CIB2 is essential for autophagy in the retinal pigment epithelium and visual function

Abstract

Purpose : Down-regulation of Calcium and integrin binding protein 2 (CIB2) in drosophila significantly reduced photoresponse amplitudes and caused light-dependent retinal degeneration. To assess the role of CIB2 in mammalian retina we developed whole-body and tissue specific Cib2 deficient mice (Cib2^ko).

Methods : We generated retinal pigment epithelium using VMD2-Cre and rod photoreceptor using rhodopsin–iCre75, as well as body-wise knockout (Cib2^ko) mutant mice. Temporal electroretinogram (ERG) was used to assess vision. While light and transmission electron microscopy was used to interrogate retinal structure and RPE/PR interface. Further, we assessed the function of CIB2 in the process of autophagy and related molecular pathway modulation using biochemical and imaging assays. Molecular partners of CIB2 interactome in the retina were validated using immunoprecipitation and nanoSPD assays. Statistics: One-way ANOVA or student’s t-test.

Results : Here we show that deficiency of CIB2 within murine RPE causes an AMD-like phenotype, including RPE vacuolization and/or sub-RPE drusen formation, lipid accumulation, aberrant clearance of photoreceptor outer segments, and attenuated electroretinogram amplitudes, which can be rescued using exogenous retinoids. Mice with ubiquitous or RPE-specific loss of Cib2 displayed reduced autophagy and lysosomal proteins, reduced number of functional lysosomes, and autophagy flux – ex vivo. In vivo, mutant mice starved for 24 hrs to induce autophagy showed that their P62/SQSTM1 and LC3-II levels doubled as compared to those in WT age-matched controls. Also, overexpressing CIB2 markedly increased autophagic flux. Similar to Cib2 mutant mice, in RPE/choroid tissues from humans affected with age-related macular degeneration (AMD), we found significantly decreased autophagy flux. We investigated the molecular mechanism of autophagy defects and found dysregulation of a master signaling cascade in the Cib2 deficient mouse RPE and also in human AMD RPE/choroid samples. This work will be presented at the meeting.

Conclusions : Our study links autophagy defects and specific signaling defects in AMD-like mouse model and human AMD patient samples. Further, mechanically we find that CIB2 regulates an essential signaling cascade controlling autophagy which has broad relevance beyond just AMD.

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