Di-retinoid-pyridinium-ethanolamine (A2E) Accumulation and the Maintenance of the Visual Cycle Are Independent of Atg7-mediated Autophagy in the Retinal Pigmented Epithelium

Bhubanananda Sahu,Akiko Maeda,Krzysztof Palczewski,Carlos S. Subauste,Yun-Zheng Le,Yu Chen,Lindsay Perusek,Tanu Parmar,Hiroshi Maeno,Eisuke Arai

doi:10.1074/jbc.m115.682310

Abstract

Autophagy is an evolutionarily conserved catabolic mechanism that relieves cellular stress by removing/recycling damaged organelles and debris through the action of lysosomes. Compromised autophagy has been implicated in many neurodegenerative diseases, including retinal degeneration. Here we examined retinal phenotypes resulting from RPE-specific deletion of the autophagy regulatory gene Atg7 by generating Atg7(flox/flox);VMD2-rtTA-cre+ mice to determine whether autophagy is essential for RPE functions including retinoid recycling. Atg7-deficient RPE displayed abnormal morphology with increased RPE thickness, cellular debris and vacuole formation indicating that autophagy is important in maintaining RPE homeostasis. In contrast, 11-cis-retinal content, ERGs and retinal histology were normal in mice with Atg7-deficient RPE in both fasted and fed states. Because A2E accumulation in the RPE is associated with pathogenesis of both Stargardt disease and age-related macular degeneration (AMD) in humans, deletion of Abca4 was introduced into Atg7(flox/flox);VMD2-rtTA-cre+ mice to investigate the role of autophagy during A2E accumulation. Comparable A2E concentrations were detected in the eyes of 6-month-old mice with and without Atg7 from both Abca4(-/-) and Abca4(+/+) backgrounds. To identify other autophagy-related molecules involved in A2E accumulation, we performed gene expression array analysis on A2E-treated human RPE cells and found up-regulation of four autophagy related genes; DRAM1, NPC1, CASP3, and EIF2AK3/PERK. These observations indicate that Atg7-mediated autophagy is dispensable for retinoid recycling and A2E deposition; however, autophagy plays a role in coping with stress caused by A2E accumulation.

Highlights

Because A2E accumulation in the retinal pigmented epithelium (RPE) is associated with pathogenesis of both Stargardt disease and age-related macular degeneration (AMD) in humans, deletion of Abca4 was introduced into Atg7flox/flox;VMD2-rtTA-cre؉ mice to investigate the role of autophagy during A2E accumulation
Atg7 is a required component of the autophagosome maturation pathway and is ubiquitously expressed in the retina, in the RPE, inner segment of photoreceptors (IS), inner plexiform layer (IPL), and the ganglion cell layer (GCL) (Fig. 1B, left)
Cell hypertrophy resulting from cellular stress was observed in Atg7-deficient RPE as early as 6 weeks of age

Summary

Background

We examined retinal phenotypes resulting from RPE-specific deletion of the autophagy regulatory gene Atg by generating Atg7flox/flox;VMD2-rtTA-cre؉ mice to determine whether autophagy is essential for RPE functions including retinoid recycling. The retinal pigmented epithelium (RPE) is a single layer of post mitotic cells located in the back of the eye directly adjacent to the photoreceptor layer of the retina This layer is responsible for maintaining many functions needed to preserve retinal health including retinoid recycling, nutrient exchange, ion balance, blood-retinal-barrier integrity, and phagocytosis of daily shed photoreceptor outer segments (POS) [1, 2]. We used a RPE-specific Atg7-deficient mouse Atg7flox/flox;VMD2-rtTA-creϩ (abbreviated to Atg7flox/flox;creϩ) to address whether autophagy-deficiency results in RPE dysfunction including impaired POS phagocytosis and abnormal A2E accumulation during aging, which could lead to retinal degeneration (28 –31)

Experimental Procedures

Results

Discussion