Abstract
Purpose :
Retinal degenerative diseases are inherited disorders that cause blindness. Defects in the prominin-1 (prom1) gene result in retinitis pigmentosa, cone-rod dystrophy, and Stargardt-like macular dystrophy; how this occurs is unknown. In tadpoles, loss of prom1 results in dysmorphic and functionally-impaired photoreceptors, but no cell death. Adult frogs (0.5-2 yrs) maintain dysmorphic photoreceptors but have growing accumulations of cellular debris in the outer segment layer. We hypothesize that these deposits mimic human reticular pseudodrusen (RPD) and that prom1 mutations can cause retinal degeneration via RPE toxicity, instead of direct effects on photoreceptor morphogenesis.
Methods :
Cas9 mRNA, GFP, and prom1-specific guide RNA were injected into fertilized eggs to knock down prom1 via random non-homologous end joining. At 2 days post-fertilization, embryos were screened for GFP expression to confirm injections of non-degraded RNA and Sanger sequencing was performed to confirm successful prom1 gene editing. Resultant phenotypes in prom1 null animals were analysed by histology, ERG, fundus photography, and OCT.
Results :
At 2 weeks, prom1 null rod outer segments are shortened and bulbous; cone outer segments are severely elongated and fragmented. At 6 weeks, dysmorphic photoreceptors remain partially functional. At 6 months, dysmorphic photoreceptors persist and accumulation of cellular debris in the outer segment layer begins. At 1-2 yrs, photoreceptor and RPE defects are visible by OCT and fundus imaging; outer segment deposits are large (ca. 35 µm), autofluorescent, stain positively for nuclear material, stain negatively for neutral lipids, and contain RPE pigment granules.
Conclusions :
Similar to humans, and unlike mice, disease progression in prom1 null X. laevis is slow and affects cones more severely than rods. Dysmorphic photoreceptors continue to synthesize and maintain outer segment discs, suggesting that prom1 is not necessary for morphogenesis but may instead play a supportive role in photoreceptor assembly and maintenance. RPD-like deposits in adult frogs suggest, for the first time, RPE-toxicity as a possible mechanism of prom1 null-mediated retinal degeneration. This finding exposes novel avenues of investigation for potential blindness-preventing therapies. RPD are not well understood as there is no suitable animal model to study them, and our prom1 null frogs could potentially address this need.
This is a 2020 ARVO Annual Meeting abstract.