June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
A Drosophila model for a Dehydrodolichyl Diphosphate Synthase (DHDDS) mutation causing Retinitis Pigmentosa in humans
Author Affiliations & Notes
  • Reut Ifrah
    Hebrew University Jerusalem, Jerusalem, Israel
  • Tal Brandwine
    Hebrew University Jerusalem, Jerusalem, Israel
  • Liliana Mizrahi-Meissonnier
    Hadassah University Hospital, Israel
  • Rachel Zaguri
    Hebrew University Jerusalem, Jerusalem, Israel
  • Tzofia Bialistoky
    Hebrew University Jerusalem, Jerusalem, Israel
  • Vladimir Katanaev
    University of Lausanne, Switzerland
  • Dror Sharon
    Hadassah University Hospital, Israel
  • Offer Gerlitz
    Hebrew University Jerusalem, Jerusalem, Israel
  • Baruch Minke
    Hebrew University Jerusalem, Jerusalem, Israel
  • Footnotes
    Commercial Relationships   Reut Ifrah, None; Tal Brandwine, None; Liliana Mizrahi-Meissonnier, None; Rachel Zaguri, None; Tzofia Bialistoky, None; Vladimir Katanaev, None; Dror Sharon, None; Offer Gerlitz, None; Baruch Minke, None
  • Footnotes
    Support  Israel Science Foundation (ISF)
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 4431. doi:
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      Reut Ifrah, Tal Brandwine, Liliana Mizrahi-Meissonnier, Rachel Zaguri, Tzofia Bialistoky, Vladimir Katanaev, Dror Sharon, Offer Gerlitz, Baruch Minke; A Drosophila model for a Dehydrodolichyl Diphosphate Synthase (DHDDS) mutation causing Retinitis Pigmentosa in humans. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4431.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The main pathology of patients carrying specific mutations in DHDDS is Retinitis Pigmentosa (RP), although the DHDDS enzyme is ubiquitously expressed. We hypothesize that knocking down the human orthologue of DHDDS in Drosophila can mimic human DHDDS mutations and serve as animal model for this disease.

Methods : We generated several transgenic lines of Drosophila, expressing RNAi that reduced the expression of the Drosophila orthologue of mammalian DHDDS. Genetic analysis included crossing a Drosophila line encoding the DHDDS-RNAi with strains encoding tissue specific promoters that drive expression of the DHDDS-RNAi to the whole body, wings or retina. Transmission electron microscopy (TEM), Electroretinogram (ERG) and Western blot analysis were applied to examine effects of DHDDS-RNAi expression on retinal structure, function and on rhodopsin level.

Results : Knockdown of fly-DHDDS in total body using the actin promoter (act5 and act5c), or in the wings using the nub promoter resulted in lethality, or wings absence respectively. Knockdown of fly-DHDDS at early retinal development using the GMR promoter resulted in a complex phenotype. At the structural level, serial thin TEM retinal sections of DHDDS-RNAi flies revealed a unique pattern of retinal degeneration, where photoreceptors #2 and #5 revealed nearly normal rhabdomeral structure, but only at depth of 40-55 μm from corneal surface (i.e. in the region of the nucleus). Statistical analyses at this depth revealed a significant difference in rhabdomeral area between WT and DHDDS-RNAi flies (n=10), while all other photoreceptors at the same retinal depth and elsewhere showed strongly degenerative rhabdomeres. Western blot analysis showed a drastic reduction in rhodopsin expression level in DHDDS flies suggesting an impaired N-glycosylation, a key step in rhodopsin maturation.

Conclusions : N-glycosylation is known to affect fly development, while knockdown of DHDDS is expected to affect N-glycosylation and therefore fly development. Indeed, driving DHDDS-RNAi to whole body or wings resulted in lethality or wings absence. Thus, knocking down Drosophila DHDDS did not mimic human mutations in DHDDS. Nevertheless, driving the fly DHDDS-RNAi to early retinal development induced a unique pattern of photoreceptor degeneration reminiscent of RP.

This is a 2020 ARVO Annual Meeting abstract.

 

Transmission Electron Microscopy of Drosophila DHDDS-RNAi and WT retinae

Transmission Electron Microscopy of Drosophila DHDDS-RNAi and WT retinae

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