Abstract
Purpose :
Different mutations in the mitophagy adaptor Optineurin (OPTN) are associated with either glaucoma or Amyotropic Lateral Sclerosis (ALS). The purpose here was to investigate whether there is a difference between wild type (Wt) and the glaucoma- or ALS-associated human OPTN variants in the movement of either OPTN or the autophagy receptor LC3b within retina ganglion cell (RGC) axons in the optic nerve (ON).
Methods :
Transgenes encoding GFP-LC3 and human mCherry-OPTN variants (Wt, glaucoma-associated mutations E50K and M98K, and ALS-associated mutations F178A and E478G) where generated by recombineering and expressed selectively in Xenopus RGCs in a tetracycline inducible manner using the zebrafish Isl2b promoter. The ONs of 4-7 young tadpoles per transgene were live imaged using a Dragonfly spinning disc confocal as either 1-minute single plane t-series acquired at 1-hz, or z-series imaging the full thickness of the ON. Imaging data were analyzed using ImageJ and IPLab scripts and the movement of LC3 and OPTN measured through kymographs.
Results :
Wt OPTN was found to move ortho- and retro-gradely in comparable numbers, both at ~ 5um/sec, with very few (~2%) of puncta stalled within the ON, and in its presence only 1/3 of LC3 puncta moved retrogradely also with very few (~1%) of puncta stalled. The most stricking differences in the mutants are very large increases in stalled OPTN (>50%) and LC3 (>30%) for E478G, M98K and E50K mutants.
Conclusions :
OPTN mutations associated with ALS and glaucoma affect the movement of both OPTN and LC3 within the ON, resulting in a high fraction of stalled OPTN and LC3. The possibility that axonal OPTN may be found outside axons is currently being analyzed in the concurrently acquired z-stacks.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.