Selective ablation of dehydrodolichyl diphosphate synthase (Dhdds) expression in RPE alters retinal structure and function

Abstract

Purpose : Mutations in the human gene encoding dehydrodolichyl diphosphate synthase (DHDDS), which is required for N-linked protein glycosylation, cause retinitis pigmentosa (RP). We generated a conditional Dhdds knockout mouse model to study the effects of cell type-specific ocular Dhdds deficiency.

Methods : A Dhdds conditional knockout construct was obtained from KOMP (UC Davis), then introduced into murine ES cells, confirmed by PCR and then FRT was used to excise a LacZ cassette. Mouse lines were established and bred to homozygosity. Mice were crossed to RPE-Cre mice (Yun Le, OUHSC), and were assessed by OCT, ERG, histology, immunofluorescence, and TEM at 1, 2, and 3 mo. postnatal.

Results : OCT analysis of RPE-Dhdds^-/- mice showed a significant reduction (vs. WT) in ONL and total retinal thickness at all ages analyzed. No OCT changes were observed in RPE-Dhdds^+/- mice. Histologic analysis showed panretinal degeneration affecting the RPE and photoreceptor layers. TEM of 3-mo old homozygous mice revealed ectopic RPE migration and displacement of the ELM. Cre-dependent GFP reporter expression indicated that >90% of RPE cells expressed Cre by 3 mo of age; however, at 1 mo, expression was barely detectable. In 1-mo old RPE-Dhdds^+/- mice, no differences from WT were observed in the scotopic ERG. However, RPE-Dhdds^-/- mice exhibited significant reduction (42%; p <0.01) in b-wave amplitudes compared to WT, without altered a-wave. By 2 mo of age, hets (RPE-Dhdds^+/-) showed a significant reduction in both the a-wave (17%; p <0.05) and b-wave (44%; p <0.001) amplitudes. These deficits were even more pronounced in RPE-Dhdds^-/- mice, with significant reductions in both the a-wave (42%; p <0.01) and b-wave (52%; p <0.001) amplitudes.

Conclusions : RPE-specific Dhdds heterozygous and homozygous knockout mice have been generated, using Cre-lox technology. Homozygous knockout mice exhibit progressive retinal degeneration, with structural and functional deficits, suggesting that RPE protein N-glycosylation is required for retinal function and viability. Functional deficits in heterozygous mice predict that human RP carriers of DHDDS mutations may exhibit a haploinsufficiency phenotype.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.