Late-Onset Cone Photoreceptor Degeneration Induced by R172W Mutation in Rds and Partial Rescue by Gene Supplementation

A product of the retinal degeneration slow (RDS) gene, Rds is a tetraspanning membrane protein expressed in the disc rim of rods and cones.1 It has been extensively studied for the past 25 years, and more than 80 disease-causing-mutations in this gene have been identified (http://www.retina-international.com/sci-news/rdsmut.htm). One of the most common Rds mutations is a substitution of tryptophan for arginine at position 172 (R172W).2–5 This mutation has been described by multiple laboratories and occurs in a large number of families. Affected persons typically have faltering visual acuity in the third to fourth decade of life.2 Children and young adults carrying the mutation have been reported as asymptomatic5 but may exhibit macular changes. Functionally, patients carrying this mutation have normal rod, but diminished cone, electroretinographic (ERG) amplitudes and elongated implicit times. Rds was first identified in association with a naturally occurring mouse model called retinal degeneration slow (rds−/−), which does not form outer segments and has no detectable rod or cone ERG response. The hemizygous (rds+/−) mouse exhibits a haploinsufficiency phenotype associated with decreased rod and cone ERG responses and whorl-like, malformed outer segments.6 Rds is a structural protein present as a homodimer or a heterodimer complex, or as both, with its homologue, Rom-1 (rod outer segment membrane protein-1).7,8 Results of in vitro studies indicate that the large extracellular D2 loop of Rds (containing more than 70% of disease-causing-mutations) is the site of interactions to form Rds/Rom-1 complexes,7 whereas the C terminus of Rds protein is responsible for outer segment targeting of the protein.9 Rds is thought to have several hypothesized roles in the outer segment: (1) Rds/Rom-1 complexes function to keep the “hairpin rim” region of the rod outer segment disks intact10–12; (2) Rds/Rom-1 complexes potentially keep the disks connected to the rod outer segment plasma membrane through interactions with GARP (glutamic acid-rich protein) and the cyclic nucleotidegated ion channels of the plasma membrane13; (3) the C terminus of Rds directs complex targeting to the outer segment and helps align outer segment disks9; and (4) the C terminus of Rds may mediate disc membrane fusion,14–17 possibly in direct cooperation with other binding partners.18 Recent evidence from our group has suggested that Rds does not function the same way in cone photoreceptors that it does in rod photoreceptors. This observation came from studies of the cone-dominant Nrl/rds double knockout mouse,19 whose phenotype is distinct from that of both the Nrl−/− (neural retina leucine zipper)20 and the rds−/− retina. In contrast to the rds−/− retina, the all-cone Nrl−/−/rds−/− retina contains cone outer segments that, despite grossly aberrant morphology, are capable of robust phototransduction. Structurally, these outer segments are tubular and do not form lamellae. The reasons for the difference in Rds function in cones and rods are not fully understood, but this observation is supported by the clinical findings that RDS mutations can cause either rod-dominant (retinitis pigmentosa) or cone-dominant (macular dystrophy) retinal degeneration.21 Although the R172W mutation occurs within the D2 loop region of Rds, it is not located in the area required for complex assembly.7 In fact, it is unclear why the R172W mutation causes this distinct cone-associated phenotype. What is clear is the importance of arginine at position 172 to cone structure. Specifically, each of the three known substitutions at position 172 (R172W, R172G, R172Q) causes a cone- or a fovea-dominant defect in patients.4,21,22 In contrast, the N244K Rds mutation causes retinitis pigmentosa, whereas the N244H mutation causes macular dystrophy.23,24 To study the mechanism underlying the pathogenesis associated with the R172W mutation, we have generated and characterized multiple transgenic mouse lines carrying this mutation on different rds genetic backgrounds. In our previous work,25 we reported the biochemical characterization of the R172W protein in a high-expressing transgenic line (H-R172W), with a level corresponding to approximately 75% of the endogenous Rds. We showed that R172W Rds associates with Rom-1 and exhibits no defects in complex formation.25 These data were supported by findings by Molday et al.8 indicating that recombinant R172W protein was glycosylated and could properly form complexes in vitro. However, our in vivo study25 with the higher expresser line demonstrated that the R172W protein was more susceptible to partial tryptic digestion than wild-type (WT) Rds, suggesting a slight conformational change in complexes containing R172W Rds. In the present study, we characterized a new R172W model that closely mimics the human retinal phenotype and tested the efficacy of gene transfer therapy as a strategy to combat diseases associated with RDS mutations. As reported previously, the H-R172W line exhibits an early-onset dominant cone-rod dystrophy that is not characteristic of the retinal degeneration observed in human patients.25 Here we present additional data obtained with this model indicating that genetic supplementation with WT Rds provides partial, long-term rescue of rod function but no permanent rescue of cone function. Although functional dominance of the R172W allele is the most likely explanation for the lack of persistent cone rescue, conclusions regarding the potential usefulness of gene transfer as a treatment are limited by the severity of the H-R172W phenotype and its lack of correlation with the human disease phenotype. Therefore, we also have characterized an alternative R172W model (L-R172W), which expresses a lower amount of R172W protein. L-R172W mice exhibit late-onset cone degeneration without manifesting a dominant effect on rods, closely resembling the human clinical phenotype. Thus, the L-R172W line offers a useful model for future studies of the mechanism of late-onset macular dystrophy.