the Golgi to the apical inner segment membrane.13 Transgenic Xenopus have also been used to examine the distribution of rhodopsin tail–GFP fusion proteins, uncovering transport signals in the last 44 amino acids that are sufficient for outer segment localization, and specifically, the last eight amino acids directed a nonpolarized protein exclusively to the outer segments.14 In addition to delocalizing, a fraction of GFP-tail fusion proteins lacking these eight C-terminal amino acids was present within the outer segment, despite removal of its transport signal. The authors suggest this may occur through cotransport with full-length rhodopsin. This mechanism has been independently suggested by another group based on observations that outer segments failed to form in photoreceptors expressing truncated rhodopsin on a rhodopsin-knockout background.15 However, this hypothesis has yet to be studied in detail and warrants further examination, to understand the transport mechanism of truncated rhodopsin in rod photoreceptors. Aberrant transport and mislocalization of truncated rhodopsin may have a detrimental effect on photoreceptors’ health and survival. Similar to transgenic Q344ter rhodopsin mice, transgenic animals expressing S334ter rhodopsin lacking the terminal 15 amino acids mislocalize a fraction of truncated opsin to ectopic photoreceptor membranes and undergo a progressive retinal degeneration.8,15,16 Various studies, however, have documented that overexpression of rhodopsin itself causes photoreceptor cell death and may induce photoreceptor cell loss in transgenic animals expressing truncated rhodopsin on a wild-type (WT) genetic background.11,17,18 Therefore, it is still unknown whether truncated rhodopsin is harmful to photoreceptors in these transgenic animals. With the creation of rhodopsin-knockout mice,19,20 it is now possible to lower the concentration of endogenous rhodopsin in vivo by genetically removing one or both rhodopsin allele(s), and this technique has been used in several studies.15,21,22 In one, Concepcion et al.15 placed S334ter transgenic mice on a +/− endogenous rhodopsin genetic background, and did not observe greater photoreceptor degeneration than in control mice. However, outer nuclear layer (ONL) thickness was observed only up to P50, and long-term effects on photoreceptors were not assessed. In the current report, we expanded on the work of Concepcion et al.15 by completing a long-term time-course study to determine whether truncated rhodopsin induces photoreceptor degeneration. We also performed detailed immunoelectron microscopy after the subcellular trafficking of S334ter rhodopsin in the absence of endogenous rhodopsin to determine whether the truncated form reaches the outer segment independent of WT rhodopsin. Truncated rhodopsin was found to affect rod function and increased the rate of photoreceptor degeneration. In addition, our data indicate that a fraction of truncated rhodopsin reaches the outer segment without requiring cotransport along with normal rhodopsin.

Purpose To identify H-2 Kb/Db-binding immunogenic peptides derived from retinal proteins. Methods Computer-based prediction was used to identify potentially H-2 Kb/Db-binding peptides derived from the interphotoreceptor retinol-binding protein (IRBP), soluble retinal antigen (S-antigen), recoverin, phosducin, and pigment epithelium-derived factor (PEDF). The affinity of the peptides was analyzed by their abilities to upregulate the expression of major histocompatibility complex (MHC) class I on TAP-deficient cells (RMA-S cells) with flow cytometry. C57BL/6 mice were immunized subcutaneously, with individual peptides in incomplete Freund's adjuvant (IFA). Eight days after immunization, splenocytes were isolated for cytotoxic T-lymphocyte (CTL) analysis. A 51chromium-release assay was used to detect specific CTL reactivity generated in the cultures. Eyes were enucleated for histopathological analysis on day 21 after immunization with IRBP or IRBP and the immunogenic peptides. Results All the 21 predicted peptides were found to upregulate expression of H-2 Kb/Db on RMA-S cells. Five peptides, the two IRBP-derived peptides IRBP89-96 and IRBP(101-108), and the three PEDF-derived peptides, PEDF389-397, PEDF139-147, and PEDF272-279, induced specific CTL responses in vivo, whereas the remaining 16 peptides, including 5 IRBP-derived peptides, 5 S-antigen-derived peptides, 1 recoverin-derived peptide, 1 phosducin-derived peptide, and 4 PEDF-derived peptides, did not induce specific CTL reactivity. The immunogenic peptides alone did not induce inflammation in the eyes, but they could enhance severity of uveitis induced by IRBP. Conclusions Five of 21 H-2 Kb/Db-binding retinal protein-derived peptides were found to be immunogenic, suggesting that these peptides could function as autoantigenic epitopes in the development of inflammatory eye diseases, such as uveitis.

