Claudin-1 deficiency in retinal pigment epithelium leads to pigmentary abnormalities and age-related extracellular deposits in mice

Abstract

Purpose : We previously reported that claudin-1 is expressed in tight junctions in mouse retinal pigment epithelial (RPE) cells (Annual meeting of Japanese Ophthalmological Society, 2022). In this study, we generate RPE-specific claudin-1 conditional knockout mice (Cldn1 cKO mice) and report the phenotype to investigate the role of claudin-1 in the RPE.

Methods : Cldn1^flox/flox mice were crossed with Best1-Cre^+/- mice to generate Cldn1 cKO mice. The choroidal thickness were evaluated by swept-source optical coherence tomography (OCT). The average of RPE cell area for each visual field and the coefficient of variation (CV) were calculated from RPE flat mounts stained with ZO-1. The pathological morphology of retina, Bruch’s membrane (BM) and choriocapillaris were examined by transmission electron microscopy (TEM) images and retina/choroid/sclera cross-sectional immunohistochemistry (F4/80).

Results : 16-week-old Cldn1 cKO mice had significantly thinner choroid (Control:61.9 ± 11.5 vs Cldn1 cKO:47.8 ± 7.30 μm, p<0.001, n=13, n=19). The representative Cldn1 cKO mouse showed a double layer sign on OCT image. In 20-week-old mouse RPE flat mounts, Cldn1 cKO mice showed significantly larger RPE cell area in peripheral area (Control:330 ± 49 vs Cldn1 cKO:410 ± 65μm², p<0.05, n=5) and larger CV in central area (Control:0.36 ± 0.013 vs Cldn1 cKO:0.41 ± 0.133, p<0.05, n=6) and equatorial area (Control:0.36 ± 0.043 vs Cldn1 cKO:0.48 ± 0.12, p<0.05, n=6) . In 20-week-old mouse TEM images, RPE pigmentary abnormalities such as ectopic pigment granules or multi-layered RPE were observed in 3 of 6 Cldn1 cKO mouse eyes. In addition, subretinal drusenoid deposits (SDD) formed accumulations of round heterogeneous vacuoles were observed in 4 of 6 Cldn1 cKO mouse eyes and basal lamellar deposits (BlamD) was detected in 6 of 6 Cldn1 cKO mouse eyes. The macrophages adhered to thicken the BM or within the BM were observed in 5 out of 6 Cldn1 cKO mouse eyes. The macrophage accumulation within choriocapillaris weas also presented by immunohistochemistry.

Conclusions : Cldn1 cKO mice showed intermediate age-related macular degeneration-like phenotype defined by RPE morphological abnormalities and age-related extracellular deposition such as SDD and BlamD. Our study provides new insights into the possible association of tight junctions molecules with lipid metabolism and cellular aging in the RPE.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.