Abstract
Purpose :
Juvenile onset macular degeneration, Stargardt (STGD) disease, affects nearly 1:8,000 people and age-related macular degeneration (AMD) is the leading cause of vision loss in people over the age of 50. Both diseases arise primarily from the loss of cone photoreceptors. Recent advances in genome engineering technology and the cone-rich retina of the zebrafish model organism provides the powerful and unique opportunity to create macular degeneration disease models for developing new therapies to slow, prevent, or reverse cone photoreceptor degeneration and prolong vision.
Methods :
STGD disease is most commonly caused by mutations in ABCA4. Using CRISPR/Cas9, we created double null mutants for the two zebrafish abca4 homologs (abca4a and abca4b) that also include transgenic fluorescent reporters for retinal pigmented epithelium (RPE) and UV cones. We examined aged retinas labeled with cone subtype specific markers and visualized lipofuscin.
Results :
Our analyses of aged (17 month) abca4 double mutants reveals all cone outer segment (COS) subtypes are dysmorphic: green COSs appear longer and thinner, crumpled with ragged outlines; UV COS appear longer and thinner; red COSs are much longer; and blue COSs are distinctly dysmorphic. Rod outer segments appear normal in the mutants. RPE phagocytosis of all COS subtypes is altered, with fewer green, red, and blue phagosomes, but more UV phagosomes present in RPE cell bodies (while none are in wildtype (wt) RPE). abca4 double mutant RPE is dystrophic; showing loss of microvillar alignment and gaps/holes. COS quantification reveals reduced numbers and phagosome quantification (per COS) shows diminished RPE phagocytosis.
Lipofuscin accumulation is observed in STGD disease, but its causal relationship to degeneration is unclear. To examine lipofuscin in the zebrafish Stgd mutants, we used 4-channel laser scanning confocal (LSC) and Fluorescence lifetime imaging microscopy (FLIM). Few, if any, structures in the wt retina are consistent with fluorescence profiles expected for lipofuscin but in the abca4 double mutant, there are several large and bright structures in both the 'lipofuscin' confocal channel and FLIM.
Conclusions :
This zebrafish Stgd model provides the unique opportunity to begin to identify and understand the cellular and molecular processes that contribute to cone degeneration in STGD and perhaps to AMD.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.