The R172W mutation in peripherin/rds causes a cone-rod dystrophy in transgenic mice

Abstract

Peripherin/rds (P/rds) is a membrane glycoprotein essential for the photoreceptor outer segment disc morphogenesis and maintenance. More than half of the disease-causing mutations in P/rds have been linked to different forms of macular dystrophy; the most common one is substitution of tryptophan for arginine at position 172 (R172W). Here we confirm the patient phenotype associated with the expression of R172W mutation in transgenic mice. Functional, structural and biochemical analyses showed that, while R172W P/rds is appropriately localized, a direct correlation exists between transgene expression levels and the onset/severity of the phenotype. In the wild-type background, both cone and rod photoreceptors' structure and function were significantly diminished, which indicates a dominant-negative, cone-rod defect. Whereas rds(+/-) mice maintained the normal cone function at early ages, cone responses in R172W/rds(+/-) mice were diminished to 41% of the wild-type level signifying a preferential damaging effect of the mutation on cones. Conversely, R172W/rds(+/-) mice showed a significant rescue of rod function and improvement of rod outer segment structure. Although rds(-/-) mice have no detectable rod or cone responses, R172W/rds(-/-) animals retained 30% of wild-type structure and rod function, but no significant rescue of cone function was detected at 1 month of age. No biochemical abnormalities were observed in complex formation and association with Rom-1; however, R172W protein was more sensitive to tryptic digestion, indicative of a change in protein conformation, possibly contributing to the cone-dominated phenotype. As the first animal model for P/rds-associated cone-rod dystrophy, R172W mice provide a valuable tool for studying the pathophysiology of P/rds-associated human retinal dystrophies and the development of therapeutic strategies to intervene in these diseases.

Differences in RDS trafficking, assembly and function in cones versus rods: insights from studies of C150S-RDS

“…To confirm that these mice stably expressed the transgene in the appropriate cells, paraffin-embedded retinal sections taken from eyes collected at postnatal (P) day 30 were co-labeled with monoclonal antibody (mAB) 3B6 and our well-characterized RDS-CT polyclonal antibody. mAB 3B6 recognizes only transgenic protein (Materials and Methods) (13,21,22) while RDS-CT recognizes both endogenous and transgenic protein (13,23). As shown in Supplementary Material, Figure S1, the COP-T protein is not ectopically expressed, and is detected panretinally in the photoreceptor cell layers of the COP-T/ rds +/+ /nrl 2/2 .…”

“…Rabbit polyclonal RDS-CT, ROM1-CT and S-opsin antibodies were generated in house, characterized previously (3,13,23) and used at dilutions of 1 : 1000 for western blot (WB) and immunofluorescence (IF) and 1 : 10 for EM/immunogold. RDS-CT recognizes both endogenous and, to a lesser extent, transgenic RDS (21,22).…”

“…Insoluble protein was removed by a 25 min centrifugation at 54 000 rpm in a Sorvall Discovery M150 ultracentrifuge using a fixed angle rotor after 1 h incubation at 48C. Reducing and non-reducing SDS -PAGE/WB (20 mg protein for detection of RDS, ROM1 and S-opsin or 75 mg for detection of M-opsin, which is present at lower levels in the nrl 2/2 than RDS, ROM1 and S-opsin) and IP (300 mg protein) were undertaken as described previously (13,23). WB densitometry was completed on at least three blots per protein imaged using a Kodak Image Station 4000r and Kodak MI Software v.4.0.3 (Carestream Health, Rochester, NY, USA).…”

“…ERGs were performed as described previously (3,23,25). Briefly, following overnight dark-adaption mice were anesthetized by intramuscular injection of 85 mg/kg ketamine and 14 mg/kg xylazine (Pharmaceutical Systems, Inc., Tulsa, OK, USA).…”

Retinal Degeneration Slow (RDS) Glycosylation Plays a Role in Cone Function and in the Regulation of RDS·ROM-1 Protein Complex Formation

“…Over 80 individual mutations of the RDS/PRPH2 gene in humans have been associated with significant diseases of the retina that can range from the rod-dominant retinitis pigmentosa to the cone-dominant diseases cone-rod dystrophy and macular degeneration (12). Additionally, extensive studies in mouse models have clearly demonstrated the importance of the RDS protein to retinal structure and function (2,(13)(14)(15)(16)(17)(18). In mice heterozygous for the Rds gene (rds ϩ/Ϫ ), the OSs become highly disorganized, and the discs adopt a bizarre whorl-like morphology.…”