Ablation of CRB2 in Rod Photoreceptors with Concomitant Loss of CRB1 in Müller Glial Cells Mimics Retinitis Pigmentosa

Abstract

Purpose : Mutations in the Crumbs homologue-1 (CRB1) gene cause either early-onset Retinitis pigmentosa (RP) or Leber’s congenital amaurosis. In the adult mouse retina, CRB1 localizes in Müller glial cells (MGCs), while CRB2 localizes in MGCs and cone and rod photoreceptors, at the subapical region above the adherens junctions. Crb1-null retinas show moderate degeneration limited to one quadrant while in retinas lacking CRB2 the entire retina is affected. We generated two new mouse models for CRB1-retinal dystrophy by ablating Crb2 in rod photoreceptors or by ablating Crb2 in rod photoreceptors and Crb1 in MGCs. Here, we describe the morphological and functional phenotype of these retinas.

Methods : Crb2^flox/floxRho-iCre and Crb1^-/-Crb2^flox/floxRho-iCre mice were generated by crossing (Crb1^-/-)Crb2^flox/flox with Rho-iCre mice driving the expressing of improved Cre (iCre) in rod photoreceptors. Animals from 1-, 3-, 6- and 9-months of age were analysed. In vivo analysis of retinal function was performed using electroretinography (ERG). Visual acuity was tested in 4-months old Crb1^-/-Crb2^flox/floxRho-iCre by the Optomotry system. Morphology of the retina was analysed using toluidine stained ultra-thin plastic sections and immunohistochemistry.

Results : Mice lacking CRB2 from rods showed retinal function impairment as measured by ERG at 9-months of age. At this age, the scotopic a-wave was decreased, while no differences were found in scotopic or photopic b-wave. The retinal morphology was grossly preserved. Mice with loss of CRB1 and retinal CRB2 showed normal retinal function at 1-month of age, but reduced amplitudes (scotopic a- and b-wave and photopic b-wave) from 3-months of age onwards. Four months old double knockout mice showed similar visual acuity compared to the littermate controls (Crb1^-/-Crb2^flox/flox) (0.386 vs 0.387 cycles per degree, respectively). These retinas showed a thinner outer nuclear layer, sporadic ectopic photoreceptor nuclei in the subretinal space and disruptions of the outer limiting membrane.

Conclusions : Ablation of CRB2 in rod photoreceptors resulted in a mild phenotype leading to retinal function impairment at 9-months of age. Moreover, additional loss of CRB1 from MGCs lead to an exacerbation of the Crb2^flox/floxRho-iCre phenotype. The retinal phenotype of the double knockout mice mimics early-onset RP with early ERG deficit and loss of photoreceptors.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.