Reduction of CRB2 Specifically in Mouse Photoreceptors with Concomitant Loss of CRB1 in Müller Glial Cells Mimics Early-onset Retinitis Pigmentosa

Abstract

Purpose : Mutations in the Crumbs homologue-1 (CRB1) gene cause early-onset Retinitis pigmentosa (RP) in children. Mice lacking CRB1 show moderate retinal degeneration limited to one quadrant of the retina while in retinas lacking CRB2 the entire retina is affected. In the mouse retina, CRB1 is localized in Müller glial cells (MGCs) and CRB2 in MGCs and photoreceptors, at the subapical region, at the outer limiting membrane (OLM). We generated and analysed the phenotype of a new CRB1-RP mouse model with reduced levels of Crb2 in photoreceptors and complete ablation of Crb1 in MGCs, which can be used to test the efficacy of adeno-associated-viral (AAV) CRB1 and CRB2 gene augmentation vectors.

Methods : Crb1^-/-Crb2^flox/+Crx-Cre mice were generated by crossing Crb1Crb2 with Crx-Cre mice driving the expressing of Cre in photoreceptors. Animals from embryonic day (E)15.5 till 8 months of age were analysed. To investigate the possible ectopic localization of proteins and cell types immunohistochemistry (IHC) was performed. Morphology of the retina was analysed using toluidine stained ultra-thin sections. In vivo analysis of retinal function was performed using electroretinography (ERG).

Results : Retinal disorganization was observed as early as E17.5. Progressive retinal disorganization was detected at early post-natal days (10 and 14), characterized by gaps at the OLM, photoreceptor rosettes and ectopic photoreceptor nuclei in the subretinal space. At a later stage (1M), disorganization and degeneration affected mainly the ventral area of the retina and the outer retina. IHC experiments showed loss of localization of members of the Crumbs complex at regions with disrupted OLM. At 3 month of age, severe degeneration was observed in the ventral area of the retina, where only very few photoreceptors remained. Increased levels of GFAP and CD11b were also observed suggesting gliosis and activated microglia. ERG performed at 1 month of age showed an attenuation of the a-wave, while no effect was observed in b-wave and flicker responses.

Conclusions : Reduction of CRB2 levels in photoreceptors with concomitant loss of CRB1 from MGCs leads to an exacerbation of the Crb1 phenotype. The retinal phenotype of these mice mimics early-onset RP. This new mouse model has a suitable therapeutic window to test efficacy of new CRB1 and CRB2 gene therapy vectors in photoreceptors.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.