Murine models of RPGR-mediated X-linked Retinitis Pigmentosa (RP) suggest a common disease mechanism across allelic variants

Abstract

Purpose : Mutations in RPGR account for 15% of RP, causing untreatable blindness. RPGR's function in the photoreceptor is yet to be determined. Human disease severity varies according to allelic heterogeneity. We, and others, have previously shown that RPGR appears to regulate the photoreceptor actin cytoskeleton. This study sought to better define RPGR’s role in health and disease.

Methods : Two Rpgr mutant mice strains were engineered by pronuclear injection of CRISPR/Cas9 constructs. One, Rpgr^Ex3D8, had an 8bp deletion in exon 3 with a null protein product. The other, Rpgr^ORFD5, had a 5bp deletion in the disordered repetitive domain of the retinal specific splice variant with a predicted truncated protein. Mice were analysed over time.

Results : On electroretinographic (ERG) testing, Rpgr^Ex3D8 mice had a significant reduction in cone-mediated flicker at 3 months (23.49uV v 18.03uV; p=0.0248) and scotopic a-wave amplitude at 6 months (161.2 uV v 120.6uV; p=0.0042). At 12 months Rpgr^ORFD5 had a significantly increased b/a wave ratio (2.462 v 3.123; p = 0.0001) but no significant reduction in flicker or scotopic a wave. Prior to ERG changes, both mice displayed increased GFAP immunolabelling of radial glial cells in the outer nuclear layer, indicating photoreceptor stress. Opsin and rhodopsin were mislocalised to the inner segments prior to ERG changes, with mass spectrometry analysis of both mutant retinas highlighting global dysregulation of trafficking pathways. Interestingly, transmission electron microscopy of both Rpgr^Ex3D8 and Rpgr^ORFD5 lines revealed loss of disc compaction and shortened outer segments. This could be in keeping with either a reduction in disc formation or abnormal disc structure.

Conclusions : Human RPGR mutations can lead to RP, cone dystrophy or cone-rod dystrophy. Efforts have been made to determine genotype-phenotype correlation. The above data suggests that, in mice, different mutations in RPGR cause a common disease pathogenesis. This could be due to a reduction in disc formation or abnormal trafficking of structural disc proteins. Disc morphogenesis is believed to occur via an actin-dependent mechanism and in the Spata7-/- mouse, where RPGR is mislocalised away from the site of disc formation, a similar disc phenotype occurs. Further work will be required to dissect RPGR’s role in photoreceptor health and disease.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.