Lack of Overt Retinal Degeneration in a K42E Dhdds Knock-In Mouse Model of RP59

Abstract

Dehydrodolichyl diphosphate synthase (DHDDS) is required for protein N-glycosylation in eukaryotic cells. A K42E point mutation in the DHDDS gene causes an autosomal recessive form of retinitis pigmentosa (RP59), which has been classified as a congenital disease of glycosylation (CDG). We generated K42E Dhdds knock-in mice as a potential model for RP59. Mice heterozygous for the Dhdds K42E mutation were generated using CRISPR/Cas9 technology and crossed to generate Dhdds K42E/K42E homozygous mice. Spectral domain-optical coherence tomography (SD-OCT) was performed to assess retinal structure, relative to age-matched wild type (WT) controls. Immunohistochemistry against glial fibrillary acidic protein (GFAP) and opsin (1D4 epitope) was performed on retinal frozen sections to monitor gliosis and opsin localization, respectively, while lectin cytochemistry, plus and minus PNGase-F treatment, was performed to assess protein glycosylation status. Retinas of Dhdds K42E/K42E mice exhibited grossly normal histological organization from 1 to 12 months of age. Anti-GFAP immunoreactivity was markedly increased in Dhdds K42E/K42E mice, relative to controls. However, opsin immunolocalization, ConA labeling and PNGase-F sensitivity were comparable in mutant and control retinas. Hence, retinas of Dhdds K42E/K42E mice exhibited no overt signs of degeneration, yet were markedly gliotic, but without evidence of compromised protein N-glycosylation. These results challenge the notion of RP59 as a DHDDS loss-of-function CDG and highlight the need to investigate unexplored RP59 disease mechanisms.

Structural basis of heterotetrameric assembly and disease mutations in the human cis-prenyltransferase complex

“…Intriguingly, the different pathogenic mutations in h cis -PT seem to have diverse effects on cellular glycosylation. For example, while the missense mutation in the RxG motif of NgBR led to reduced glycosylation in patients fibroblasts 16 , and the patient suffering from the fatal glycosylation disorder displayed hypoglycosylation of serum glycoproteins 15 , the arRP mutation in DHDDS does not seem to have any significant effect on glycosylation in a knock-in mouse model 18 , 19 , and some patients with DHDDS-related developmental epileptic encephalopathy display normal glycosylation assay results 14 . Thus, the interplay between the genotype and cellular phenotype may be more complex than originally thought and awaits further exploration.…”

Inherited Retinal Degeneration Caused by Dehydrodolichyl Diphosphate Synthase Mutation–Effect of an ALG6 Modifier Variant

“…#M7132) in BioRad iCycler™ or MyCycler™ PCR machines. Methods for PCR analysis were essentially as described in detail previously [76]. Prior to DNA sequencing, all PCR products were gel-purified using a GeneJET Gel Extraction ® kit (Thermo Fisher Scientific, Cat.…”

Cells Special Issue: “The Molecular and Cellular Basis of Retinal Diseases”

“…Ramachandra Rao et al [2] describe the initial characterization of a newly developed mouse model of retinitis pigmentosa-59 (RP59; OMIM #613861), an autosomal recessive form of RP that involves progressive, irreversible dysfunction, and degeneration of retinal rod and eventually cone photoreceptors. The most prevalent form of this disease involves a K42E point mutation in the enzyme dehydrodolichyl diphosphate synthase (DHDDS), which, along with its binding partner Nogo-B receptor (NgBR), comprises the cisprenyltransferase (CPT) enzyme complex.…”