June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Glaucoma-associated human Optineurin mutations increase transmitophagy in the Xenopus laevis tadpole optic nerve
Author Affiliations & Notes
  • Yaeram Jeong
    Ophthalmology, University of California Davis, Davis, California, United States
  • Nicholas Marsh-Armstrong
    Ophthalmology, University of California Davis, Davis, California, United States
  • Footnotes
    Commercial Relationships   Yaeram Jeong None; Nicholas Marsh-Armstrong None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 9. doi:
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    • Get Citation

      Yaeram Jeong, Nicholas Marsh-Armstrong; Glaucoma-associated human Optineurin mutations increase transmitophagy in the Xenopus laevis tadpole optic nerve. Invest. Ophthalmol. Vis. Sci. 2022;63(7):9.

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

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Abstract

Purpose : Different mutations in the mitophagy adaptor Optineurin (OPTN) are associated with either glaucoma or Amyotropic Lateral Sclerosis (ALS). The purpose of this research was to investigate whether disease-associated OPTN variants alter the behavior of mitochondria, OPTN or the autophagy receptor LC3b within retina ganglion cell (RGC) axons in the optic nerve (ON).

Methods : Transgenes encoding GFP-LC3b and human mCherry-OPTN variants (Wt, glaucoma-associated mutations E50K, M98K and H486R, ALS-associated mutations E478G, and synthetic mutations F178A and D474N affecting LC3b or ubiquitin binding) were expressed selectively in Xenopus RGCs in a tetracycline inducible manner using the zebrafish Isl2b RGC-specific promoter 3 days prior to imaging. For sparse-labeling studies, small groups of cells were transplanted from transgenic donors to non-transgenic hosts early in eye development. The ONs of 4-6 tadpoles per transgene were live imaged using a spinning disc confocal as 1-minute single plane t-series acquired at 1 Hz, and Z-scan imaging the full thickness of the ON. Imaging data were analyzed using ImageJ, IPLab scripts and Imaris and the movement of mitochondria, OPTN and LC3b measured using kymographs. Vision was assessed in a dot-avoidance assay.

Results : Wt OPTN and LC3b were found to move ortho- and retro-gradely in comparable numbers with very few (~2%) of puncta stalled within the ON, whereas 23-88% of OPTN and LC3b were found stalled after expression of all OPTN mutants. In addition, a greater fraction (~65%) of axonal mitochondria were also found stationary in E50K mutants. In the imaging of sparsely labeled axons, E50K OPTN expression resulted in increased OPTN (5.7% in Wt→21.8% in E50K) and mitochondria (15.2% in Wt→35.8% in E50K) outside of RGC axons. Tadpoles expressing E50K OPTN had normal vision at the time of live imaging but developed vision impairment a week later.

Conclusions : OPTN mutations associated with ALS and glaucoma as well as synthetic mutants perturbing its interaction with damaged mitochondria or autophagosomal machinery all affect the movement of mitophagy machinery (OPTN and LC3). The glaucoma-associated mutations in addition increased the amount of axonal mitochondria and OPTN outside of axons. Visual impairment in the E50K animals subsequent to increased transmitophagy suggests that the increased or dysregulated transmitophagy may be detrimental.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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