June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Mutant rhodopsins associated with sector RP primarily localize to rod photoreceptor outer segments in transgenic Xenopus laevis
Author Affiliations & Notes
  • Aaron D Loewen
    Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, British Columbia, Canada
  • Beatrice M Tam
    Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, British Columbia, Canada
  • Ross T Scharbach
    Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, British Columbia, Canada
  • Colette N Chiu
    Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, British Columbia, Canada
  • Orson L Moritz
    Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, British Columbia, Canada
  • Footnotes
    Commercial Relationships   Aaron Loewen, None; Beatrice Tam, None; Ross Scharbach, None; Colette Chiu, None; Orson Moritz, None
  • Footnotes
    Support  NSERC (RGPIN-2015-04326), CIHR (PJT-155937 and PJT-156072), and Fighting Blindness Canada
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 1552. doi:
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    • Get Citation

      Aaron D Loewen, Beatrice M Tam, Ross T Scharbach, Colette N Chiu, Orson L Moritz; Mutant rhodopsins associated with sector RP primarily localize to rod photoreceptor outer segments in transgenic Xenopus laevis. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1552.

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

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Abstract

Purpose : Over 200 mutations in rhodopsin (RHO) have been associated with retinitis pigmentosa (RP); however, the relationship between many of these mutations and their cell death mechanism is unknown. To broaden our understanding of the diversity of possible mechanisms and phenotypes, we have investigated the effects of sector RP-associated RHO mutations on rhodopsin localization and retinal degeneration (RD) in Xenopus laevis. We further investigated the effects of dark rearing and chromophore binding on the recently identified sector RP-associated N-terminal RHO mutation L31Q, for which minimal mechanistic information is available.

Methods : We generated transgenic X. laevis by injecting wildtype (WT) oocytes with sperm nuclei and human RHO transgenes (either WT or containing missense mutations) using the REMI method. We also introduced a second RHO mutation that eliminates chromophore binding (K296R). Tadpoles were raised in either cyclic light or constant dark for 14 days before euthanization. Tadpole eyes were enucleated and were either cryosectioned and immunolabelled using anti-mammalian rhodopsin antibodies for confocal microscopy or analysed for rhodopsin levels using a dot blot assay.

Results : We observed RD associated with expression of nine different RHO mutations expressed in our transgenic X. laevis model. Mutant rhodopsins were primarily localized to rod outer segments with some mutant rhodopsins also displaying some rod inner segment localization. We observed RD in L31Q RHO transgenic tadpoles reared in cyclic light; however, dark rearing or preventing chromophore binding via K296R in L31Q RHO transgenic tadpoles mitigated RD.

Conclusions : We have created a number of novel RP models using transgenic X. laevis. Previously, only glycosylation-deficient mutated rhodopsins such as T4K and T17M were known to be trafficked robustly to outer segments. Our data shows that a significant proportion of sector RP-associated mutant rhodopsins traffic to outer segments. Furthermore, RD associated with expression of the non-glycosylation-deficient mutant rhodopsin L31Q was mitigated by dark rearing or by preventing chromophore binding, similar to our previous findings for RD caused by T4K and T17M rhodopsins. Our results suggest cell death mechanisms involving photoactivation-induced instability may be responsible for a significant fraction of sector RP.

This is a 2020 ARVO Annual Meeting abstract.

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