We have demonstrated that a rhodopsin point mutation, a c.553T>C for a p.C185R substitution, causes dominant RD in the R3 mouse line. The gene symbol for R3 mutation is designated as RhoR3. The Rho-C185R mutant protein fails to properly traffic in the rods and accumulates in inner segments of the photoreceptor cells. Downstream effects of abnormally accumulated mutant opsin proteins lead to a rapid loss of photoreceptor cells.
More than 100 human rhodopsin mutations, most of which are point mutations, have been reported to cause dominant RP. However, only a few rhodopsin mutations have been identified in animals thus far, including one naturally occurring rhodopsin mutation, T4R, in English Mastiff dogs22
and two chemically induced mouse rhodopsin mutations, C110Y23
and C185R. These three mutations closely mimic some of the phenotypic characteristics of human RP and are useful for investigating the biochemical mechanisms for RP caused by mutant opsins.24,25
It is unclear why such a small number of naturally occurring rhodopsin mutations have been identified in animals though rhodopsin mutations are prevalent in humans. Perhaps there is a strong evolutionary pressure against the reproduction of animals with vision loss, especially with early-onset blindness, in the natural environment.
Previous studies have demonstrated multiple mechanisms underlying retinal dysfunction caused by rhodopsin mutations, including responses of misfolded or mistrafficked proteins25–29
and the generation of constitutively active forms of rhodopsin through mutation of phosphorylation and arresting-binding shutoff mechanisms.30–32
The C185R mutant shares similarities with the C110Y and T4R mutants that affect the “plug” at the intradiscal (extracellular) side of the rhodopsin protein, which is responsible for protecting the chromophore from bulk water access.33,34
The structure in the extracellular, intradiscal domain of rhodopsin surrounding the Cys110-Cys187 disulfide bond has been shown to be important for the correct folding of this protein.35
residue also contributes to the formation of abnormal Cys185-Cys187 disulfide bonds detected in the mutant Rho-P23H human rhodopsin.
Opsin mutations account for 30% to 40% of identified autosomal dominant RP (adRP), and the P23H mutation is the most prevalent, causing approximately 10% of adRP in US Caucasians and showing a variable progression of RP in humans and in transgenic mice. The P23H mutation results in a misfolded apoprotein opsin, defined by a deficiency in its ability to bind 11-cis
retinal to form rhodopsin. Further studies suggest how abnormal Cys185-Cys187 disulfide bonds affect protein function. A detailed biochemical study of the C185A mutant protein, which loses its ability to form this abnormal disulfide bond, has revealed that the mutant protein is less thermally stable and has a slow rate for binding 11-cis
retinal. These results indicate that the C185A mutation alone destabilizes the open-pocket conformation of opsin.25
Structural modeling of the C185R mutant protein suggests a disruption of proper folding of the intradiscal domain. The accumulation of mutant proteins in inner segment reflects the mistrafficking of denatured proteins. Therefore, the C185R mutation likely destabilizes the open-pocket conformation and results in a misfolded rhodopsin protein.
Comparative studies of the rate of loss of photoreceptor cells in heterozygous (R3/+), homozygous (R3/R3), and compound mutant retinas (R3/−) with one null (knockout) allele provide valuable insight about the variable RD phenotypes in both humans and animal models. Genetic background and expression dosage variations in rhodopsin transgenic mice lead to a complex situation for the comparison of the rate of loss of photoreceptor cells between various animal models. The C185R mutation leads to rapid degeneration of rods resulting in a flat ERG, whereas only a single row of cones remains in R3/R3 mutant mice by P21. The R3/− mutant mice with one null allele, however, displayed less severe degeneration than homozygous mutant mice. Heterozygous R3/+ mutant mice with one wild-type allele developed the retinal degeneration phenotype more slowly (). Clearly, the presence of a wild-type rhodopsin allele delays but does not prevent photoreceptor loss because the R3/+ mutant mice had only a single outer nuclear layer, presumably the cone photoreceptors, by P42. The absence of outer segments, as observed in ultrastructure studies of R3/R3 homozygous mutant retinas at all ages examined, suggests that mutant rhodopsin proteins are improperly trafficked and sorted in inner segments and fail to form outer segments.
In conclusion, these results indicate that a dosage-dependent accumulation of C185R mutant proteins in photoreceptor cell inner segments likely triggers or promotes the death of photoreceptors. The ENU-mutagenized R3 mutant mouse line may have advantages over existing transgenic and knockout mouse models of retinal degeneration. Given that the mutation occurs in an endogenous rhodopsin gene, studies in this mutant mouse line avoid problems of transgene copy number and position effects in other genetically engineered animal models. Thus, the R3 mouse line is a useful model for dissecting the downstream stress signaling pathways activated by dosage-dependent accumulation of mutant proteins in rod photoreceptors.