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Proof-of-concept for a successful adeno-associated virus serotype 5 (AAV5)-mediated gene therapy in X-linked juvenile retinoschisis (XLRS) has been demonstrated in an established mouse model for this condition. The initial studies concentrated on early time-points of treatment. In this study, we aimed to explore the consequences of single subretinal injections administered at various stages of more advanced disease. By electroretinogram (ERG), functional improvement in treated versus untreated eyes is found to be significant in retinoschisin-deficient mice injected at the time-points of 15 days (P15), 1 month (PM1), and 2 months (PM2) after birth. In mice treated at 7 months after birth (PM7), an age previously shown to exhibit advanced retinal disease, ERG responses reveal no beneficial effects of vector treatment. Generally, functional rescue is paralleled by sustained retinoschisin expression and significant photoreceptor survival relative to untreated eyes. Quantitative measures of photoreceptors and peanut agglutinin–labeled ribbon synapses demonstrate rescue effects even in mice injected as late as PM7. Taken together, AAV5-mediated gene replacement is beneficial in slowing disease progression in murine XLRS. In addition, we show the effectiveness of rescue efforts even if treatment is delayed until advanced signs of disease have developed. Human XLRS patients might benefit from these findings, which suggest that the effectiveness of treatment appears not to be restricted to the early stages of the disease, and that treatment may prove to be valuable even when administered at more advanced stages.

A 240-kDa protein exhibiting immunochemical cross-reactivity with red blood cell spectrin has been shown to be directly associated with the 63-kDa cGMP-gated channel of bovine rod outer segments. When detergent-solubilized, chromatographically purified channel preparations were treated with Sepharose beads coupled to either an anti-240-kDa monoclonal antibody (PMs 4B2) or an anti-63-kDa channel monoclonal antibody (PMc 1D1), both the 240-kDa protein and the 63-kDa channel protein were concomitantly immunoprecipitated as analyzed by Western blotting of sodium dodecyl sulfate gels. Both of these antibody-Sepharose matrices also removed cGMP-gated channel activity as measured by functional reconstitution. In control studies anti-rhodopsin monoclonal antibody (Rho 1D4)-Sepharose beads removed residual rhodopsin, but not the 63/240-kDa complex or channel activity. Western blotting of purified rod outer segment disk and plasma membrane fractions and immunogold-dextran labeling of lysed rod outer segments indicated that the 240-kDa polypeptide, like the 63-kDa channel, is preferentially localized to the plasma membrane as visualized by electron microscopy. The 240-kDa protein does not appear to be directly involved in the cGMP-gated channel activity, but it may be part of a cytoskeletal system that serves to maintain the organization of the 63-kDa channel complex within the rod outer segment plasma membrane.

The cGMP-gated channel and the peripherin/rds-rom-1 complex are two oligomeric membrane proteins that play key roles in the structure and function of photoreceptor outer segments. The channel is localized on the plasma membrane where it controls the flow of Na+ and Ca2+ into the outer segment in response to light-induced changes in cGMP. The rod channel consists of two homologous subunits, designated alpha and beta, which assemble into a heterotetrameric complex. Both subunits contain a core structural unit consisting of six transmembrane segments, a pore region and a cGMP binding domain. The alpha subunit is the dominant functional subunit since it forms a functional channel by itself. The beta subunit does not assemble into a functional channel by itself, but modulates the activity of the channel. The peripherin/rds-rom-1 complex is localized along the rim region of disk membranes where it plays a crucial role in disk morphogenesis. This complex consists of two peripherin/rds and two rom-1 subunits that interact non-covalently to form a heterotetramer. Peripherin/rds is the dominant subunit since, in the absence of rom-1, it self-assembles into a homotetramer that effectively supports outer segment disk formation and structure. Rom-1 on its own does not initiate outer segment formation. Instead, it plays a minor role in fine tuning disk structure. Recently, peripherin/rds-containing tetramers have been shown to form disulfide-mediated higher-order oligomers. This novel oligomerization is suggested to play a central role in outer segment disk formation.

