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T Michael Redmond, Todd Duncan, Yichao Li, Haohua Qian, Lijin Dong, Yan Li; P25L knock-in of mouse Rpe65 shows little change in visual function under normal low light conditions but confers protection against acute high intensity light damage. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3471.
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© ARVO (1962-2015); The Authors (2016-present)
RPE65 is the RPE visual cycle retinol isomerase and various mutations of RPE65 in human lead to the retinal dystrophy termed Leber congenital amaurosis 2 (LCA2) that result in blindness. One such mutation is the homozygous P25L found in a LCA2 patient with mild phenotype including well-preserved cone function. We previously found that the hypomorphic RPE65 P25L missense mutant enzyme retains about 8% of wildtype (WT) activity in vitro. We generated the Rpe65/P25L knock-in mouse model to understand how this missense mutation affects retinal biochemistry and physiology.
Retinal/RPE integrity of the homozygous Rpe65 /P25L knock-in mice (referred as KI/KI) and WT siblings (both Leu at aa450) were studied by optical coherence tomography (OCT) in vivo, and by histology. Visual function was examined by single flash ERG at different stimulus intensities. The ocular retinoid levels of KI/KI and WT mice were compared by reverse-phase HPLC. KI/KI mice and WT siblings were subjected to acute light damage treatment (10, 000 lux for 30 minutes) and consequent retinal degeneration was monitored.
Both histology and OCT showed no significant morphological differences between KI/KI and WT mice maintained under standard colony management protocols. HPLC retinoid analysis revealed comparable levels of 11-cis retinal and other retinoids in KI/KI and WT retinae and eyecups. Scotopic ERG responses of KI/KI mice also were similar to those observed in WT mice. The recovery of dark adaptation after light bleach, however, was slightly delayed in KI/KI mice, suggesting altered kinetics of visual cycle and/or rhodopsin regeneration. Significantly, P25L KI/KI mice showed increased resistance to light-induced retinal damage compared to WT animals.
Taken together, our data suggest that under modest light intensities (<100 lux; such as experienced in standard mouse husbandry), mice with the P25L mutation have ample time to replenish chromophore, which thus manifests in a minimal phenotype in the KI/KI mice. Only when the retina is stressed with acute high light intensity is the biochemical effect of this mutation seen at the physiological level, when the reduced expression and catalytic activity of the hypomorphic P25L RPE65 protects the retina from the successive cycles of opsin regeneration that drive the light damage outcome.
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