PURPOSE. To characterize rod and cone distribution, organization, and phagocytosis in the diurnal mouse-like rodent Arvicanthis ansorgei. METHODS. Retinas of adult A. ansorgei were processed for histology, electron microscopy and immunohistochemistry using rod- and mouse cone-specific antibodies. For phagocytosis studies, retinas were sampled every 3 hours under a 12-hour light- dark cycle and processed for double-label immunohistochemistry. The number of phagosomes in the retinal pigmented epithelium were quantified with a morphometric system. RESULTS. A. ansorgei retinas were composed of 33% cones and 67% rods, approximately 10 times more cones than mice and rats. Cones were arranged in two cell layers at the scleral surface, distributed uniformly across the entire retina. Cone arrestin was distributed throughout the dark-adapted cones, from outer segments to synapses, whereas short- and mid-wavelength cone opsins were restricted to outer segments. Short-wavelength cone density was mapped in wholemounted retinas, in a significantly higher number in the central region. Rhodopsin immunopositive (rod) phagosomes showed a small peak late in the dark phase, then a large burst 1 to 2 hours after light onset, after decreasing to low baseline levels by 12 AM. Mid-wavelength cone opsin immunopositive (cone) phagosomes were 10 times less numerous than rods, and demonstrated a broad peak 1 to 2 hours after light onset. CONCLUSIONS. The diurnal rodent A. ansorgei possesses a large number of cones, organized in a strict anatomic array. Rod and cone outer segment phagocytosis and shedding can be monitored simultaneously and show similar profiles but different amplitudes. This species may constitute a valuable novel animal model for investigating cone pathophysiology.

To test the hypothesis that inter- and intramolecular spreading to S-antigen (S-Ag) and interphotoreceptor retinoid binding protein (IRBP)-derived epitopes occurs in a spontaneous model of recurrent uveitis in the horse.The immune response of eight horses with equine recurrent uveitis (ERU) was compared with that of five control horses with healthy eyes. Lymphocytes derived from peripheral blood (PBLs) were tested every 8 weeks for their reactivity against S-Ag and various S-Ag and IRBP-derived peptides for 12 to 39 months (median, 22 months). During uveitic episodes, additional blood samples were analyzed.Intermolecular epitope spreading was detectable in all ERU cases during the study. Intramolecular spreading occurred in seven (of eight) horses with ERU. Fourteen relapses were analyzed during the observation period. Ten uveitic episodes were accompanied by neoreactivity to S-Ag or IRBP-derived peptides during the relapse. Shifts in the immune response profile were also detectable without any clinical signs of inflammation. Eye-healthy control horses were negative at all time points in the in vitro proliferation assays.Inter- and intramolecular spreading was detectable in a spontaneous model of recurrent uveitis. The shifts in immunoreactivity could account for the remitting-relapsing character of the disease.

PURPOSE.—Light-driven protein translocation is responsible for the dramatic redistribution of some proteins in vertebrate rod photoreceptors. In this study, the involvement of microtubules and microfilaments in the light-driven translocation of arrestin and transducin was investigated. METHODS.—Pharmacologic reagents were applied to native and transgenic Xenopus tadpoles, to disrupt the microtubules (thiabendazole) and microfilaments (cytochalasin D and latrunculin B) of the rod photoreceptors. Quantitative confocal imaging was used to assess the impact of these treatments on arrestin and transducin translocation. A series of transgenic tadpoles expressing arrestin truncations were also created to identify portions of arrestin that enable arrestin to translocate. RESULTS.—Application of cytochalasin D or latrunculin B to disrupt the microfilament organization selectively slowed only transducin movement from the inner to the outer segments. Perturbation of the microtubule cytoskeleton with thiabendazole slowed the translocation of both arrestin and transducin, but only in moving from the outer to the inner segments. Transgenic Xenopus expressing fusions of green fluorescent protein (GFP) with portions of arrestin implicates the C terminus of arrestin as an important portion of the molecule for promoting translocation. This C-terminal region can be used independently to promote translocation of GFP in response to light. CONCLUSIONS.—The results show that disruption of the cytoskeletal network in rod photoreceptors has specific effects on the translocation of arrestin and transducin. These effects suggest that the light-driven translocation of visual proteins at least partially relies on an active motordriven mechanism for complete movement of arrestin and transducin. Transport of molecules is critical to the proper functioning of cells, but even more so for polarized cells. In neurons, perhaps the epitome of polarized cells, molecular transport uses both fast and slow components to renew membrane lipids and protein elements in the membrane and cytosol. Rod photoreceptors are arguably one of the most highly polarized cells with regard to both structure and function. Structurally, rods are divided into two segments, the rod outer segment (ROS) and the rod inner segment (RIS), joined by a narrow connecting cilium. The ROS contains a highly elaborate system of stacked, disc-shaped membranes that are densely packed with the visual pigment rhodopsin, whereas the RIS is more typical of the

