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M. Haeri, P. D. Calvert, B. E. Knox; A Novel Mechanism for Retinal Degeneration: Photoreceptor Membrane and Structure Destabilization Caused by Rhodopsin Aggregation. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4056.
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© ARVO (1962-2015); The Authors (2016-present)
RhoP23H is the most common rhodopsin mutation in humans. Current animal models that express RhoP23H in rods exhibit retinal degeneration. In cultured mammalian cells, RhoP23H was found in aggresomes containing chaperones (Saliba et al. 2002, J. Cell. Sci. 115, 2907) suggesting this could also be a cellular fate of RhoP23H in retina. Thus, we examined rods for potential RhoP23H oligomers or protein aggregates by quantitative live cell imaging.
We generated transgenic Xenopus that expressopsin (Rho-EGFP) or P23H mutant (RhoP23H-EGFP) cDNAs with EGFP fused to its carboxyl terminus. We utilized high-resolution live cell imaging and fluorescence recovery after photobleaching to reveal the localization and dynamics of RhoP23H-EGFP within the photoreceptor.
Live imaging of transgenic rods showed that RhoP23H-EGFP appears in concentrated fluorescent foci both in inner and outer segments. Based on dynamic FRAP analysis, we found that the outer segment foci were immobile aggregates. RhoP23H-EGFP inner segment foci were mobile, with fluorescence recovery rates after photobleaching that were similar to non-aggregated Rho-EGFP. The immobile fluorescent foci were more abundant toward the distal end of the outer segment, suggesting a time-dependence for foci formation. Fluorescent foci were more abundant in rods with high RhoP23H-EGFP expression levels, and were commonly found in disk incisures in contrast to Rho-EGFP. Rods co-expressing both RhoP23H-EGFP and Rho-mCherry showed exclusion of Rho-mCherry from immobile fluorescent foci. Retina from transgenic Xenopus expressing RhoP23H-EGFP showed abnormal vesicotubular structures in rod outer segment membranes in electron micrographs, which were not observed in transgenic Xenopus expressing Rho-EGFP.
We have shown, for the first time, that RhoP23H-EGFP forms aggregates in the outer segment of transgenic rods. In addition, we observed abnormal vesicotubular structures in the outer segment membranes of these animals. These results suggest a new mechanism for destabilizing the rod: mutant protein aggregation. Formation of such protein aggregates has been shown in a number of neurodegenerative diseases such as Alzheimer’s disease. Further studies will address the nature of these aggregates and their efficacy to trigger photoreceptors degeneration.
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