Abstract
Purpose :
Retinal dehydrogenase 12 (RDH12) is a photoreceptor NADPH-dependent retinal reductase enzyme, which converts all-trans-retinal to all-trans-retinol. Mutations in RDH12 are primarily associated with severe early onset autosomal recessive (AR) Leber congenital amaurosis and in rare cases mild late onset autosomal dominant (AD) retinitis pigmentosa. While RDH12 mouse and zebrafish models do not recapitulate the early-onset human phenotype, we developed retinal organoid models derived from human induced pluripotent stem cells (hiPSC), obtained from skin biopsies of one RDH12 AR, one RDH12 AD patients and one unrelated unaffected individual (WT).
Methods :
Wild-type, RDH12 AR and RDH12 AD hiPSC were differentiated into retinal organoids and characterised at different stages of differentiation by immunostaining (n=3 per line) with retinal- and photoreceptor-specific markers (CRX, OTX2, RECOVERIN, RHO, LM-opsin), autophagy (LC3), oxidative (8-OHdG, Acrolein) and ER stress (CHOP/DDIT3, BiP/HSPA5) markers and TUNEL assay for cell death. Mature RDH12 mutant retinal organoids were stressed with 100 µM all-trans-retinal for 24 hours and assessed for cell death, autophagy, oxidative and ER stress (n=3 per condition).
Results :
Immunofluorescence studies revealed 9-week-old RDH12 mutant retinal organoids produced less CRX, RECOVERIN and OTX2 than wild-type organoids. Both wild-type and RDH12 retinal organoids were grown to week 44, they all developed rod and cone photoreceptors but there was no significant difference in cell death, autophagy, oxidative and ER stress. Moreover, RDH12 mutant organoids were resistant to all-trans-retinal induced stress.
Conclusions :
Our study is the first to characterise human RDH12 retinal organoid models but there were few differences between WT and mutants at later stages of photoreceptor differentiation. Further work is required to study the ultrastructure and transcriptomic signature for any intracellular and molecular pathophysiology associated with RDH12-related retinopathies.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.