Sriganesh Ramachandra Rao, Lara A. Skelton, Fuguo Wu, Agnieszka Onysk, Grzegorz Spolnik, Witold Danikiewicz, Mark C. Butler, Delores A. Stacks, Liliana Surmacz, Xiuqian Mu, Ewa Swiezewska, Steven J. Pittler, Steven J. Fliesler | iScience | Vol 23, Issue 6 | June 2020 26 | doi.org/10.1016/j.isci.2020.101198
Summary
Dehydrodolichyl diphosphate synthase (DHDDS) catalyzes the committed step in dolichol synthesis. Recessive mutations in DHDDS cause retinitis pigmentosa (RP59), resulting in blindness. We hypothesized that rod photoreceptor-specific ablation of Dhdds would cause retinal degeneration due to diminished dolichol-dependent protein N-glycosylation. Dhddsflx/flx mice were crossed with rod-specific Cre recombinase-expressing (Rho-iCre75) mice to generate rod-specific Dhdds knockout mice (Dhddsflx/flx iCre+). In vivo morphological and electrophysiological evaluation of Dhddsflx/flx iCre+ retinas revealed mild retinal dysfunction at postnatal (PN) 4 weeks, compared with age-matched controls; however, rapid photoreceptor degeneration ensued, resulting in almost complete loss of rods and cones by PN 6 weeks. Retina dolichol levels were markedly decreased by PN 4 weeks in Dhddsflx/flx iCre+ mice, relative to controls; despite this, N-glycosylation of retinal proteins, including opsin (the dominant rod-specific glycoprotein), persisted in Dhddsflx/flx iCre+ mice. These findings challenge the conventional mechanistic view of RP59 as a congenital disorder of glycosylation.
Introduction
Retinitis pigmentosa (RP) represents a large class of inherited retinal dystrophies caused by mutations in several families of genes, leading to pigmentary retinopathy and progressive, irreversible blindness. Typically, RP is characterized by the initial loss of rod photoreceptors (PRs), deposition of pigment granules, and peripheral vision loss (Ferrari et al., 2011, Hamel, 2006). Defective asparagine-linked glycosylation (N-glycosylation) of proteins in rod cells, particularly the visual pigment rhodopsin (RHO), results in progressive, irreversible rod cell degeneration and death, with concomitant loss of vision (Murray et al., 2009, Murray et al., 2015, Kaushal et al., 1994, Fliesler et al., 1984a). Successful glycosylation of RHO is necessary for its vectorial trafficking through the inner segment (cell body) of the rod cell to the site of rod outer segment (ROS) membrane assembly at the base of the ROS. Retinal degeneration has been observed in patients harboring RHO mutations involving the N-glycosylation consensus sites, and in animal models involving comparable RHO mutations (Van Den Born et al., 1994, Zhu et al., 2004, Sullivan et al., 1993, Murray et al., 2015, Iwabe et al., 2016), as well as by tunicamycin-induced and genetic inhibition of global/RHO N-glycosylation (Fliesler and Basinger, 1985, Fliesler et al., 1985, Sabry et al., 2016, Thompson et al., 2013, Murray et al., 2015). Protein N-glycosylation involves the following steps (schematic representation, Figure 1): generation of dolichol (Dol, an important isoprenoid arising from the mevalonate pathway) and dolichyl phosphate (Dol-P, the obligate glycan carrier necessary for N-linked glycosylation, O-mannosylation, and C-mannosylation) (Burda and Aebi, 1999, Endo et al., 2003, Park et al., 2014, Cantagrel et al., 2010, Burton et al., 1979, Maeda et al., 2000, Doucey et al., 1998), generation of complex Dol-P-linked oligosaccharides (DLO) (Krasnova and Wong, 2016, Gandini et al., 2017, Behrens and Leloir, 1970), and transfer of those oligosaccharides from DLO to the N-glycosylation consensus site on the target polypeptide (Welply et al., 1983). Genetic defects affecting the glycosylation mechanism constitute a large family of syndromes termed “congenital disorders of glycosylation” (CDGs), with more than 150 causative genes (Ng and Freeze, 2018, Sparks and Krasnewich, 1993). A family of genetic diseases pertaining to Dol synthesis is classified as CDG-I (the class of CDG involving defective glycan assembly and/or their transfer in the endoplasmic reticulum [ER]) due to the requirement of DLO for N-glycosylation. Common clinical features of CDGs include failure to thrive, retarded development, protein-losing enteropathy, early-onset encephalopathy, as well as retinopathies such as RP (Sparks and Krasnewich, 1993, Thompson et al., 2013, Hamdan et al., 2017, Morava et al., 2009).
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