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. Author manuscript; available in PMC: 2014 Jul 18.
Published in final edited form as: Cell. 2013 Jul 18;154(2):365–376. doi: 10.1016/j.cell.2013.06.012

Non-canonical Autophagy Promotes the Visual Cycle

Ji-Young Kim 1,5, Hui Zhao 1,5, Jennifer Martinez 2, Teresa Ann Doggett 1, Alexander V Kolesnikov 1, Peter H Tang 3, Zsolt Ablonczy 3, Chi Chao Chan 4, Zhenqing Zhou 1, Douglas R Green 2,6,*, Thomas A Ferguson 1,6,*
PMCID: PMC3744125  NIHMSID: NIHMS497035  PMID: 23870125

SUMMARY

Phagocytosis and degradation of photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE) is fundamental to vision. Autophagy is also responsible for bulk degradation of cellular components but its role in POS degradation is not well understood. We report that the morning burst of RPE phagocytosis coincided with the enzymatic conversion of autophagy protein LC3 to its lipidated form. LC3 then associated with single membrane phagosomes containing engulfed POS in an Atg5 dependent manner that required Beclin1 but not the autophagy pre-initiation complex. The importance of this process was verified in mice with Atg5-deficient RPE cells that showed evidence of disrupted lysosomal processing. These mice also exhibited decreased photoreceptor responses to light stimuli and decreased chromophore levels that were restored with exogenous retinoid supplementation. These results establish that the interplay of phagocytosis and autophagy within the RPE are required for both POS degradation and the maintenance of retinoid levels to support vision.

INTRODUCTION

The retinal pigment epithelium (RPE) is single layer of non-regenerating cells essential to homeostasis in the retina and the preservation of vision. Its important functions include supplying nutrients and O2 to the retina, the metabolism of vitamin A for the visual cycle, regulation of fluid and ion balance, maintenance of the blood retinal barrier, and daily phagocytosis of shed photoreceptor outer segments (POS). The RPE is post-mitotic and must carry out these essential functions throughout the life of an individual without the possibility of regeneration (Strauss, 2005). It is not surprising that defects in the RPE are found in a number of degenerative diseases of the eye and that disruption of the RPE can have serious consequences for vision (Davidson et al., 2011; Gal et al., 2000).

RPE cells also play an important role in the integrity of the visual axis through their phagocytic function. Photoreceptors continuously renew their outer segments; a process regulated by circadian rhythms. Early morning outer segment tip shedding precedes a burst of RPE phagocytosis which rapidly clears POS from the retina. Defects in this process in several models (Duncan et al., 2003; Edwards and Szamier, 1977; Nandrot et al., 2007; Nandrot et al., 2006; Nandrot et al., 2008) have demonstrated its importance for the function and longevity of the photoreceptors. Degradation of POS following phagocytosis by RPE takes place following the delivery of lysosomal enzymes to the phagosome and the activation of proteolytic enzymes during acidification of this compartment. Degradation products are then removed from the cell by transport to the blood while some materials are recycled to the photoreceptors to replenish necessary components (Strauss, 2005). Disruption of RPE phagocytosis has been linked to disease phenotypes such as retinitis pigmentosa and rod/cone dystrophies (Gal et al., 2000) however the precise mechanisms are not yet clear.

Another important process for removing cellular debris and damaged organelles is macroautophagy (hereafter autophagy). Constitutive autophagy has been shown to function as a cell-repair mechanism that is particularly important for long-lived post-mitotic cells (Degterev et al., 2005; Komatsu et al., 2006; Nishiyama et al., 2007) (Hartleben et al., 2010). Autophagy declines with age in many tissues (Cuervo et al., 2005) and defects in autophagy have been linked to neurodegenerative syndromes such as Alzheimer’s disease (Boland et al., 2008), Parkinson’s disease, Huntington’s disease and amyotrophic lateral sclerosis (ALS) (Wong and Cuervo, 2010). Autophagy proteins are strongly expressed in the eye (Krohne et al., 2010; Kunchithapautham and Rohrer, 2007b; Wang et al., 2009a) and cellular stresses such as intense light, oxidative conditions, and mitochondrial damage can increase autophagic activity in ocular cells (Kunchithapautham and Rohrer, 2007a; Reme et al., 1999; Wang et al., 2009b) supporting the idea that autophagy is critical to the health of the RPE and retina. In addition, the byproducts of biochemical processes that accumulate with age such as oxidized lipids and lipofuscin are known to interfere with lysosomal degradative processes in cultured RPE cells (Kopitz et al., 2004; Vives-Bauza et al., 2008) suggesting that interference with autophagy might be one mechanism for cellular dysfunction and, by inference, disease progression. The relationship of autophagy to POS phagocytosis and degradation is currently unknown.

