Conditional late deletion of IRBP causes loss of retinal function and a slow retinal degeneration without myopic eye elongation

Abstract

Purpose : Germline knockout of IRBP (i.e., RBP3) results in severe myopia starting early in development (P7) followed much later by retinal degeneration (P25+). Here, we test the hypothesis that late deletion of IRBP expression should cause loss of photoreceptor function and retinal degeneration without an increase in eye axial length.

Methods : To conditionally delete IRBP late in rod photoreceptors specifically, we used Rho-iCRE75. Littermate controls without the Rho-iCRE75 driver, but with the RBP3^lox/lox gene, were used for comparison. IRBP mRNA levels were measured by digital droplet RT-PCR. Retina function was measured with electroretinograms (ERGs). Fundus imaging and SD-OCT evaluated retina morphology in vivo. Eye axis and circumference dimensions were measured with noncontact micrometers at sub-micron accuracy and precision. ONL counts were conducted on H&E stained sections.

Results : By P30, IRBP gene expression levels were significantly decreased by about 90% in the Rho-iCRE75 RBP3^lox/lox mice vs the RBP3^lox/lox mice (p<.0001). At P60, the ERGs showed significant decreases in a-wave (~34%; p<0.01) and c-waves (~21%; p<0.05) for the Rho-iCRE75 RBP3^lox/lox mice, while the b-wave trended about 13% lower but was not statistically significant. Fundus photographs and SD-OCT imaging showed no obvious pathology in any group. Eye axial length, equatorial width, fit diameter, and roundness were not significantly different between genotypes at either age group. Retinal arc lengths were not different at either age group. Interestingly, outer nuclear layer counts at P30 showed an 8% significant decrease of nuclei in Rho-iCRE75 RBP3^lox/lox versus RBP3^lox/lox mice (p<0.05).

Conclusions : 1. Loss of IRBP late (after rapid early eye growth is mostly complete) was not associated with myopic eye growth. 2. The eye may already be incompetent to experience abnormal eye elongation when IRBP was lost late. 3. Selectively early IRBP expression was sufficient to allow normal emmetropia in the mouse eye. 4. However, the late loss of IRBP profoundly decreased photoreceptor number and function soon after IRBP mRNA was lost. This suggests that IRBP independently controls photoreceptor maintenance and emmetropic eye growth, implying at least two different and independent mechanistic roles for IRBP.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.