Retinoschisin or RS1 is a discoidin domain-containing protein encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration characterized by a splitting of the retina. Retinoschisin, expressed and secreted from photoreceptors and bipolar cells as a homo-octameric complex, associates with the surface of these cells where it serves to maintain the cellular organization of the retina and the photoreceptor-bipolar synaptic structure. To gain insight into the role of retinoschisin in retinal cell adhesion and the pathogenesis of XLRS, we have investigated membrane components in retinal extracts that interact with retinoschisin. Unlike the discoidin domain-containing blood coagulation proteins Factor V and Factor VIII, retinoschisin did not bind to phospholipids or retinal lipids reconstituted into unilamellar vesicles or immobilized on microtiter plates. Instead, co-immunoprecipitation studies together with mass spectrometric-based proteomics and Western blotting showed that retinoschisin is associated with a complex consisting of Na/K ATPase (alpha3, beta2 isoforms) and the sterile alpha and TIR motif-containing protein SARM1. Double labeling studies for immunofluorescence microscopy confirmed the co-localization of retinoschisin with Na/K ATPase and SARM1 in photoreceptors and bipolar cells of retina tissue. We conclude that retinoschisin binds to Na/K ATPase on photoreceptor and bipolar cells. This interaction may be part of a novel SARM1-mediated cell signaling pathway required for the maintenance of retinal cell organization and photoreceptor-bipolar synaptic structure.

X-linked retinoschisis (RS) is an inherited recessive macular degeneration that affects between 1 in 5000 and 1 in 25,000 males early in life (George et al., 1995; Sieving, 1998; Tantri et al., 2004). It is characterized by a loss in central vision, splitting of the retina with the appearance of spoke-like cystic cavities radiating from the parafoveal region of the retina, a loss in the b-wave of the electroretinogram (ERG), and progressive atrophy of the macula. In about 50% of the cases, bilateral schisis is observed in the peripheral retina with some loss in peripheral vision. During the course of the disease, complications can arise which include retinal detachment, vitreal hemorrhage and choroidal sclerosis.

Purpose: Gene-based therapies are making a comeback, as evidenced by the clinical success of Glybera, rAAV2. RPE65, and rAAV2. REP1. These therapies utilize ubiquitous promoters, however future gene therapies may benefit from promoters that can direct tissue- and cell-type specific expression. Capturing the expression of a human gene in a promoter small enough to use in AAV is challenging. Paired box six (PAX6) encodes a transcription factor with a complex and potentially therapeutically-useful retinal-expression pattern. Also, PAX6 has several previously published discrete regulatory regions (RRs), and therefore may contain modular regulation suitable for MiniPromoter design. Finally, mutations in PAX6 cause the vision-loss disorder aniridia, and a MiniPromoter capable of recapitulating the expression of PAX6 would be a useful tool for an aniridia gene therapy.Methods: PAX6 RRs were predicted using Hi-C, UCSC Genes, CAGE, 100 vertebrate phastCons, transcription factor binding site (TFBS), Segway and ChromHMM data. MiniPromoters were cloned into AAV genomes containing an emGFP reporter and WPRE, packaged into AAV2(Y272F, Y444F, Y500F, Y730F, T491V), and administered to postnatal day 14 mice by intravitreal injection. Ocular tissue was collected 30 days after injection, emGFP expression was evaluated by epifluorescent imaging of mouse retinas, and staining with antibodies against PAX6, Brn3a, syntaxin, and calbindin. MiniPromoters driving PAX6-like expression were examined for unique TFBSs using oPOSSUM 3.Results: A PAX6 containing highly interactive domain was revealed within which most previously published PAX6 RRs are located. Within this domain, 31 RRs were bioinformatically predicted, nine of which were selected to construct seven MiniPromoters. Of these MiniPromoters, two did not drive interesting emGFP expression in the retina, and five drove expression in the two retinal layers that express PAX6 (ganglion cell and inner nuclear). Twenty-one TFBSs were found to be unique to these MiniPromoters. Interestingly, one of the five (Ple255) not only drives expression in the same cell layers that express PAX6, but also in all the same cell types (ganglion, amacrine, and horizontal).Conclusions: Bioinformatic approaches can be used to design MiniPromoters capturing the endogenous expression pattern of a gene for a specific tissue. The resulting MiniPromoters may be important tools for future retinal gene therapy, with Ple255 being especially applicable to gene therapy for aniridia.

X-linked juvenile retinoschisis (RS) is a common cause of juvenile macular degeneration in males. RS is characterized by cystic spoke-wheel-like maculopathy, peripheral schisis, and a negative (b-wave more reduced than a-wave) electroretinogram (ERG). These symptoms are due to mutations in the RS1 gene in Xp22.2 leading to loss of functional protein. No medical treatment is currently available. We show here that in an Rs1h-deficient mouse model of human RS, delivery of the human RS1 cDNA with an AAV vector restored expression of retinoschisin to both photoreceptors and the inner retina essentially identical to that seen in wild-type mice. More importantly, unlike an earlier study with a different AAV vector and promoter, this work shows for the first time that therapeutic gene delivery using a highly specific AAV5–opsin promoter vector leads to progressive and significant improvement in both retinal function (ERG) and morphology, with preservation of photoreceptor cells that, without treatment, progressively degenerate.

