June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Characterization of autophagy in normal and degenerating rod photoreceptors of Xenopus laevis using dually fluorescent LC3 markers.
Author Affiliations & Notes
  • Runxia Wen
    Ophthalmology and Visual Science, University of British Columbia, Vancouver, British Columbia, Canada
  • Paloma Stanar
    Ophthalmology and Visual Science, University of British Columbia, Vancouver, British Columbia, Canada
  • Orson L Moritz
    Ophthalmology and Visual Science, University of British Columbia, Vancouver, British Columbia, Canada
  • Footnotes
    Commercial Relationships   Runxia Wen, None; Paloma Stanar, None; Orson Moritz, None
  • Footnotes
    Support  CIHR-MOP64400;Foundation Fighting Blindness – Canada
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 5374. doi:
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      Runxia Wen, Paloma Stanar, Orson L Moritz; Characterization of autophagy in normal and degenerating rod photoreceptors of Xenopus laevis using dually fluorescent LC3 markers.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5374.

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

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Abstract

Purpose : Previous results suggest that enhanced autophagy is a feature of retinal degeneration (RD) caused by the P23H rhodopsin mutation in Xenopus laevis models of retinitis pigmentosa (RP). We therefore established transgenic Xenopus laevis lines expressing fluorescent reporters of autophagy and used them to further characterize autophagy in wild-type and degenerating rods.

Methods : We established transgenic Xenopus laevis lines expressing dually fluorescent mRFP-eGFP-LC3 under control of either the Xenopus Rod Opsin promoter (XOP) for rod-specific expression, or the hsp70 promoter (hsp70) for inducible expression. F1 animals were sacrificed at various time-points under various lighting conditions. We also generated LC3 reporter animals co-expressing a bovine P23H rhodopsin transgene. Animals were also exposed to potential inducers of autophagy, including HDAC inhibitors.
Cryosectioned eyes were examined by confocal microscopy, and fluorescent puncta were quantified.

Results : Autophagic structures were observed in the inner segments of wildtype rods, suggesting autophagy is a normal feature of rod photoreceptors.
Experiments using heat-shock induction of fluorescent LC3 demonstrated that in rods, autophagosomes persist for 6 to 8 hours before fusing with lysosomes and acidifying. Acidified autolysosomes persist for 28 hours at least.
We observed diurnal non-circadian variation in the number of autophagic structures, with more autophagosomes generated under light illumination; the number of autophagic structures increased 1.9 fold after 10 hours of 1700 lux light illumination.
In rods expressing bP23H rhodopsin, the number of autophagic structures increased by 52% compared to that of wildtype rods.
The histone deacetylase (HDAC) inhibitors VPA and CI994, which can rescue retinal degeneration associated with P23H rhodopsin, promoted autophagy in rods by 23% and 37% respectively.

Conclusions : Autophagy was enhanced in rods expressing P23H rhodopsin, suggesting it plays a role in retinal degeneration; autophagy induced by light may be a related phenomenon associated with decreased stability of WT rhodopsin.
HDAC inhibitor treatment, especially CI994, promoted autophagy in rods. Upregulation of autophagy is a candidate protective mechanism by which HDAC inhibitor treatment may prevent RD associated with P23H rhodopsin in X. laevis models of RP.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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