The neural retina leucine zipper (Nrl) gene encodes a Maf-family transcription factor that regulates the expression of several rod photoreceptor genes.1–3 Missense mutations in NRL are associated with autosomal dominant retinitis pigmentosa in humans.4 Deletion of Nrl in mice (Nrl−/−) results in the complete absence of rods in the retina, as revealed by histology, immunocytochemistry, electrophysiology, and gene expression analysis. Instead, there is a concomitant increase in short-wave–sensitive photoreceptor cells, apparently generated by switching of rod to cone cell fate during development.5 In the absence of Nrl, the mouse retina lacks a scotopic response and has a short-wavelength sensitivity enhanced sixfold in its photopic electroretinogram.5 These results suggest that the Nrl−/− mouse may be a useful mammalian model for the investigation of cone function, cone biochemistry, and cone-specific disease, but the initial investigation proposed that Nrl−/− photoreceptors are cone–rod intermediates (or “cods”), which function as cones but do not elaborate their full differentiation program.5 The potential of the Nrl−/− mouse as a preparation for the investigation of cone function and disease cannot be fully realized until the classification of the Nrl−/− photoreceptors is resolved. To make a definitive characterization of Nrl−/− photoreceptors, we undertook a thorough analysis of the photoreceptor layer of the Nrl−/− mouse with light and electron microscopy, of the levels of proteins known to be expressed specifically in cones, and of the electrical responses of the photoreceptors in vivo by using single- and paired-flash methods to record and analyze the a-wave of the ERG. The comparison with results from WT mice showed Nrl−/− photoreceptors to exhibit a large set of molecular, ultrastructural, histochemical, and kinetic features highly distinct from the corresponding features of WT rods, but which correspond, with noted exceptions, to the features of WT cones.

PURPOSE. Biochemical analysis of age-related macular degeneration (AMD) at distinct stages of the disease will help further understanding of the molecular events associated with disease progression. This study was conducted to determine the ability of a new grading system for eye bank eyes, the Minnesota Grading System (MGS), to discern distinct stages of AMD so that retinal region-specific changes in rod photoreceptor protein expression from donors could be determined. METHODS. Donor eyes were assigned to a specific level of AMD by using the MGS. Expression of the rod photoreceptor proteins rhodopsin and arrestin was evaluated by Western immunoblot analysis in the macular and peripheral regions of the neurosensory retina from donors at different stages of AMD. RESULTS. A significant linear decline in both arrestin and rhodopsin content correlated with progressive MGS levels in the macula. In contrast, the peripheral region showed no significant correlation between MGS level and the content of either protein. CONCLUSIONS. The statistically significant relationship between decreasing macular rod photoreceptor proteins and progressive MGS levels of AMD demonstrates the utility of the clinically based MGS to correspond with specific protein changes found at known, progressive stages of degeneration. Future biochemical analysis of clinically characterized donor eyes will further understanding of the pathobiochemistry of AMD. (Invest Ophthalmol Vis Sci. 2005;46:769‐775) DOI:10.1167/ iovs.04-0810

