Characterization of rhodopsin P23H-induced retinal degeneration in a Xenopus laevis model of retinitis pigmentosa.
OBJECTIVE
To investigate the pathogenic mechanisms that underlie retinal degeneration induced by the rhodopsin mutation P23H in a Xenopus laevis model of RP.


METHODS
Transgenic X. laevis were generated that expressed the rhodopsin mutants rhoP23H and rhoP23H/K29R (a variant incapable of transducin activation). Using quantitative dot blot assay, transgenic rhodopsin levels and the extent of retinal degeneration were determined. The contribution of rhodopsin signal transduction to cell death was assessed by comparison of rhoP23H and rhoP23H/K296R effects and by dark rearing of rhoP23H tadpoles. Intracellular localization and the oligomeric state of rhoP23H were determined by confocal immunofluorescence microscopy and Western blot analysis.


RESULTS
RhoP23H induced retinal degeneration in a dose-dependent manner whereas expression of a control rhodopsin did not, indicating that rod photoreceptor death was specific to the P23H mutation and was not caused by the overexpression of rhodopsin. Neither abolishment of rhoP23H photosensitivity and ability to activate transducin nor dark rearing rescued rod viability. RhoP23H was localized primarily to the endoplasmic reticulum (ER) of inner segments. Western blot analysis of transgenic retinas showed that rhoP23H was prone to form dimers and higher molecular weight oligomers. However, aggresomes were not observed in rhoP23H transgenic retinal sections, despite their being reported in cultured cells expressing rhoP23H.


CONCLUSIONS
These results support a role for rhoP23H misfolding and inner segment accumulation in rod death, possibly by ER overload or other cellular stress pathways rather than by altered rhodopsin signal transduction or aggresome formation.
Tam
Beatrice M
BM
Department of Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, Canada.
Moritz
Orson L
OL
D000818:Animals; D030801:Animals, Genetically Modified; D015153:Blotting, Western; D016923:Cell Death; D002460:Cell Line; D003623:Dark Adaptation; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D019084:Fluorescent Antibody Technique, Indirect; D007668:Kidney / Q000196:embryology; D018613:Microscopy, Confocal; D009154:Mutation; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism / Q000473:pathology; D012162:Retinal Degeneration / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D012174:Retinitis Pigmentosa / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D012243:Rhodopsin / Q000235:genetics* / Q000378:metabolism; D015293:Transducin / Q000378:metabolism; D014162:Transfection; D019076:Transgenes; D014982:Xenopus laevis
D012243:Rhodopsin; D015293:Transducin