The role of a novel scaffold protein in mediating RPE phagocytosis of photoreceptor outer segment

Abstract

Purpose : The efficient diurnal RPE-mediated phagocytosis of photoreceptor outer segment (OS) relies on a tightly coordinated cascade of extra- and intracellular events that includes OS binding and internalization, followed by phagosome maturation and degradation. However, many aspects of phagocytic machinery, including mechanisms linking cytoskeleton-dependent temporal events with maturation of newly formed phagosomes remain elusive. We used previously established model systems of defective phagocytosis (cBEST1 and MREG/LC3B) to uncover molecular factors contributing to RPE phagocytic clearance in health and disease.

Methods : A combination of LC-MS/MS, Western blot, and co-IP analyses were used to explore the molecular signature of RPE and validate protein-protein interactions. In vivo (UHR-OCT) and ex vivo (H&E/IHC) assessments were performed to characterize retinal phenotype of Ahnak^-/- mouse. Subcellular localization, spatial and temporal association of AHNAK with OS were determined in vitro in primary human and mouse RPE cells using phagosome uptake and maturation assays.

Results : Expression of a giant protein AHNAK (~630kDa) was confirmed across mammalian retina, co-localizing with actin filaments at the RPE apical aspect, suggesting AHNAK’s involvement in regulating RPE cell membrane cytoarchitecture. In vivo imaging revealed a spectrum of abnormalities in Ahnak^-/- eyes, predominantly associated with RPE-photoreceptor and RPE/choroid complexes, which was further confirmed by H&E, IHC, and ex vivo study in Ahnak ^-/- retinal flat-mounts. The involvement of AHNAK in OS processing was examined using in vitro OS uptake model, which showed AHNAK-OS phagosome co-localization at the apical surface of hRPE and accumulation of phagosomes in the RPE of Ahnak ^-/- model. Notably, proteomic study exposed association of AHNAK with MREG and its binding partner LC3B, linking temporal regulation of OS to MREG/LC3B-dependent machinery mediating OS phagosome maturation.

Conclusions : We report a new molecular regulator of RPE phagocytic machinery, AHNAK, and show an essential role of this scaffold protein in temporal regulation of OS phagocytosis and MREG/LC3B-mediated phagosome maturation. Further study is required to better understand the extensive signaling network of the AHNAK-MREG nexus, which ultimately will lead to identification of new therapeutic targets for inherited as well as age-related retinopathies.

This is a 2021 ARVO Annual Meeting abstract.