Abstract
Abstract: :
Purpose: Previous studies have indicated the C–terminal tail of rhodopsin is an important sequence for proper transport to the photoreceptor outer segment (Deretic, Li, Tam). However, others have shown that a subset of opsins missing the critical 5 terminal residues still traffic to the outer segment (Sung, Chen). To study the transport mechanism of this mutant opsin we generated C–terminal truncated mutant mice on a rhodopsin knockout (KO) background. Rhodopsin mutants were also placed on a half KO background (1 wt opsin allele : 1 mutant) to determine if the mis–sorted rhodopsin is toxic to photoreceptors. Transgenic animals expressing truncation mutants also exhibit cell loss but this may be due to an overexpression defect. Methods: s334ter transgenic mice were crossed to rhodopsin (rho) knockout mice and their pups were mated together to generate animals on various KO backgrounds. Immunochemistry, Westerns, and IEM microscopy were performed on animals using opsin and rom–1 antibodies at P5–30. TEM and SEM microscopy were also performed. Retinal function of s334ter +, rho +/– mutants at ages 90 and 180 days postnatal were measured using full flash ERGs. ONL thickness was measured morphometrically. Wildtype, +/– rho KO, and –/– rho mice were used as controls. Results: Photoreceptors of s334ter +, rho –/– expressing low amounts of mutant opsin develop rudimentary membranes at the distal tip of the connecting cilium. Rom–1 is restricted to this outer segment domain while mutant opsin localizes to both inner segment and distal tip membranes. Prior to membrane accumulation, rom–1 and s334ter concentrate at the tip of the connecting cilium. Photoreceptors of s334ter +, rho +/– exhibit a decrease in A wave amplitudes upon flash stimulation with short ISI. The rate of photoreceptor degeneration was found to be faster in these animals compared to littermate heterozyote KO controls as measured by ERG and histology. Conclusions:A subset of C–terminally truncated opsin is able to traffic to the outer segment via a mechanism independent of its last 15 amino acids. Additionally, this mutant opsin does not need to co–transport with full–length opsin in order to be inserted into the outer segment. Another disk membrane protein, rom–1 is not affected by opsin mislocalization. Expression of truncated opsin was found to have a dominant negative affect in photoreceptors likely due to a toxic buildup of mutant opsin in the plasma membrane. Mice expressing truncated rhodopsin on a half knockout background will serve as an animal model that more closely mimics human ADRP.
Keywords: photoreceptors • retinal degenerations: cell biology • transgenics/knock–outs