Abstract
Purpose :
To evaluate retinal degeneration (RD) phenotypes, retinomotor functions and visual behaviors in abca4b-/- zebrafish model.
Methods :
The abca4b-/- zebrafish model was generated by CRISPR-Cas9 gene-editing. To assess the impact of abca4b knockout on retinal functions, retinomotor movements were evaluated by measuring parameters like melanosomes migration, photoreceptor (PR) inner and outer segment (OS) and rod nuclear layer thickness (n=3). The accumulation of auto-fluorescing lipofuscin deposits was checked using confocal microscope. Visual behaviour of 12M mutants was assessed based on their ability to capture feed using different maze paradigms (n=20). An optokinetic response (OKR) drum with sine wave gratings of 0.3 cycles/degree with 100% contrast sensitivity was designed to record the saccadic eye movements of mutants.
Results :
The abca4b-/- mutants exhibited normal day-night retinomotor movements, but displayed significant reduction in inner and outer segment thickness and early disintegration of cone OS and a gradual loss of rod and cone PRs, suggesting progressive RD. Upon dark adaptation, the arborization of apical microvilli of RPE was significantly reduced in mutants and the cells were hyperpigmented under light-adaptation, suggesting an increased photosensitivity of RPE and impaired absorption of photons by PRs. PCNA+ proliferating precursors at ciliary marginal zone (CMZ) of mutants was significantly reduced at 6M and absent at 12M, suggesting accelerated loss of retinal progenitor reserve, due to the ongoing degenerative stress and repair processes in mutants. The mutants also displayed accumulation of lipofuscin deposits within RPE and choroidal layer, indicating RPE cell stress. When the mutants were tracked for feed capture response, the heat maps indicated random movements. Frame-based quantification and polar plots displayed a higher latency, restricted directional movements and turn angles during feed capture in mutants. OKR evaluations of mutants displayed a random or totally extinguished saccadic eye movements.
Conclusions :
The abca4b-/- mutants recapitulated the Stargardt’s macular dystrophy displaying accelerated PR degeneration, hyperpigmentation, and increased photosensitivity of RPE with lipofuscin deposits. The loss of retinal precursor in the CMZ accelerated retinal degeneration leading to diminished visual function in abca4b-/- mutants.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.