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 orthologue 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.

中文翻译：

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果蝇视网膜中脱氢花青素二磷酸合酶 (DHDDS) 的敲除导致了一种独特的视网膜变性模式

Dehydrodolichyl 二磷酸合酶 (DHDDS) 是一种普遍表达的酶，可催化顺式异戊二烯链延长以产生多萜醇的聚异戊二烯主链。它出现在包括神经系统在内的所有组织中，它是一种高度保守的酶，可以在所有动物物种中找到。在 DHDDS 基因中具有双等位基因错义突变的个体会出现潜在机制未知的非综合征性色素性视网膜炎。我们使用果蝇模型来破坏 DHDDS 直向同源基因 (CG10778)，以寻找细胞和分子机制，当有缺陷时，可能会导致这种视网膜疾病。Gal4/UAS 系统用于通过 RNAi 介导的敲低在各种组织中抑制 CG10778 的表达。使用 q-RT-PCR 评估所得表型，透射电子显微镜 (TEM)、视网膜电图、抗体染色和蛋白质印迹分析。使用肌动蛋白启动子在早期胚胎中或使用 nub 启动子在发育中的翅膀中靶向敲低 CG10778-mRNA，分别导致致死或翅膀丢失。在幼虫眼盘和蛹视网膜中使用玻璃多报告基因 (GMR)-Gal4 驱动程序 (GMR-DHDDS-RNAi) 靶向表达 CG10778-RNAi 导致复杂的表型：a) TEM 视网膜切片显示了一种独特的视网膜 -变性，其中光感受器 R2 和 R5 表现出其信号室（横纹肌）的几乎正常结构，但仅在细胞核区域，而所有其他光感受器在所有区域都显示出视网膜变性。b) 蛋白质印迹分析显示 GMR-DHDDS-RNAi 果蝇中视紫质水平急剧下降，TEM 切片显示内质网 (ER) 异常积累。总而言之，在发育中的视网膜中损害 DHDDS，同时允许视网膜的形成，导致了一种独特的视网膜变性模式，其特征是视紫质蛋白水平显着降低和感光细胞中 ER 膜的异常积累，从而表明DHDDS 对正常的视网膜形成至关重要。