Cone structure and function in dogs with RPGRIP1 mutation

Abstract

Purpose: A 44 bp insertion (^ins/ins) in exon 2 of RPGRIP1 is thought to be causal for canine cone-rod dystrophy. In a research colony of dogs segregating RPGRIP1^ins/ins, we observed that dogs having RPGRIP1^ins/ins mutation show different cone ERG function phenotypes that vary from normal to absent cone ERG responses when examined under photopic conditions. Interestingly, none of these dogs are visually impaired for photopic vision. In this study we have examined cone cell structure and protein expression in dogs having abnormal cone ERGs even though photopic vision is present.

Methods: Based on serial photopic flicker ERGs using full field ganzfeld stimulation and recorded with the Espion system and ColorDome stimulator, we categorized the dogs into RPGRIP1^ins/ins with either absent or normal cone ERG, RPGRIP1^ins/- with normal cone ERG and WT control. Retinal structure was assessed in PF fixed and OCT embedded tissues by H&E staining. Relevant proteins of the retina viz. hCAR, L/M opsin, S-opsin, RHO, GNB3, synaptophysin, SNAP-25, Go-alpha, PKC-alpha and GFAP were examined by immunohistochemistry (IHC).

Results: All the dogs had normal retinal structure as evident from H&E staining. There was no stress response in any of the retinae based on GFAP immunolabeling. There was no observable change in any of the groups studied for SNAP-25. The absent cone ERG RPGRIP1^ins/ins retina showed fewer numbers of cone outer segments. The cone inner segments were also reduced in number, short and stubby as evident from hCAR and GNB3 labeling. Both L/M and S- opsin labeled OS were decreased in number. Interestingly, there was a mild delocalization of RHO. Synaptophysin labeling showed a thinner OPL. Go-alpha labeling showed that fewer bipolar cells were labeled, and had retracted dendritic processes. There was also decreased labeling of ON-bipolar cells with PKC-alpha antibody.

Conclusions: Our preliminary results suggest that in absent cone ERG RPGRIP1^ins/ins dogs there was a reduced number of cones, and labeling of bipolar cells was decreased. While the remaining cells may be adequate for functional day light vision, they are insufficient to elicit recordable cone ERG signals.