top of page

Knockdown of Dehydrodolichyl Diphosphate Synthase in the Drosophila Retina Leads to a Unique Pattern of Retinal Degeneration

Jul 4, 2021

Dehydrodolichyl diphosphate synthase (DHDDS) is a ubiquitously expressed enzyme that catalyzes cis-prenyl chain elongation to produce the poly-prenyl backbone of dolichol. It appears in all tissues including the nervous system and it is a highly conserved enzyme that can be found in all animal species. Individuals who have biallelic missense mutations in the DHDDS gene are presented with non-syndromic retinitis pigmentosa with unknown underlying mechanism. We have used the Drosophila model to compromise DHDDS ortholog gene (CG10778) in order to look for cellular and molecular mechanisms that, when defective, might be responsible for this retinal disease. The Gal4/UAS system was used to suppress the expression of CG10778 via RNAi-mediated-knockdown in various tissues. The resulting phenotypes were assessed using q-RT-PCR, transmission-electron-microscopy (TEM), electroretinogram, antibody staining and Western blot analysis. Targeted knockdown of CG10778-mRNA in the early embryo using the actin promoter or in the developing wings using the nub promoter resulted in lethality, or wings loss, respectively. Targeted expression of CG10778-RNAi using the glass multiple reporter (GMR)-Gal4 driver (GMR-DHDDS-RNAi) in the larva eye disc and pupal retina resulted in a complex phenotype: (a) TEM retinal sections revealed a unique pattern of retinal-degeneration, where photoreceptors R2 and R5 exhibited a nearly normal structure of their signaling-compartment (rhabdomere), but only at the region of the nucleus, while all other photoreceptors showed retinal degeneration at all regions. (b) Western blot analysis revealed a drastic reduction in rhodopsin levels in GMR-DHDDS-RNAi-flies and TEM sections showed an abnormal accumulation of endoplasmic reticulum (ER). To conclude, compromising DHDDS in the developing retina, while allowing formation of the retina, resulted in a unique pattern of retinal degeneration, characterized by a dramatic reduction in rhodopsin protein level and an abnormal accumulation of ER membranes in the photoreceptors cells, thus indicating that DHDDS is essential for normal retinal formation.


Introduction

The identification of a founder mutation in the dehydrodolichyl diphosphate synthase (DHDDS) gene in Ashkenazi Jews (AJ) with non-syndromic retinitis pigmentosa (RP) was reported a decade ago (Zelinger et al., 2011; Züchner et al., 2011). A single-nucleotide mutation in the DHDDS gene c.124A > G was found in an AJ family. The p.K42E mutation affects a highly conserved region of the DHDDS protein, which is located in close proximity to a binding site of farnesyl diphosphate. This mutation has subsequently been confirmed in other similar patients and is found in 10–20% of autosomal recessive RP in AJ population (Zelinger et al., 2011). The clinical phenotype of patients who are homozygous for the p.K42E mutation is within the spectrum often described in autosomal recessive RP (Zelinger et al., 2011; Züchner et al., 2011). Patients harboring mutations in DHDDS demonstrated fundus findings at a relatively early age. Clinically, they demonstrated waxy appearance of the optic nerve head, attenuation of retinal blood vessels and retinal atrophy in the mid and far periphery combined with significant bone spicule-like pigmentation, starting already in their 20s. The atrophic changes spread into the macular area and the pigmentary changes became denser with age (Kimchi et al., 2018). Kinetic visual fields revealed reduced peripheral function in the youngest patients studied and only small central islands of vision remaining later in life. Electroretinogram responses were not detectable in most patients (Zelinger et al., 2011). In the human retina, DHDDS is expressed in the inner segment of photoreceptors, where dolichol biosynthesis is predicted to occur (Zelinger et al., 2011). A possible link between insufficient DHDDS activity and photoreceptor degeneration was investigated in zebrafish, in which the expression of DHDDS was knocked down by morpholino oligonucleotides injected into zebrafish one cell embryos. The results demonstrated that suppression of DHDDS expression in zebrafish led to loss of photoreceptor outer segments and visual function (Wen et al., 2014). The results thus support the hypothesis that insufficient DHDDS function can lead to retinal degeneration. However, an additional study describing a patient with a severe multisystem disease associated with DHDDS deficiency shows that RP is not the only clinical sign in cases of DHDDS deficiency (Sabry et al., 2016).



 

References

Kimchi, A., Khateb, S., Wen, R., Guan, Z., Obolensky, A., Beryozkin, A., et al. (2018). Nonsyndromic retinitis pigmentosa in the ashkenazi jewish population: genetic and clinical aspects. Ophthalmology 125, 725–734. doi: 10.1016/j.ophtha.2017.11.014

Sabry, S., Vuillaumier-Barrot, S., Mintet, E., Fasseu, M., Valayannopoulos, V., Héron, D., et al. (2016). A case of fatal Type I congenital disorders of glycosylation (CDG I) associated with low dehydrodolichol diphosphate synthase (DHDDS) activity. Orphanet J. Rare Dis. 11:84.

Wen, R., Dallman, J. E., Li, Y., Züchner, S. L., Vance, J. M., Peričak-Vance, M. A., et al. (2014). Knock-down DHDDS expression induces photoreceptor degeneration in zebrafish. Adv. Exp. Med. Biol. 801, 543–550. doi: 10.1007/978-1-4614-3209-8_69

Zelinger, L., Banin, E., Obolensky, A., Mizrahi-Meissonnier, L., Beryozkin, A., Bandah-Rozenfeld, D., et al. (2011). A missense mutation in DHDDS, encoding dehydrodolichyl diphosphate synthase, is associated with autosomal-recessive retinitis pigmentosa in ashkenazi jews. Am. J. Hum. Genet. 88, 207–215. doi: 10.1016/j.ajhg.2011.01.002

Züchner, S., Dallman, J., Wen, R., Beecham, G., Naj, A., Farooq, A., et al. (2011). Whole-exome sequencing links a variant in DHDDS to retinitis pigmentosa. Am. J. Hum. Genet. 88, 201–206. doi: 10.1016/j.ajhg.2011.01.001


Comments

bottom of page