PURPOSE. Cone arrestin (CAR) is highly expressed in all cone photoreceptors of the retina and a subset of pinealocytes in the pineal gland. This study was initiated to examine the ciselements responsible for the cell-specific expression pattern of CAR. METHODS. Mutagenesis and specific deletions of known ciselements in the proximal promoter of the mouse CAR (mCAR) gene were introduced and analyzed in vitro and in vivo. A series of mCAR promoter-luciferase reporter constructs were transiently transfected into COS-7 or Weri-Rb-1 retinoblastoma cells and tested in in vitro promoter assays. Transgenic Xenopus laevis were created with deletional or mutated promoter fragments driving an enhanced green fluorescent protein (EGFP) reporter gene. The resultant EGFP expression pattern in the transgenic animals was analyzed by fluorescence microscopy and immunocytochemistry. RESULTS. A significant decrease in in vitro transcriptional activities was observed when the minimal 215-bp promoter fragment was mutated in each of the four cone-rod homeobox (CRX)-binding elements (CBEs) or in either of the two TATAelements. The 215-bp mCAR proximal promoter drove EGFP expression to cone photoreceptors and pinealocytes in transgenic Xenopus laevis; however, the truncated 147-bp fragment drove expression to both cone and rod photoreceptors. Transgenic tadpoles carrying a single mutation in either the TATA-box, the TATA-element or the proximal CBE had undetectable EGFP expression in the retina. However, when one of the other three CBEs was mutated, EGFP expression was observed in muscle and brain tissues, in addition to the eyes. Also, when both TATA elements were mutated, transgenic animals had EGFP expression in all photoreceptors. Because no reporter activity was observed when either a 3.2-kb 5 extended region or the first intron of the mCAR gene was tested in Weri-Rb-1 cells, neither construct was examined in vivo. CONCLUSIONS. The data demonstrate that the regulatory functions of the known cis-elements in the mCAR promoter are highly dependent on location and nucleotide sequence conservation. The TATA elements and CBEs are crucial for driving both basal transcriptional activity and tissue specificity to cone photoreceptors and pinealocytes. (Invest Ophthalmol Vis Sci. 2004;45:3877‐3884) DOI:10.1167/iovs.04-0663

Retinal cell culture has been a useful tool for ocular research. Although it does not replace the intact eye, retinal cell cultures provide convenient experimental systems for the assessment of numerous retinal processes. As with any in vitro systems, cell culture offers great advantages, but not without potential limitations. Advantages include controllable conditions that allow for the assessment of isolated cellular functions, a more affordable system when compared with the more costly animal research, and time course flexibility. Potential limitations include loss of native tissue architecture, lack of functional feedback from other retinal cell types, and a questionable correlation between in vitro and in vivo findings. However, for a great number of research applications, the advantages offered by in vitro systems outweigh the limitations. Retinal cell culture can be routinely used to determine the cell specificity of promoter sequences,1 the effect of mutations on the structure and function of retinal proteins,2 or the role of multiple domains on the function of retinal proteins.3 Furthermore, retinal cell culture has been applied in studies of cell growth, death, differentiation, and cytotoxicity (for review, see Ref. 4). Photoreceptor cells are terminally differentiated, specialized neuronal cells with a limited capacity for cell division. Therefore, to establish a line of photoreceptor cells, it is essential to transform them, possibly with a virus. Immortalized cell lines of several ocular cell types currently exist, including Muller,5 ganglion,6 corneal endothelial,7 and RPE cells.8 A cell line expressing retina-specific genes, including interphotoreceptor retinol-binding protein (IRBP) and cone transducin, has been isolated from a mouse ocular tumor.9 In addition, Y-7910 and WERI-Rb11 are immortalized human retinoblastoma cell lines available for the study of photoreceptors. Initially, it was thought that the Y-79 cells were of cone cell origin,12 but more recently these cells have been shown to express rod-specific antigens, such as opsin, transducin, phosphodiesterase, and recoverin.13,14 Primary retinal cultures have been created from several vertebrate retinas, including those of humans.15 These types of cultures, in addition to being tedious to prepare, are not adequate for some types of studies, because of their heterogeneity, limited cell division, and special conditions for growth as monolayers. Thus, there is a need for additional photoreceptor cell models that are homogeneous, passageable, and easily grown as a monolayer by using standard tissue culture techniques. Herein, we describe a mouse photoreceptor-derived cell line (661W) immortalized by the expression of simian virus (SV)40 T antigen (T-ag) under control of the human IRBP promoter.16 Cellular, and molecular analyses show that these cells express cone but not rod photoreceptor markers, which suggests that the cells arise from a cone photoreceptor lineage. For this reason, the 661W cell line should contribute significantly to the study of cone photoreceptor cell function and of diseases affecting cone photoreceptor cells, including mechanisms of photoreceptor cell death in various retinal dystrophies.

