Dhdds Knock-down Induces Photoreceptor Degeneration In Zebrafish

Abstract

Purpose: : We have identified the K42E mutation in DHDDS (dehydrodolichol diphosphate synthase) as the cause of retinitis pigmentosa (RP) phenotype in a single family with non-syndromic recessive RP. The present work examines whether insufficient DHDDS activity induces photoreceptor degeneration in zebrafish.

Methods: : Morpholino oligonucleotides (MO) designed against exon1/intron1 of the zebrafish DHDDS gene were injected into fertilized eggs. Embryos were allowed to develop for four days. Wild type and DHDDS morphants were tested for responses to light on-off switches with a high-speed camera recording at 60 frames per second. After behavioral analysis, semi-thin plastic sections of fish eyes were prepared and examined by light microscopy. Cone outer segments were labeled by PNA (peanut agglutinin) and examined by confocal microscopy.

Results: : Four-day-old wild type larvae reliably responded to abrupt reductions in light intensity with bursts of swimming. In contrast, MO-injected fish failed to react to light on-off switches. In retinas of MO-injected fish, the photoreceptor outer segments were very short or completely missing. PNA staining clearly showed that cone outer segments were almost non-existing in the retinas of MO-injected fish.

Conclusions: : Our findings are consistent with DHDDS being a key protein for the generation and continuous renewal of photoreceptor outer segments. DHDDS catalyzes the formation of dehydrodolichol diphasphate as the first step of biosynthesis of dolichol, the critical lipid carrier in the dolichol pathway for N-linked glycosylation. Insufficient DHDDS activity would affect the efficiency of protein glycosylation. Photoreceptors produce large amount of glycoprotein opsin or rhodopsin each day to renew outer segments. Our results thus highlight the importance of protein glycosylation in photoreceptor outer segment generation and renewal and support the notion that deficiency in protein glycosylation can result in retinal degeneration. Additional experiments are in progress to characterize changes in photoreceptors in the DHDDS knock-out and DHDDS K42E knock-in mice.

Keywords: retinal degenerations: cell biology • mutations • glycoconjugates/glycoproteins