The visual cycle is a series of biochemical reactions within the RPE and retina that is fundamental to vision (Tang et al., 2013; Travis et al., 2007). The RPE utilizes all-trans retinol (ROL, also known as vitamin A) to synthesize the chromophore 11-cis retinal (RAL), which is then shuttled across the interphotoreceptor matrix by the interphotoreceptor retinoid binding protein (IRBP) to rod and cone outer segments. Within the POS, 11-cis RAL is bound to G-protein coupled receptors (opsins) to form light-sensitive visual pigment. Exposure to light isomerizes the 11-cis RAL to all-trans configuration altering the 3-dimensional structure of the opsin protein and thereby activating the phototransduction signaling cascade. All-trans RAL is then released from the opsin, reduced to all-trans ROL, and transported back to the RPE for recycling back to 11-cis RAL.

Aging is known to cause a progressive loss of night vision and vitamin A supplementation can be beneficial for treating night vision problems in the elderly (Owsley et al., 2006; Wahl, 1994). One hypothesis suggests that a decline in available retinoids is a factor in the decrease in visual function that can accompany aging (Jackson et al., 2002). Moreover, inadequate 11-cis RAL production has been shown to be associated with cone degeneration; however, the mechanisms are currently not well understood (Rohrer et al., 2005; Tang et al., 2010; Znoiko et al., 2005). The primary route by which the RPE recovers the majority of all-trans ROL from the POS has traditionally been attributed to the activity of IRBP shuttling retinoids across the interphotoreceptor matrix (Bok, 1993; Edwards and Adler, 2000); however, more recent studies have challenged this notion (Liou et al., 1998; Palczewski et al., 1999; Ripps et al., 2000). Although the RPE can also take up bis-retinoids (Finnemann et al., 2002; Katz et al., 1987), 11-cis RAL bound to opsin, and probably other forms of retinoids from the photoreceptors through daily phagocytosis, little is known about how retinoid products recovered by this route affect photoreceptor function and/or survival.

In the current study, we provide data to support a link between the autophagy machinery, POS phagocytosis and the regeneration of chromophore by the visual cycle, without which the RPE cannot support optimal visual function. Our results show that the autophagy protein LC3 associates with single membrane phagosomes containing engulfed POS in an Atg5-dependent manner that also requires Beclin1 but not Ulk1, Atg13, or Fip200 of the autophagy pre-initiation complex. The importance of this process was verified in mice with Atg5-deficient RPE cells that showed evidence of disrupted POS degradation, diminished chromophore levels, and decreased visual function. Supplementation with exogenous chromophore restored visual function, further establishing the connection between phagocytosis and the visual cycle. Thus, in contrast to conventional autophagy, our evidence suggests that a non-canonical form of autophagy functions to support chromophore regeneration through the efficient processing of POS by the RPE.

RESULTS

Activity of the autophagy pathway is cyclically engaged in the RPE

We evaluated whether autophagy is engaged in the eye by examining the conversion of microtubule-associated light chain-3 (LC3) to its lipidated form (LC3-II) (Klionsky et al., 2012) in the retina and RPE of 12 and 28 week old mice (Figure 1A). While LC3-II was readily detected in the retinas of adult mice throughout the day, we noted that this product of the autophagy pathway was most abundant in RPE at 7:00 AM (1 h after lights on) but much less LC3-II was observed when western blots were performed on RPE harvested at 7:00 PM (1 h into the dark cycle). Thus, under basal conditions RPE cells have significant levels of LC3-II conversion that is linked to the time of day.

Figure 1. Autophagy in the mouse eye.

Figure 1

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A. RPE and retinae were isolated from 12 and 28 week old C57BL/6J mice at 7:00 AM and 7:00 PM. Western blotting was performed to determine the amount of the non-lipidated (LC3-I) and lipidated (LC3-II) LC3 present. β-actin was used as the loading control. B. RPE and retinae were isolated at 8:00 AM from control (Atg5f/f) and ATG5ΔRPE mice at 8 and 16 weeks. Western blotting was performed to determine the amount of Atg5 and LC3. β-actin was used as the loading control. C. Posterior cups were harvested at 8 AM from Atg5ΔRPE and littermate controls. Retinae were removed and RPE flat mounts were prepared and then placed in Chloroquine (CLQ) for 1 h. RPE were isolated and western blotting for LC3 was performed. D. Atg5ΔRPE mice were crossed to the GFP-LC3 reporter mouse and RPE flat mounts (harvested at 8:00 AM) were examined for GFP-LC3 punctae.

