Complete rescue of photoreceptor dysplasia and degeneration in transgenic retinal degeneration slow (rds) mice

Travis, Gabriel H.; Groshan, Karen; Lloyd, Monte; Bok, Dean

doi:10.1016/0896-6273(92)90226-4

Cited by 112 publications

(62 citation statements)

References 19 publications

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“…1A. The rom/D2 coding unit was assembled into a transgene construct containing 6.5 kilobase pairs from upstream of the mouse rhodopsin gene as a transcriptional regulator and the SV40 t-intron and early polyadenylation signal as a transcriptional terminator, similar to what we have used in other transgenic studies (21,24,25). The construct was confirmed by DNA sequence analysis prior to oocyte injection.…”

Section: Methodsmentioning

confidence: 99%

Analysis of the rds/peripherin·rom1 Complex in Transgenic Photoreceptors That Express a Chimeric Protein

Kędzierski

Weng

Travis

1999

Journal of Biological Chemistry

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Mice homozygous for the retinal degeneration slow (rds) mutation completely lack photoreceptor outer segments. The rds gene encodes rds/peripherin (rds), a membrane glycoprotein in the rims of rod and cone outer segment discs. rds is present as a complex with the related protein, rom1. Here, we generated transgenic mice that express a chimeric protein (rom/D2) containing the intradiscal D2 loop of rds in the context of rom1. rom/D2 was N-glycosylated, formed covalent homodimers, and interacted non-covalently with itself, rds, and rom1. The rds⅐rom/D2 interaction was significantly more stable than the non-covalent interaction between rds and rom1 by detergent/urea titration. Analysis of mice expressing rom/D2 revealed that rds is 2.5-fold more abundant than rom1, interacts non-covalently with itself and rom1 via the D2 loop, and forms a high order complex that may extend the entire circumference of the disc. Expression of rom/D2 fully rescued the ultrastructural phenotype in rds؉/؊ mutant mice, but it had no effect on the phenotype in rds؊/؊ mutants. Together, these observations explain the striking differences in null phenotypes and frequencies of diseasecausing mutations between the RDS and ROM1 genes.Rod and cone photoreceptors contain a structure, called the outer segment, comprising a stack of flattened membranous discs. These discs are the sites of photon capture and reactions of visual transduction. rds is a 36-kDa glycoprotein with four transmembrane segments located in the rims of rod and cone outer segment discs (1, 2). Although the function of rds is not known, the phenotype resulting from a null mutation in its gene has been described. Photoreceptors in retinal degeneration slow (rdsϪ/Ϫ) 1 mice completely fail to develop outer segments (3-5). Instead, rhodopsin-containing vesicles are present in the subretinal space between the photoreceptor cell bodies and retinal pigment epithelium (6 -8). Disc addition at the base of the outer segment is an ongoing process in mature photoreceptors, to compensate for the diurnal shedding of distal outer segments (9). The rdsϪ/Ϫ phenotype suggests that outer segment membranes are synthesized in these mutants but cannot fold into discs without rds. rdsϩ/Ϫ heterozygotes have a partial phenotype of short and grossly disorganized outer segments, which results from haploinsufficiency (10, 11).More than 50 mutations in the human RDS gene are responsible for several dominantly inherited retinal degenerations including retinitis pigmentosa (RP) (reviewed in Refs. 12 and 13). Most RDS mutations cause single-residue substitutions within the large intradiscal D2 loop of rds between transmembrane segments three and four (Fig. 1A). An unusual form of RP with digenic inheritance has been described (14). Affected individuals in three pedigrees were heterozygous for both a mutation in RDS, causing an L185P substitution in the D2 loop, and an early frameshift mutation in the unlinked gene for rom1 (14). Neither mutation alone caused disease.rom1 is a protein homologous to rds with an...

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Section: Methodsmentioning

confidence: 99%

Analysis of the rds/peripherin·rom1 Complex in Transgenic Photoreceptors That Express a Chimeric Protein

Kędzierski

Weng

Travis

1999

Journal of Biological Chemistry

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“…We introduced a T to C transition into codon 185, resulting in L185P. The construct was otherwise identical to a previously described transgene encoding normal rds (16). Fertilized oocytes of hybrid strain B6 ϫ DBA mice were microinjected with this construct.…”

Section: Methodsmentioning

confidence: 99%

“…Although the disrupted gene is transcribed in rdsϪ͞Ϫ photoreceptors (protected band visible in Fig. 1), no protein product is made (16,21). Thus, the spontaneous rds allele is null.…”

Section: Levels Of the Rds And Rom1 Mrnas In Transgenic Retinasmentioning

confidence: 99%

Deficiency of rds/peripherin causes photoreceptor death in mouse models of digenic and dominant retinitis pigmentosa

