Very Low Expression of Rho(P23H) Causes Aberrant Trafficking, Mislocalization of Mutant Rhodopsin and Dysfunction of Xenopus Photoreceptors

Abstract

Purpose: : To better understand the molecular basis for retinal degeneration in retinitis pigmentosa, we studied the subcellular localization and trafficking of the most common disease–related rhodopsin mutant, Rho(P23H).

Methods: : We prepared transgenic Xenopus tadpoles that expressed wild–type rhodopsin–EGFP (Rho–EGFP) and Rho(P23H)–EGFP fusion proteins in rod photoreceptors. Live and fixed retinas were examined by high resolution and quantitative confocal imaging at various stages of development to determine the localization and quantity of the fusion proteins. Visual function of the transgenic animals was measured electrophysiologically (by electroretinogram) and behaviorally.

Results: : On average 3 – 6 attomoles of Rho–EGFP or Rho(P23H)–EGFP were expressed per photoreceptor; this is ∼ 1/1000th of the mass of endogenous rhodopsin. In stage 40–43 tadpoles both Rho–EGFP and Rho(P23H)–EGFP were found in the outer segments (OS), and within the inner segment (IS) and in its plasma membrane. After stage 45, Rho–EGFP localized exclusively to the rod outer segment, whereas Rho(P23H)–EGFP continued to be detectable in both IS and OS. The fluorescence levels in the OS varied as a function of distance along the long axis of the rod with two distinct spatial frequencies, indicating variation in rhodopsin transport to this compartment. The high frequency pattern is reminiscent of the "Kaplan" banding pattern found with birefringence measurements (Kaplan, Vis. Res. 24:1163–8, 1984). Interestingly, this banding pattern was attenuated in Rho(P23H)–EGFP expressing cells. Rho(P23H) rods had shorter OS compared to those expressing Rho–EGFP, and there was an age–dependent loss of total retinal fluorescence accompanied by a 10–fold loss in retinal sensitivity by stage 55.

Conclusions: : 1. Quantitative analysis of EGFP transgene expression shows for the first time that expression levels as low as 1 Rho(P23H) per 1000 normal rhodopsin molecules is sufficient to induce retinal dysfunction. 2. That most Rho(P23H)–EGFP is transported to the outer segment establishes that Rho(P23H) is not excluded by the sorting and transport mechanisms that deliver rhodopsin to the outer segment.

Keywords: retinal degenerations: cell biology • photoreceptors • imaging/image analysis: non-clinical