Abstract
Purpose :
Oxidative stress is considered a hallmark of aging and is purported to play a role in many retinal diseases. However, many in vivo models of oxidative stress and damage are limited due to systemic effects or a lack of inducibility. This study aimed to generate an inducible zebrafish model of cone photoreceptor-specific oxidative stress using the KillerRed protein, which generates reactive oxygen species when photobleached.
Methods :
We injected plasmid containing mitochondrially-targeted KillerRed under the cone gnat2 promoter into embryos to generate Tg(gnat2:mito-KillerRed) zebrafish. For live imaging of cone mitochondria, Tg(gnat2:mito-KillerRed) fish were crossed with Tg(gnat2:mito-TagBFP) fish and stimulated with 561 nm laser.
An optogenetic stimulus array was constructed using 565nm LED Stars, an aluminum heatsink, an LED driver, and a power supply. LEDs were attached to the heatsink in an array to fit under the 12 wells of a standard 48-well plate fitted to 3D-printed housing. Zebrafish larvae were subject to 15 minute intervals of stimulation from 4 to 6 days of age.
Results :
Confocal timelapse stimulation of gnat2:mito-KillerRed in fish co-expressing gnat2:mito-BFP shows an induction of cone mitochondrial migration, a previously reported response to mitochondrial stress (FigA). LED stimulation similarly increases the total number of mislocalized mitochondria in gnat2:mito-KillerRed fish relative to control fish lacking KillerRed and unstimulated gnat2:mito-KillerRed fish (FigB). LED stimulation of gnat2:mito-KillerRed fish caused a specific disruption in mitochondrial morphology in cones while normal morphology of photoreceptors and other retinal cells was maintained (FigC).
Conclusions :
Our results demonstrate that the Tg(gnat2:mito-KillerRed) zebrafish model can be used as an inducible tool for monitoring photoreceptor responses to mitochondrial oxidative stress. Future work with this model will evaluate its potential for studying chronic oxidative stress as animals age.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.