Kędzierski

Nusinowitz

Birch

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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Retinitis pigmentosa (RP) is a group of inherited blinding diseases caused by mutations in multiple genes including RDS. RDS encodes rds͞peripherin (rds), a 36-kDa glycoprotein in the rims of rod and cone outer-segment (OS) discs. Rom1 is related to rds with similar membrane topology and the identical distribution in OS. In contrast to RDS, no mutations in ROM1 alone have been associated with retinal disease. However, an unusual digenic form of RP has been described. Affected individuals in several families were doubly heterozygous for a mutation in RDS causing a leucine 185 to proline substitution in rds (L185P) and a null mutation in ROM1. Neither mutation alone caused clinical abnormalities. Here, we generated transgenic͞knockout mice that duplicate the amino acid substitutions and predicted levels of rds and rom1 in patients with RDS-mediated digenic and dominant RP. Photoreceptor degeneration in the mouse model of digenic RP was faster than in the wild-type and monogenic controls by histological, electroretinographic, and biochemical analysis. We observed a positive correlation between the rate of photoreceptor loss and the extent of OS disorganization in mice of several genotypes. Photoreceptor degeneration in RDS-mediated RP appears to be caused by a simple deficiency of rds and rom1. The critical threshold for the combined abundance of rds and rom1 is Ϸ60% of wild type. Below this value, the extent of OS disorganization results in clinically significant photoreceptor degeneration. R etinitis pigmentosa (RP) is a family of inherited retinal diseases characterized by progressive night blindness and loss of peripheral vision (1). Pathologically, RP is associated with degeneration of rod photoreceptors. Heterozygous mutations in the RDS gene are a common cause of autosomal dominant RP (2, 3). An example is the RDS P216L-allele (4). RDS encodes rds͞peripherin (rds), a 36-kDa glycoprotein in the rims of rod and cone outer-segment (OS) discs (5, 6). These stacked membranous structures are the sites of photon-capture and reactions of visual transduction. Rom1 is a related disk-rim protein with 37% overall identity and similar membrane topology to rds (7). Mice homozygous for a knockout mutation in the rom1 gene displayed mild OS dysplasia (8), in contrast to complete absence of OS in rdsϪ͞Ϫ mice (9-11). Unlike RDS, no mutations in the ROM1 gene alone have been convincingly associated with human retinal disease (12, 13). However, an unusual digenic form of RP has been described. Affected individuals in four pedigrees were doubly heterozygous for a mutation in RDS causing a leucine 185 to proline substitution in rds (L185P) and a second presumptive null mutation in the unlinked ROM1 gene (13,14). Neither mutation alone caused significant abnormalities.The phenotype in rdsϪ͞Ϫ mutant mice (9, 10) indicates a critical role for rds in the formation of OS. Expression of a chimeric protein in transgenic mice on an rdsϪ͞Ϫ geneticbackground established that rds is 2.5-fold more abundant than rom1, and that the rds-rom1 in...

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“…Germline transgenesis has been used to complement two mouse retinal degeneration mutations (15,16), but rat transgenic technology is more costly and not as widely used as that for mouse. We therefore chose to address the problem by somatic gene transfer with a viral vector.…”

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Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk

Vollrath

Feng

Duncan

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

264

188

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The Royal College of Surgeons (RCS) rat is a widely studied animal model of retinal degeneration in which the inability of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segments leads to a progressive loss of rod and cone photoreceptors. We recently used positional cloning to demonstrate that the gene Mertk likely corresponds to the retinal dystrophy (rdy) locus of the RCS rat. In the present study, we sought to determine whether gene transfer of Mertk to a RCS rat retina would result in correction of the RPE phagocytosis defect and preservation of photoreceptors. We used subretinal injection of a recombinant replication-deficient adenovirus encoding rat Mertk to deliver the gene to the eyes of young RCS rats. Electrophysiological assessment of animals 30 days after injection revealed an increased sensitivity of treated eyes to low-intensity light. Histologic and ultrastructural assessment demonstrated substantial sparing of photoreceptors, preservation of outer segment structure, and correction of the RPE phagocytosis defect in areas surrounding the injection site. Our results provide definitive evidence that mutation of Mertk underlies the RCS retinal dystrophy phenotype, and that the phenotype can be corrected by treatment of juvenile animals. To our knowledge, this is the first demonstration of complementation of both a functional cellular defect (phagocytosis) and a photoreceptor degeneration by gene transfer to the RPE. These results, together with the recent discovery of MERTK mutations in individuals with retinitis pigmentosa, emphasize the importance of the RCS rat as a model for gene therapy of diseases that arise from RPE dysfunction.T he Royal College of Surgeons (RCS) rat strain displays an unusual retinal phenotype in which shed photoreceptor (PR) outer segment (OS) debris accumulates in the subretinal space (1). PR disk shedding normally commences at postnatal day (P)12 in the rat (2), and an OS debris layer is readily apparent in the eyes of RCS animals at P20. Genetic chimera studies suggest that the defect is a cell-autonomous function of the RPE, not the PRs (3). Indeed, in vivo (4) and RPE cell culture studies (5) revealed a defect in the ability of RCS RPE to phagocytize shed OS membranes. Despite the RPE-specific nature of the defect, death strikes the PRs first, through the process of apoptosis (6). The time course of PR degeneration is rapid, beginning around P20, with few PR nuclei remaining in the outer nuclear layer (ONL) by P60. Thinning and atrophy of the RPE begins after onset of PR degeneration. The cause of PR cell death is not well understood; the debris zone acting as a diffusion barrier to metabolites (7), disruption of the interphotoreceptor matrix (8), and PR hypoxia due to diminished oxygen diffusion (9) have been proposed as explanations.In contrast to the extensively studied retinal phenotype of the RCS rat, the nature of the genetic defect has remained obscure until recently. Early genetic linkage studies showed that the phenotype is complet...

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Complete rescue of photoreceptor dysplasia and degeneration in transgenic retinal degeneration slow (rds) mice

Cited by 112 publications

References 19 publications

Analysis of the rds/peripherin·rom1 Complex in Transgenic Photoreceptors That Express a Chimeric Protein

Analysis of the rds/peripherin·rom1 Complex in Transgenic Photoreceptors That Express a Chimeric Protein

Deficiency of rds/peripherin causes photoreceptor death in mouse models of digenic and dominant retinitis pigmentosa

Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk

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