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Rong Wen, Ying Li, Zhengying Wang, Byron Lam, Yiwen Li; Genetic ablation of DHDDS induces photoreceptor degeneration. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5954.
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© ARVO (1962-2015); The Authors (2016-present)
We have identified the K42E mutation in DHDDS (dehydrodolichol diphosphate synthase) as the cause of retinitis pigmentosa (RP), which accounts for about 12% of autosomal recessive RP cases in patients of Ashkenazi Jewish origin. Previously we reported that global ablation of the DHDDS was embryonically lethal. In the present work, the DHDDS gene was conditionally knocked out in photoreceptors.
Exon4 of DHDDS, a critical exon, was floxed to result in DHDDSfl/fl mice. Conditional knockout of DHDDS was achieved by subretinal injection of AAV-Rho-Cre (adeno-associated virus carrying Cre recombinase driven by a 500 bp murine rhodopsin promoter). Conditional knockout of DHDDS in photoreceptors was also accomplished by crossing the DHDDSfl/fl mice with the LMOP-Cre mice, animals expressing cre recombinase under the control of the long-murine opsin promoter. Eyes were collected at endpoints and semi-thin plastic sections were cut to display the entire retina. Loss of photoreceptors was characterized morphologically by light microscopy.
DHDDSfl/fl mice were injected subretinally with 1.5 µl of AAV-Rho-Cre (7.2x1012 GC/ml) to the left eyes and with 1 µl of AAV-Rho-GFP (4.5x1013 GC/ml) to the right eyes in adult animal. Animals were killed one month later and the eyes were collected. Photoreceptors in the AAV-Rho-GFP injected eyes appeared having normal outer and inner segments. The outer nuclear layer (ONL) had 10 or more rows of nuclei in the injected area. In the AAV-Rho-Cre injected eyes, the outer segments of photoreceptors became short and the ONL was reduced to less than half of the original thickness in the injected area, with near 4 rows of photoreceptor nuclei; but photoreceptors and the ONL in the area away from the injection site were normal. No photoreceptor degeneration was found in wild-type retinas injected with AAV-Rho-Cre. Loss of photoreceptors is also evident when the DHDDSfl/fl mice were crossed with the LMOP-Cre mice. At the age of 4 months, most eyes lost more than half of photoreceptors with about 4-5 rows of nuclei in the ONL and shortening of photoreceptor outer segments. No such loss was seen in animals with either DHDDSfl/fl mice without cre, or LMOP-Cre mice without DHDDSfl/fl.
These results demonstrate that DHDDS plays a vital role in photoreceptors. The loss of the functions of DHDDS cannot be compensated by other proteins or mechanisms.
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