Abstract
Purpose :
Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) gene cause retinitis pigmentosa, an inherited retinal dystrophy resulting in sight loss that has no treatment. RPGR localises to the photoreceptor connecting cilium and has previously been implicated in regulating the cell's actin cytoskeleton. Identifying RPGR's exact role in the photoreceptor, and the pathways it helps control, may help with developing novel therapies. To this end, we generated novel, humanised Rpgr-mutant mice to interrogate disease pathogenesis.
Methods :
We performed transmission electron microscopy, super-resolution imaging and novel, live imaging experiments of retinal slice cultures of 6 week old Rpgr mutant mice to define actin cytoskeletal dyamics within the connecting cilium. Conventional immunoprecipitation and immunoblotting were used to identify novel Rpgr interactors. Rescue experiments were performed by intravitreal injection of various modulators of actin nucleation pathways.
Results :
Rpgr mutant mice develop retinal stress prior to undergoing loss of visual function and a slow photoreceptor degeneration, significant at 18 months. Prior to this, regulation of actin turnover at the site of disc morphogenesis is disturbed, with excessive actin polymerisation and reduced actin dynamics. Outer segment discs are dysmorphic, with vesicles shed from the photoreceptor at the base of the outer segment. In the retina, Rpgr exists in complex with the actin severing protein Cofilin and its activity is dysregulated when Rpgr is perturbed. In keeping, Rpgr mutant mice display longer actin microfilaments at the site of disc formation and their structure is altered. Manipulation of photoreceptor actin partially rescues the phenotype seen in Rpgr mutant photoreceptors.
Conclusions :
This work helps to define Rpgr's role in the photoreceptor connecting cilium, where we propose it facilitates outer segment disc formation. Rescue studies show it is possible to target the pathways for therapeutic gain, raising the possibility that novel treatments could be developed for this devastating disease.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.