MPP4 and MPP5 are closely related members of the p55-subfamily of membrane-associated guanylate kinases (MAGUKs) known to mediate the assembly of protein complexes at the plasma membrane of cell-cell junctions. Both MPP4 and MPP5 have been implicated in retinal function; however, their specific roles in the cellular mechanisms underlying vision are largely unknown. Here, we generated specific poly- and monoclonal antibodies against the two proteins and show that MPP4 and MPP5 are localized at distinct sites of cell-cell contact in the mouse retina. While MPP4 is a component of the synaptic terminals of photoreceptors, MPP5 exclusively localizes to apical membrane domains of the outer limiting membrane (OLM) junctions. The vertebrate homologs of Caenorhabditis elegans lin-7, Veli1, -2, and -3, have previously been identified as putative binding partners of MPP5. In this study, we show that MPP4 directly interacts with the Veli proteins via L27 heterodimerization in vitro. In addition, two of the three Veli isoforms, Veli1 and -3, are demonstrated to be expressed in the mouse retina. Immunofluorescence microscopy reveals extensive colocalization of Veli3 with both MPP4 and MPP5. This association of Veli3 with either MPP4 or MPP5 suggests that the MAGUKs recruit Veli3 and its binding partners to different cellular regions of the retina where they may participate in the organization of specialized intercellular junctions.

Peripherin 2 is a photoreceptor-specific membrane protein implicated in outer segment disk morphogenesis and linked to various retinopathies including autosomal dominant retinitis pigmentosa (ADRP). Peripherin 2 and ROM1 assemble as a mixture of core noncovalent homomeric and heteromeric tetramers that further link together through disulfide bonds to form higher order oligomers. These complexes are critical for disk rim formation and outer segment structure through interaction with the cGMP-gated channel and other photoreceptor proteins. We have examined the role of subunit assembly in peripherin 2 targeting to disks, outer segment structure, and photoreceptor degeneration by examining molecular and cellular properties of peripherin 2 mutants in COS-1 cells and transgenic Xenopus laevis rod photoreceptors. Wild-type (WT) and the ADRP-linked P216L mutant were transported and incorporated into newly formed outer segment disks of transgenic X. laevis. The P216L mutant, however, induced progressive outer segment instability and photoreceptor degeneration possibly through the introduction of a new N-linked oligosaccharide chain. In contrast, the C214S and L185P disease-linked, tetramerization-defective mutants, were retained in the inner segment, but did not affect outer segment structure or induce photoreceptor degeneration. Together, these results indicate that peripherin 2 mutations can cause ADRP either through a deficiency in WT peripherin 2 (C214S, 1.185P) or by a dominant negative effect on disk stability (P216L).

ABCR, also known as ABCA4, is a member of the superfamily of ATP binding cassette transporters that is believed to transport retinal or retinylidene-phosphatidylethanolamine across photoreceptor disk membranes. Mutations in the ABCR gene are responsible for Stargardt macular dystrophy and related retinal dystrophies that cause severe loss in vision. ABCR consists of two tandemly arranged halves each containing a membrane spanning segment followed by a large extracellular/lumen domain, a multi-spanning membrane domain, and a nucleotide binding domain (NBD). To define the role of each NBD, we examined the nucleotide binding and ATPase activities of the N and C halves of ABCR individually and co-expressed in COS-1 cells and derived from trypsin-cleaved ABCR in disk membranes. When disk membranes or membranes from co-transfected cells were photoaffinity labeled with 8-azido-ATP and 8-azido-ADP, only the NBD2 in the C-half bound and trapped the nucleotide. Co-expressed half-molecules displayed basal and retinal-stimulated ATPase activity similar to full-length ABCR. The individually expressed N-half displayed weak 8-azido-ATP labeling and low basal ATPase activity that was not stimulated by retinal, whereas the C-half did not bind ATP and exhibited little if any ATPase activity. Purified ABCR contained one tightly bound ADP, presumably in NBD1. Our results indicate that only NBD2 of ABCR binds and hydrolyzes ATP in the presence or absence of retinal. NBD1, containing a bound ADP, associates with NBD2 to play a crucial, non-catalytic role in ABCR function.

