Subcellular localization of mutant P23H rhodopsin in an RFP fusion knockin mouse model of retinitis pigmentosa

The P23H mutation in rhodopsin (Rho), the visual pigment protein in rod photoreceptor neurons, is the most common genetic cause of autosomal dominant retinitis pigmentosa (adRP), a retinal disease that causes blindness. Despite multiple studies in animal models, the subcellular details of the fate of misfolded mutant Rho in rod photoreceptors have not been completely defined. We generated a new mouse model of adRP, in which the P23H-Rho mutant allele is fused to the fluorescent protein Tag-RFP-T (P23HhRhoRFP). In heterozygotes, outer segments formed, and WT rhodopsin was properly localized there, but mutant P23H-Rho protein was specifically mislocalized in the inner segments of rods. Despite this cellular phenotype, the P23HhRhoRFP heterozygous mice exhibited only slowly progressing retinal degeneration; in ERG recordings, scotopic a-wave amplitudes were reduced by 24% and 26% at 30 days and 90 days respectively, and the corresponding scotopic b-waves by 18% and 24%. Outer nuclear layer thickness was still 80% of WT at 90 days, but at 364 days had declined to 40% of WT. Transmission electron microscopy revealed greatly expanded membrane lamellae in the inner segment, and by fluorescence imaging, we determined that the mislocalized P23HhRhoRFP was contained in greatly expanded endoplasmic reticulum (ER) membranes. TUNEL staining revealed a slow pace of cell death involving chromosomal endonucleolytic degradation. Quantification of mRNA for markers of ER stress and the unfolded protein response revealed little or no increases in levels of messages encoding the proteins BiP, CHOP, ATF6, XBP1, PERK, Eif2 and Derlin-1, but a decreased level of total Rhodopsin (mouse + human) mRNA levels. The decline in the rate of cell death after an initial burst suggests that P23HhRhoRFP mutant rods undergo an adaptative process that prolongs survival despite gross P23HhRhoRFP protein accumulation in the ER. Because of its slowly progressing nature, and easy visualization of the mutant protein, the P23H-Rho-RFP mouse may represent a useful tool for the future study of the pathology and treatment of P23H-Rho and adRP.