The lipidation of LC3 involves a complex of the Atg5–12 conjugate and Atg16L, which together may act as an E3-like enzyme for the transfer of phosphatidylethanolamine (PE) to LC3 (Mizushima et al., 2010). We tested this in RPE cells by generating animals in which Atg5 was specifically deleted in the RPE (Atg5ΔRPE). At 8 weeks Atg5 expression was detectable (albeit less so) in the Atg5ΔRPE mouse compared to littermate control mice (Figure 1B). The low level of Atg5 at 8 weeks was reflected in our inability to detect the LC3-II form in control or Atg5ΔRPE RPE suggesting that at this early age this pathway may not be functioning maximally. However, by 16 weeks Atg5 expression was robust in control mice but was undetectable in Atg5ΔRPE RPE cells confirming that we had deleted Atg5 from RPE cells. Further, the appearance of LC3-II in the RPE of adult mice was strikingly diminished in Atg5ΔRPE mice at this time (Figure 1B). Upon treatment with chloroquine to block autophagic flux (Klionsky et al., 2012), RPE still failed to accumulate LC3-II in Atg5ΔRPE mice (Figure 1C) verifying the loss of Atg5. We then visualized the association of LC3-II with membranes through the use of animals with ubiquitous expression of a GFP-LC3 transgene (Mizushima et al., 2004) by generating ATG5ΔRPE× GFP-LC3 transgenic mice and examining RPE flat mounts. In control mice expressing Atg5, GFP-LC3 punctae were readily apparent (Figure 1D, left panel arrows), however these were undetectable in Atg5ΔRPE mice (Figure 1D, right panel).

Retinal function decreases in Atg5ΔRPE mice

As the mice matured, we noted that the absence of Atg5 in the RPE compromised the vision of Atg5ΔRPE mice as determined by scotopic and photopic electroretinography (ERG) recordings. The a-wave amplitude of scotopic ERG waveforms was used as a direct measure of rod function; whereas, the b-wave amplitude of photopic ERG waveforms was used as an indirect measure of cone function (Tang et al., 2011; Tang et al., 2010). At 12 weeks ERG responses in the Atg5ΔRPE mice were indistinguishable from littermate control animals (Figure 2A); however, at 16 weeks Atg5ΔRPE mice exhibited significantly decreased rod response to brighter light stimuli compared to their littermate controls (Figure 2B). Furthermore, cone responses from Atg5ΔRPE mice also showed comparable decreases to bright light stimuli compared to control mice (Figure 2B). Although we observed a significant reduction in ERG responses we did not observe significant loss of nuclei in the outer nuclear layer (ONL) by 24 weeks (Figure 2C) or at any time examined up to 1.5 yrs. (not shown). We also did not observe a significant reduction in cone photoreceptors when they were enumerated following PNA staining at any time point (Figure 2D, and data not shown). Thus, while Atg5 loss in the RPE results in diminished retinal function there was not a detectable decrease in the numbers of photoreceptors.

Figure 2. ERG analysis of the Atg5ΔRPE mouse.

Figure 2

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ERGs were performed on Atg5ΔRPE and littermate control mice at 12 weeks (A) and 16 weeks (B). Each point represents the average of 5 mice. C. ONL counts were performed on control and Atg5ΔRPE retina in the superior and inferior direction from H&E sections as described in the Materials and Methods. No significant differences were noted (p<.05). D. The number of cones was enumerated from PNA stained frozen cross section in 4 zones in the superior and inferior direction from the optic nerve head (ONH). No significant differences were noted (p<.05).

Atg5 and LC3 associate with single-membrane phagosomes containing POS

The apparent correspondence of LC3-II generation in the RPE with the start of morning disk shedding when RPE phagocytosis of outer segment tips is maximal (Nandrot et al., 2007; Strauss, 2005), led us to examine the relationship between phagocytosed POS in the RPE and the distribution of Atg5 and LC3. Strikingly, we found that Atg5 co-localized with engulfed POS in control mice (Figure 3A, arrows merged image) (visualized by rhodopsin staining (Nandrot et al., 2007)) as well as with LC3 in the GFP-LC3 transgenic mice (Figure 3C, arrows). Atg5 did not associate with the POS in Atg5ΔRPE RPE cells (Figure 3B). A reconstruction of z-stack images (x-z plane) of the Atg5/POS complex confirmed Atg5/POS association in control RPE (Figure 3A, inset) but not Atg5ΔRPE RPE cells (Figure 3B, inset). It was also notable that without Atg5, POS did not penetrate much beyond the apical surface of the RPE at this time point (Figure 3B, inset). Secondary effects on the expression of receptors involved in POS binding and phagocytosis (Gal et al., 2000; Nandrot et al., 2007; Nandrot et al., 2004) due to the deletion of Atg5 were not observed in Atg5ΔRPE RPE cells compared to control (Figure S1).