Deleterious mutations in RS1 encoding retinoschisin are associated with X-linked juvenile retinoschisis (RS), a common form of macular degeneration in males. The disorder is characterized by a negative electroretinogram pattern and by a splitting of the inner retina. To gain further insight into the function of the retinoschisin protein and its role in the cellular pathology of RS, we have generated knockout mice deficient in Rs1h , the murine ortholog of the human RS1 gene. We show that pathologic changes in hemizygous Rs1h −/Y male mice are evenly distributed across the retina, apparently contrasting with the macula-dominated features in human. Similar functional anomalies in human and Rs1h −/Y mice, however, suggest that both conditions are a disease of the entire retina affecting the organization of the retinal cell layers as well as structural properties of the retinal synapse.

The rod cGMP-gated channel is localized in the plasma membrane of rod photoreceptor outer segments, where it plays a central role in phototransduction. It consists of alpha- and beta-subunits that assemble into a heterotetrameric protein. Each subunit contains structural features characteristic of nucleotide-gated channels, including a cGMP-binding domain, multiple membrane-spanning segments, and a pore region. In addition, the beta-subunit has a large glutamic acid- and proline-rich region called GARP that is also expressed as two soluble protein variants. Using monoclonal antibodies in conjunction with immunoprecipitation, cross-linking, and electrophoretic techniques, we show that the cGMP-gated channel associates with the Na/Ca-K exchanger in the rod outer segment plasma membrane. This complex and soluble GARP proteins also interact with peripherin-2 oligomers in the rim region of outer segment disc membranes. These results suggest that channel/peripherin protein interactions mediated by the GARP part of the channel beta-subunit play a role in connecting the rim region of discs to the plasma membrane and in anchoring the channel.exchanger complex in the rod outer segment plasma membrane.

Publisher Summary Cyclic nucleotide-gated (CNG) channels comprise a family of cation-selective channels that are cooperatively activated or inhibited by the binding of cGMP or cAMP. In vertebrate rod and cone photoreceptor cells and olfactory receptor neurons, these channels play a central role in sensory transduction pathways by controlling the flow of Na+ and Ca2+ into the cell in response to signal-mediated changes in intracellular cyclic nucleotide levels. The physiologic role of CNG channels in nonsensory cells, however, has yet to be determined. The CNG channels of rod photoreceptors and olfactory neurons have been most extensively studied at a molecular level. This chapter describes several procedures that have been used to purify the cGMP-gated channel from ROS membranes and reconstitute the channel into lipid vesicles for functional analysis. The application of these methods to the isolation of CNG channels from other cells systems is discussed.

The cGMP-gated channel of the rod photoreceptor cell plays a key role in phototransduction by controlling the flow of Na+ and Ca2+ into the outer segment in response to light-induced changes in cGMP concentrations. The rod channel is composed of two homologous subunits designated as alpha and beta. Each subunit contains a core region of six putative membrane spanning segments, a cGMP binding domain, a voltage sensor-like motif and a pore region. In addition the beta-subunit contains an extended N-terminal region that is identical in sequence to a previously cloned retinal glutamic acid rich protein called GARP. Three spliced variants of GARP (the GARP part of the beta channel subunit; full length free GARP; and a truncated form of GARP) are expressed in rod cells and localized within the outer segments. Immunoaffinity chromatography has been used to purify the channel from detergent solubilized rod outer segments. A significant fraction of the rod Na+/Ca(2+)-K+ exchanger copurifies with the channel as measured by western blotting suggesting that the channel can interact with the exchanger under certain conditions.

Publisher Summary An important step in the analysis of the molecular structure, function, and morphogenesis of rod cell outer segment (ROS) is the development of preparative procedures to purify intact ROS from retinal tissue extracts, separate the soluble or cytosolic fraction from the ROS membrane, and isolate the disk and plasma membranes. This chapter describes the methods necessary to purify and fractionate ROS and explains the compositional analysis of defined ROS fractions. A number of methods have been developed to isolate ROS from rod-dominant retinas and, in particular, from bovine and frog retinas. Although sucrose gradients have been most widely used, other gradients, such as Ficoll-sucrose, have also produced relatively pure and intact ROS. For frog ROS preparations, Percoli gradient centrifugation is particularly effective to isolate the intact ROS. The chapter also describes the separation of ROS into soluble and membrane fractions.

