Abstract
Purpose :
Mutations in RDH12 are primarily associated with severe early onset Leber congenital amaurosis, and in rare cases autosomal dominant mutations are linked to late onset retinitis pigmentosa. Aim: to generate a rdh12 zebrafish mutant model and characterise the retinal phenotype to study disease mechanisms.
Methods :
CRISPR/Cas9 gene editing was used to generate rdh12 mutant zebrafish. Fish were characterised by retinal histology, TUNEL staining for apoptotic nuclei and immunostaining for rhodopsin and cone opsins. Levels of all-trans retinal were quantified by HPLC. Transmission electron microscopy was used to examine retinal ultrastructure. Expression of oxidative stress and autophagy markers were analysed by RT-qPCR.
Results :
The rdh12u533 mutant line was generated and carried a 7 base pair (bp) deletion in exon 1 (c.17_23del; p.(Val6AlafsTer5)), which resulted in significantly reduced expression of rdh12 mRNA transcript at 5 days post-fertilisation (5 dpf). Characterisation of rdh12u533 fish at 5 dpf did not reveal a retinal phenotype. At 12 mpf, no gross abnormalities were detected in retinal histology and TUNEL staining, however rhodopsin was mislocalised to the inner segments and TEM revealed significantly larger phagosomes in the RPE of mutant fish. Expression of autophagy marker atg12 and oxidative stress marker sod2 were also significantly reduced at 12 mpf.
Conclusions :
Mutant rdh12 fish displayed a late onset rod-predominant degeneration, with early indications of disrupted pathways in adult fish. However, stress in the retina is effectively controlled so as not to cause severe disruption. This phenotype corresponds more with the autosomal dominant RP phenotype. Fish are currently being followed up at 24 mpf to assess further degeneration.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.