Atypical Degeneration Mechanisms of the Retinal Pigment Epithelium upon Ablation of Ran-binding protein 2 (Ranbp2)

Abstract

Purpose: The manifestation of several neurodegenerative and aging-related disorders, such as age-related macular degeneration (AMD), culminates with the degeneration of the retinal pigment epithelium (RPE). However, the pathophysiological and cell-death mechanisms underlying RPE degeneration upon a variety of intrinsic and extrinsic stressors remain ill-defined. This study aims at testing the hypothesis that compared to other cell types, Ranbp2 controls unique and shared mechanisms of cell survival in the RPE.

Methods: We generated mice with selective ablation of Ranbp2 in the RPE (cre-RPE::Ranbp2^flox/flox). Longitudinal examination of RPE degeneration was carried-out by molecular, proteomic, morphometric and electrophysiological analyses between wild-type or cre-RPE::Ranbp2^+/+ and cre-RPE::Ranbp2^flox/flox. Markers of cell death were used to discern cell death mechanisms in the RPE.

Results: Ranbp2 undergoes ablation at E18, but physiological EdU labeling shows that Ranbp2 is dispensable to RPE cell proliferation and RPE degeneration ensues upon RPE cells exit mitosis at ~P4. RPE degeneration does not undergo the development of TUNEL⁺-cells or activation of caspases. Instead, ablation of Ranbp2 triggers the activation of a set of metalloproteinases in the mature RPE. This mechanism causes the escape of dying RPE cells to the subretinal space and profuse fluorescein leakage from the choriocapillaris. Compared to cre-RPE::Ranbp2^+/+, cre-RPE::Ranbp2^flox/floxmice present first a reduction of the amplitude of the light-adapted ERG; by 8- and 24-weeks of age, light and dark-adapted ERGs become reduced. The amplitude of the waveform of the direct current (dc) ERGs of the RPE was markedly reduced with all its major components (c-wave, fast oscillation, light-peak and off-response) being greatly affected at 4-weeks of age. However, there were no differences in the kinetics of dark adaptation between genotypes even though there is a decrease of the transcriptional and translational levels of Rpe65 in cre-RPE::Ranbp2^flox/flox mice. Protein profiling uncovered strong deregulation of proteostasis of ~12 proteins as early as P14 in mutant RPE.

Conclusions: These studies unveil novel mechanisms underlying the degeneration of RPE and with secondary pathological effects on neighboring tissues. These studies will contribute to our understanding of AMD pathogenesis and other aging-related disorders.

Keywords: 701 retinal pigment epithelium • 494 degenerations/dystrophies • 636 pathobiology