Localization of exon-skipping RGR-d opsin and C5b-9 deposits in Bruch’s membrane of mice

Abstract

Purpose : Age-related macular degeneration is characterized by extracellular drusen in the form of basal linear and basal laminar deposits beneath or within Bruch’s membrane. In humans, the exon-skipping isoform of RGR-opsin (RGR-d) is associated with drusen and co-localizes with vitronectin and terminal complement complex C5b-9 in extracellular deposits. Here, we studied the localization of mouse RGR-d and C5b-9 in gene-edited RGR-d mice.

Methods : Multiple RGR-d mutant mouse lines (strain: B6D2F1/J) were created by mutation of the splice site at each end of exon VI of the mouse Rgr gene. RGR knockout mice (RGR-/-) were generated previously. Eyes were fixed with 4% paraformaldehyde for frozen sections and immunofluorescence (IF) assay. Affinity-purified polyclonal antibody DE21, which is directed against human RGR-d, and a monoclonal antibody against the neoepitope of C5b-9 were used as IF probes.

Results : We have shown previously by Western blot assay that RGR-d is expressed at extremely low levels in homozygous RGR-d (d/d) mice relative to the abundance of normal RGR in wildtype (+/+) and heterozygous (+/d) mice (<1% of normal RGR). We did not detect RGR-d by immunofluorescent labeling in an 8-mon-old RGR-d (d/d), 8-mon-old wildtype (B6D2F1/J), and 2-yr-old RGR-/- knockout mice. On the other hand, a 2-yr-old RGR-d (d/d) mouse showed pronounced immunofluorescent labeling of RGR-d within RPE cells, in the basolateral plasma membrane, or close to the basal side of the RPE, while labeling of other RPE cells was negative. C5b-9 was present noticeably in the basal extracellular areas of the RPE in the 8-mon-old RGR-d (d/d) mouse, but undetectable in the age-matched wildtype (+/+) mouse. In contradistinction, the 2-yr-old RGR-d (d/d) mouse did not present significant deposits of C5b-9.

Conclusions : The steady-state accumulation of RGR-d appears to be significantly higher in older RGR-d mutant mice and has a histological distribution similar to that found in older humans, including sporadic targeting to the basolateral plasma membrane and basal areas of discrete RPE cells. Meanwhile, it is not clear whether complement activation in Bruch’s membrane of these mice is linked to RGR-d, and we did not find obvious co-localization of RGR-d and C5b-9. Ultrastructural analysis by immunoelectron microscopy will provide further insight into the phenotypic changes that occur in these mice.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.