PURPOSE. To investigate the effects of basic fibroblast and epidermal growth factor (FGF2 and EGF, respectively) on the survival and phenotypic expression of photoreceptors isolated from adult mammalian retinas. METHODS. Primary cultures highly enriched in photoreceptors were prepared from adult domestic pig retinas and maintained in chemically defined medium. Cell culture composition was characterized through the use of specific antibody markers of retinal neurons, and neuronal survival was quantified through viability assays as a function of time in the presence or absence of different doses of FGF2 and EGF. Western blot analysis of phosphotyrosine residues was used to monitor activation of FGF2 and EGF signaling pathways. RESULTS. Reproducible survival of adult pig rod and cone photoreceptors was obtained for approximately 2 weeks in vitro, with the continued expression of rod opsin, recoverin, S-antigen, cone arrestin, and neuron-specific enolase. Purity of cultures was routinely more than 95% photoreceptors, with a rod-cone ratio of 2:3.1. Photoreceptor survival was stable for the initial week, decreasing slowly during the second, with rapid cell loss occurring thereafter. In the presence of FGF2 (20 ng/mL), the percentage of photoreceptor survival during the second week in culture was statistically significantly different, at least two times higher than in control experiments. Photoreceptor survival correlated directly with increasing concentrations of FGF2, and also of EGF. Combined treatment with FGF2 and EGF did not induce higher survival than either factor alone. There was no detectable selective loss of rods or cones in the experimental model. Phosphotyrosine immunoblots after stimulation of cultures with FGF2 and EGF revealed time-dependent appearance of multiple immunoreactive bands. CONCLUSIONS. The adult pig photoreceptor culture in the current study exhibits reproducible neuronal survival in vitro and represents a useful novel experimental system for the study of potential neuroprotective effects and signaling pathways of neurotrophic factors such as FGF2 and EGF in fully adult higher mammalian retina.

PURPOSE: To elucidate the antigen recognized by monoclonal antibody (mAb) 7G6, a widely used cone-specific marker. METHODS: 7G6 immunocytochemistry was performed on sections of human, primate, and bovine retina. The antigen was immunoprecipitated from human retinal lysates and purified with protein G. Edman degradation and liquid chromatography of tryptic peptides combined with tandem mass spectrometry (LC-MS/MS) identified the antigen. RESULTS: Sequencing of peptides derived from the immunoprecipitated 7G6 antigen identified it as cone arrestin. The identity was confirmed by Western blot analysis with recombinant human cone arrestin and competition with the antibody in immunocytochemistry. Subcellular localization of cone arrestin in dark-adapted and bleached bovine retinas showed that cone arrestin accumulated in cone outer segments of light-adapted retina but was more concentrated in the inner segments of dark-adapted retina. By expression of truncated human cone arrestin mutants systematically deleting areas divergent from bovine and primate cone arrestins, the epitope of 7G6 was identified as a divergent loop exposed at the surface within the N-domain of cone arrestin. CONCLUSIONS: Several independent methods established that the 7G6 antigen is cone arrestin. The 7G6 epitope is contained in a divergent loop, the sequence of which is conserved in bovine and primates but not other vertebrate species consistent with the specificity of the antibody. The light-dependent translocation of cone arrestin suggests a role in light-dark adaptation of cones. Because of the location of its gene on the X-chromosome, cone arrestin is a candidate gene for X-linked cone dystrophies. Supported by NIH Grant R01EY08123 (WB). Additional support came from the Macular Vision Research Foundation, Research to Prevent Blindness, Inc., and a Center grant from the Foundation Fighting Blindness to the University of Utah. WB is the recipient of a Senior Investigator Award from RPB and a Ralph and Mary Tuck endowment to the Department of Ophthalmology at the University of Utah.