The outer segments of vertebrate rod photoreceptor cells consist of an ordered stack of membrane disks, which, except for a few nascent disks at the base of the outer segment, is surrounded by a separate plasma membrane. Previous studies indicate that the protein, peripherin or peripherin/rds, is localized along the rim of mature disks of rod outer segments. A mutation in the gene for this protein has been reported to be responsible for retinal degeneration in the rds mouse. In the present study, we have shown by immunogold labeling of rat and ground squirrel retinas that peripherin/rds is present in the disk rims of cone outer segments as well as rod outer segments. Additionally, in the basal regions of rod and cone outer segments, where disk morphogenesis occurs, we have found that the distribution of peripherin/rds is restricted to a region that is adjacent to the cilium. Extension of its distribution from the cilium coincides with the formation of the disk rim. These results support the model of disk membrane morphogenesis that predicts rim formation to be a second stage of growth, after the first stage in which the ciliary plasma membrane evaginates to form open nascent disks. The results also indicate how the proteins of the outer segment plasma membrane and the disk membranes are sorted into their separate domains: different sets of proteins may be incorporated into membrane outgrowths during different growth stages of disk morphogenesis. Finally, the presence of peripherin/rds protein in both cone and rod outer segment disks, together with the phenotype of the rds mouse, which is characterized by the failure of both rod and cone outer segment formation, suggest that the same rds gene is expressed in both types of photoreceptor cells.

Retinal degeneration slow (rds) is a retinal disorder of an inbred strain of mice in which the outer segment of the photoreceptor cell fails to develop. A candidate gene has recently been described for the rds defect [Travis, G. H., Brennan, M. B., Danielson, P. E., Kozak, C. & Sutcliffe, J. G. (1989) Nature (London) 338, 70-73]. Neither the identity of the normal gene product nor its intracellular localization had been determined. We report here that the amino acid sequence of the bovine photoreceptor-cell protein peripherin, which was previously localized to the rim region of the photoreceptor disk membrane, is 92.5% identical to the sequence of the mouse protein encoded by the normal rds gene. The differences between the two sequences can be attributed to species variation. Monoclonal antibodies were used with Western blot analysis to localize the wild-type mouse peripherin/rds protein to isolated mouse rod outer segments and to show that it, like bovine peripherin, exists as two subunits linked by one or more disulfide bonds. The relative amounts of peripherin/rds protein and rhodopsin in retinal extracts of normal and rds mutant mice were also compared. Identification of peripherin as the protein encoded by the normal rds gene and its localization to membranes of rod outer segments will serve as a basis for studies directed toward defining the role of this protein in the morphogenesis and maintenance of the outer segment and toward understanding the mechanism by which the rds mutation causes retinal degeneration.

Mutations in the gene encoding ABCR are responsible for Stargardt macular dystrophy. Here we show by immunofluorescence microscopy and western-blot analysis that ABCR is present in foveal and peripheral cone, as well as rod, photoreceptors. Our results suggest that the loss in central vision experienced by Stargardt patients arises directly from ABCR-mediated foveal cone degeneration.

The plasma membrane and disk membranes of bovine retinal rod outer segments (ROS) have been purified by a novel density-gradient perturbation method for analysis of their protein compositions. Purified ROS were treated with neuraminidase to expose galactose residues on plasma membrane-specific glycoproteins and labeled with ricin-gold-dextran particles. After the ROS were lysed in hypotonic buffer, the plasma membrane was dissociated from the disks by either mild trypsin digestion or prolonged exposure to low ionic strength buffer. The dense ricin-gold-dextran-labeled plasma membrane was separated from disks by sucrose gradient centrifugation. Electron microscopy was used to follow this fractionation procedure. The dense red pellet primarily consisted of inverted plasma membrane vesicles containing gold particles; the membrane fraction of density 1.13 g/cc consisted of unlabeled intact disks and vesicles. Ricin-binding studies indicated that the plasma membrane from trypsin-treated ROS was purified between 10-15-fold. The protein composition of plasma membranes and disks was significantly different as analyzed by SDS gels and Western blots labeled with lectins and monoclonal antibodies. ROS plasma membrane exhibited three major proteins of 36 (rhodopsin), 38, and 52 kD, three ricin-binding glycoproteins of 230, 160, and 110 kD, and numerous minor proteins in the range of 14-270 kD. In disk membranes rhodopsin appeared as the only major protein. A 220-kD concanavalin A-binding glycoprotein and peripherin, a rim-specific protein, were also present along with minor proteins of 43 and 57-63 kD. Radioimmune assays indicated that the ROS plasma membrane contained about half as much rhodopsin as disk membranes.

Two ricin-specific glycoproteins have been identified on neuraminidase-treated rod outer segment plasma membranes of bovine retinal photoreceptor cells. Ricin-gold-dextran particles were observed by electron microscopy to densely label the surface of neuraminidase-treated rod outer segments. Western blotting of proteins separated by SDS-gel electrophoresis indicated that two ricin-binding glycoproteins of M r 230 000 and 110 000 are specific for the plasma membrane and are not found in disk membranes. These glycoproteins can serve as specific probes for the purification of the rod outer segment plasma membrane.