Immunolocalization of RPGR and RPGRIP in Normal Dogs, and in Mutants (XLPRA1/XLPRA2) Affected with Microdeletions of RPGR Exon ORF15

Abstract

Abstract: : Purpose: Different microdeletions of canine RPGR exon ORF15 result in either late- or early-onset inherited retinal degeneration (XLPRA1, XLPRA2 respectively). To study the mechanisms of these diseases, we examined the expression and immunolocalization of RPGR and RPGRIP in normal and mutant retinas. Methods: Retinas were fixed in paraformaldehyde (PF; 4% for 48 hrs or 4% for 3 hrs/2% for 24 hrs) prior to OCT embedding and sectioning. Polyclonal antibodies directed against different human RPGR [exon ORF15 (MCW 27); exon 19 (MCW 21)] and RPGRIP (MCW4) domains were used. Results: In normal retinas, RPGR antibodies MCW 21 and 27 diffusely labeled the outer segment with a slightly more intense immunolabeling of the connecting cilium and the basal outer segments. The anti RPGRIP antibody (MCW4) yielded similar results, and also labeled amacrine cells. In the diseased retinas affected with the milder stop (XLPRA1) and more severe frameshift (XLPRA2) mutations there was an altered pattern of expression. Both the XLPRA1 and XLPRA2 retinas showed very weak to no RPGR labeling of the photoreceptor outer segments or connecting cilium. In contrast, the RPGRIP antibody was weakly positive in the connecting cilium, but not the outer segments of the XLPRA1 retina; however, amacrine cell labeling was distinct. The XLPRA2 mutant retina showed no positive photoreceptor labeling. Conclusions: Retinal expression of RPGR isoforms and RPGRIP is markedly altered in the RPGR mutant retinas. In contrast to the results observed in amacrine cells, our findings also support the role of RPGR in the proper localization of RPGRIP in outer segments. This new battery of antibodies directed against different RPGR and RPGRIP functional domains will be useful in examining cellular alterations which occur in mutant photoreceptors.

Keywords: retinal degenerations: cell biology • retinal degenerations: hereditary • animal model