Abstract
Purpose :
KCNJ13 encodes Kir7.1, a low conductance inwardly rectifying potassium channel expressed in the apical processes of human retinal pigment epithelium (RPE). Kir7.1 is involved in ion homeostasis and maintaining the RPE resting potential. Human KCNJ13 mutations lead to Leber’s Congenital Amaurosis (p.W53X, p.L241P and p.R166X). The kcnj13-/- (obetd15 p.F168L) zebrafish shows altered body pigment patterning attributed to impaired responses to melanosome dispersion signals, the ocular phenotype has not previously been investigated. The purpose of this longitudinal study was to characterise the retinal phenotype of the kcnj13-/- mutant zebrafish.
Methods :
Wild type (wt) and kcnj13-/- zebrafish were studied at day 5, then 1, 2, 3, 6 and 12 months (n=5 at each timepoint). Retinal histology and live in vivo OCT imaging were used to assess degeneration and measure retinal thickness using Image J and Bioptigen InVivoVue software, respectively (mean ± SD µm). TUNEL assays were performed to assess apoptosis. Immunostaining of the retina with zpr-1, zpr-3 and GFAP were undertaken to study the effect on cone and rod photoreceptors and muller cells.
Results :
The kcnj13-/- mutant retina develops normally until 3 months, after which mild retinal thinning (194.0 ± 5µm) is seen from retinal histology compared to the wt (220.0 ± 4µm), p<0.01 using unpaired t-test. Muller cell activation is seen at 6 months from localised increase in GFAP expression, and associated widespread retinal degeneration of the RPE and photoreceptor layers with an increase in cell death. At 12 months this progresses to gross atrophy and retinal thinning (133.0 ± 3µm) compared to age-matched wt fish (269.0 ± 4µm), p<0.01. There was a reduction in both apoptosis and GFAP expression. OCT confirmed significant retinal thinning at 1 year with en face images qualitatively demonstrating increased spacing of photoreceptors in kcnj13-/- mutant compared to wt zebrafish.
Conclusions :
There is evidence of an early onset progressive retinal degeneration from 3 months, with signs of muller cell activation and apoptosis from 6 months in the kcnj13-/- zebrafish. Disruption of ion homeostasis and melanosome dysfunction within the RPE may be contributing to the downstream pathophysiology seen in this disease model.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.