Abstract
Purpose :
Activation of retinal melatonin receptors at nighttime is thought to synchronize cyclic events that optimize homeostatic photoreceptor renewal with the predictable daily solar cycle. We hypothesize that disruption of melatonin receptor-meditated entrainment of circadian retinal functions is detrimental to long-term photoreceptor health.
Methods :
Xenopus tropicalis embryos were injected with Cas9 enzyme and single guide RNAs targeting specific exon sequences of the Mel1a melatonin receptor (mtnr1a) gene. Mature F0 were crossed with wild-type (WT) animals to generate heterozygous F1 progeny. F0 and F1 animals were genotyped by RFLP assays, T7E1 endonuclease cleavage assays, and PCR sequencing. F1 tadpoles were exposed to melatonin at night and several hours each day for 1- 2 weeks. Phenotypic analyses of F1 progeny included histological processing and immunohistochemistry.
Results :
Rod dystrophy was observed in F1 heterozygous animals expressing a nine-base pair non-frameshift deletion mutation of the mtnr1a coding region, with the severity correlating to age and/or developmental stage. The most severe phenotype was in a late-stage tadpole with delayed metamorphosis in which there was an 88.1% loss (P< 0.0001) of rod outer segments (ROS) with preservation of cone photoreceptors. Earlier stage mutants also displayed rod dystrophy that was often associated with infiltration of retinal pigment epithelial (RPE) cells into the ROS layer. Postmetamorphic froglets revealed subtle signs of earlier pathological trauma from which they had recovered, such as glial scarring, Müller cell proliferation, and RPE invasion. Acute cyclic treatment with melatonin that included several hours of light exposure induced mild ROS dystrophy in WT tadpoles, but not in mtnr1a deletion mutants.
Conclusions :
The rod dystrophy in F1 mtnr1a heterozygous mutant tadpoles suggests that the Mel1a receptor mutation may show a dominant negative effect similar to those of other G-protein coupled receptor mutants. The enhanced ability of the X. tropicalis retina to regenerate after metamorphosis may account for the less severe postmetamorphic dystrophy. The deleterious impact of melatonin signaling perturbation on long-term rod health may paradoxically protect rods from the detrimental effects of acute melatonin exposure during the daytime.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.