Rhodopsin Mutant P23H Destabilizes Rod Photoreceptor Disk Membranes

Abstract

Mutations in rhodopsin cause retinitis pigmentosa in humans and retinal degeneration in a multitude of other animals. We utilized high-resolution live imaging of the large rod photoreceptors from transgenic frogs (Xenopus) to compare the properties of fluorescently tagged rhodopsin, Rho-EGFP, and RhoP23H-EGFP. The mutant was abnormally distributed both in the inner and outer segments (OS), accumulating in the OS to a concentration of ∼0.1% compared to endogenous opsin. RhoP23H-EGFP formed dense fluorescent foci, with concentrations of mutant protein up to ten times higher than other regions. Wild-type transgenic Rho-EGFP did not concentrate in OS foci when co-expressed in the same rod with RhoP23H-EGFP. Outer segment regions containing fluorescent foci were refractory to fluorescence recovery after photobleaching, while foci in the inner segment exhibited recovery kinetics similar to OS regions without foci and Rho-EGFP. The RhoP23H-EGFP foci were often in older, more distal OS disks. Electron micrographs of OS revealed abnormal disk membranes, with the regular disk bilayers broken into vesiculotubular structures. Furthermore, we observed similar OS disturbances in transgenic mice expressing RhoP23H, suggesting such structures are a general consequence of mutant expression. Together these results show that mutant opsin disrupts OS disks, destabilizing the outer segment possibly via the formation of aggregates. This may render rods susceptible to mechanical injury or compromise OS function, contributing to photoreceptor loss.

Inherent Instability of the Retinitis Pigmentosa P23H Mutant Opsin

“…Thus, irregular disc orientations have been reported in P23H/ϩ knock-in mice (24). In addition, aggregated punctate fluorescence was observed in the discs of P23H-EGFP TG Xenopus, but not WT-rhodopsin-EGFP TG Xenopus, indicating that P23H opsin aggregation could occur in the ROS discs (58). Possible strategies for treatment could involve: 1) efficient supplementation of the chromophore with its analog; 2) novel compounds that accelerate pigment regeneration by stabilizing P23H opsin via an allosteric binding site and accelerate pigment regeneration; 3) novel compounds that enhance degradation of P23H opsin, to promote the maintenance of the ROS structure and visual function by the remaining WT allele; and 4) some combination of the above.…”

“…The P23H transgene has also been expressed in Xenopus, revealing an inner segment accumulation (10). The P23H-EGFP fusion protein expressed in Xenopus showed both ROS and inner segment accumulation, but cross-section imaging of ROS showed P23H-EGFP aggregates distributed unevenly on the disc membrane (58). In TG C. elegans, we expressed the P23H opsin in neurons with no endogenous visual function.…”

“…Genetic ablation of retinoid storage by knocking out lecithin retinal acyltransferase in P23H/ϩ knock-in mice significantly accelerates the progression of retinal degeneration due to the P23H mutation (24). Despite the protective effect of endogenous chromophore, all previous animal models, including P23H TG Drosophila (14), Xenopus (10,58), rats (12), mice (11, 59 -61), pigs (13), and knock-in mice (24,25), undergo various degrees of retinal degeneration that are also reported in adRP patients carrying this mutation (24,62). This suggests that the presence of the endogenous chromophore does not suffice to prevent P23H-induced adRP.…”

Rhodopsin Trafficking and Mistrafficking

“…30 Though quantitative assessment of OS targeting efficacy has been difficult in the past, FPs can be fused to rhodopsin and accurately calibrated to known concentrations of soluble FPs. 26,31 This approach was effectively used to compare the trafficking efficacy of a rhodopsin-FP fusion either containing or lacking the VXPX motif. Consistent with the redundancy in the cilia-targeting signals, rhodopsin lacking the VXPX motif can still reach the rod OSs.…”

“…171,172 By calibrating rhodopsin-FP fusions with known quantities of soluble FPs, this approach allows accurate determination of the quantity and concentration of rhodopsin-FP in subcellular compartments of rod photoreceptors. 26,31 Furthermore, several methods were developed to increase the contrast for recently synthesized rhodopsin-FP. For example, by expressing rhodopsin-FP under the regulation of specific promoters which are inducible by small molecule or heat shock, 79,173 it is possible to visualize and track the movement of newly synthesized rhodopsin-FP.…”