Degenerative phenotype observed in retinal pigment epithelium of Prpf31^A216P/+ mice is caused by an abnormal pre-mRNA splicing of Rpe65 gene

Abstract

Purpose: Mutations in ubiquitously expressed pre-mRNA splicing-factor genes PRPF3, PRPF8, and PRPF31 cause autosomal dominant retinitis pigmentosa (adRP). It has been reported that transgenic mouse models carrying mutations in these genes exhibit morphological changes in the retinal pigment epithelium (RPE) (Graziotto et al. IOVS 2011). The reasons why these genes produce a degenerative phenotype in the RPE are unknown. Thus, we deeply characterized a heterozygous knock-in (KI) mouse model carrying the mutation A216P in the Prpf31 gene (Prpf31 ^A216P/+) to better understand the molecular mechanism involved in this form of adRP.

Methods: Eight-month-old C57BL/6J Prpf31 ^A216P/+ KI mice and wild type (WT) littermates were used in all experiments. Spatial vision (visual acuity and contrast sensitivity) was evaluated by optomotor test (OT) using 6 spatial frequencies from 0.031 to 0.272 cycles/degree (c/d) at 100%, 75% and 50% contrast sensitivity. The c-wave amplitude, which is originated in the RPE, was measured in a Ganzfeld electroretinogram (ERG). Retinal thickness and RPE reflectivity were quantified by optical coherence tomography (OCT). Western blotting (WB), RT-PCR, and cDNA sequencing were used to detect mRNA splicing errors in visual cycle genes. Fundus and histology of RPE were performed to detect lipofuscin accumulation.

Results: 1) OT: the percentage of positive responses at 100% to 50% contrast sensitivity in all spatial frequencies tested is diminished in the KI mice (100% contrast sensitivity, spatial frequency 0.272 c/d, WT= 70 ± 3 %, KI= 38 ± 2 %, p< 0.01). 2) ERG: the mean of c-wave amplitude is smaller in KI mice (WT= 283 ± 7 µV, KI= 190 ± 25 µV, p< 0.05). 3) OCT: the mean KI retina is thinner (WT= 220 ± 5 µm, KI= 198 ± 5 µm p< 0.05). 4) WB, RT-PCR and cDNA sequencing demonstrate an abnormal pre-mRNA splicing in exons 3 and 9 of Rpe65 gene. 5) Fundus evaluation and histology of RPE show an increase of autofluorescence and accumulation of lipofuscin granules.

Conclusions: Prpf31^A216P/+ mice have been previously characterized, without a clear phenotype of photoreceptor degeneration (Bujakowska et al. IOVS 2009). However, we have found that these mice have a visual dysfunction and degenerative phenotype in the RPE which is caused by an abnormal pre-mRNA splicing of Rpe65 gene.