Figure 3. Atg5 and LC3 associate with phagocytosed outer segments.

Figure 3

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RPE flat mounts were prepared at 8:00 AM from 16 week old (A) littermate control (Atgf/wt;cre+) and (B) Atg5ΔRPE mice, stained with anti-rhodopsin (red) and anti-Atg5 (green), and visualized by confocal microscopy. Nuclei were visualized by DAPI staining (blue). Reconstructed confocal Z-stack images were rotated 90° to visualize Atg5/rhodopsin association in control (Panel A, merged image, inset) and Atg5ΔRPE RPE cells (Panel B, merged image, inset). Bar = 20μm. C. RPE flat mounts prepared from GFP-LC3 mice were stained with anti-rhodopsin (red) to visualize association of LC3 and the POS. DAPI staining was used to visualize the nuclei (blue). D. TEM of a representative RPE from littermate control mice showing electron dense single membrane phagosomes (arrows). E–G. TEM images of a representative Atg5ΔRPE RPE cells showing degenerating phagosomes (arrowheads). Scale bar =1 μm. See also Figure S1

In conventional autophagy, LC3-II associates with double-membrane autophagosomes (Klionsky et al., 2012; Mizushima et al., 2008). However, examination of control RPE by transmission electron microscopy (TEM) failed to reveal any association of double-membrane structures with phagocytosed POS (the latter were readily seen as electron dense structures in the cytoplasm surrounded by a single membranes, white arrows) (Figure 3D). Examining POS in the RPE of Atg5ΔRPE mice revealed the accumulation of phagosomes that contain poorly processed POS (Figure 3E–G, arrowheads) suggesting disrupted lysosomal processing in the absence of Atg5. This, together with the absence of abundant double-membrane autophagosomes containing outer segments suggested an interesting alternative to a role for conventional autophagy in the process of POS uptake and degradation.

A non-canonical autophagy pathway facilitates phagosome formation and POS degradation

We previously described a process in macrophages (Martinez et al., 2011; Sanjuan et al., 2007) and plasmacytoid dendritic cells (Henault et al., 2012) in which the single membranes of phagosomes directly associate with LC3, a phenomenon we called LC3-associated phagocytosis (LAP) (Sanjuan et al., 2009). LAP is engaged by the engulfment of particles that stimulate Toll-like receptors (TLR)-1/2, 2/6, 3, or 4 (Sanjuan et al., 2007), Fc-receptors (Henault et al., 2012; Huang et al., 2009), or upon engulfment of dying cells (Martinez et al., 2011). Similarly, mammary epithelial cells that engulf living cells (entosis) (Florey et al., 2011) also engage LAP. Like conventional autophagy, LAP requires Beclin-1, Atg5, and Atg7 (Florey et al., 2011; Henault et al., 2012; Martinez et al., 2011; Sanjuan et al., 2007) but LAP proceeds independently of the autophagic pre-initiation complex, including Ulk1 (Henault et al., 2012; Martinez et al., 2011), Fip200/Atg17 (Florey et al., 2011; Henault et al., 2012), and ATG13 (Henault et al., 2012).

To determine if phagocytosis of POS by RPE induces LAP, we utilized an RPE cell line, RPE-J (Finnemann and Rodriguez-Boulan, 1999; Nandrot et al., 2007), transduced with GFP-LC3, and fed with labeled POS (Finnemann and Rodriguez-Boulan, 1999; Nandrot et al., 2007). Upon engulfment, we noted association of LC3 with the POS (Figure 4A–E, lower left panels, arrows) and that this was dependent on several autophagy proteins as determined by silencing components of the conventional autophagy pathway. After confirming successful knockdown of these proteins (Figure S2A) we found that the silencing of Atg5 (Figure 4A, lower right panel) or Beclin1 (Figure 4B, lower right) prevented POS-induced LAP as assessed visually (Figure 4A–B), or by LC3 conversion (Figure S2B). Silencing of any of these components had little effect on the extent of uptake of POS (Figure S2C), and all knockdowns disrupted conventional autophagy as assessed by rapamycin-induced GFP-LC3 punctae formation (Figure 4A–E, upper right panels) and accumulation of rapamycin-induced LC3-II (Figure S2C). Strikingly however, silencing of Fip200 (Figure 4C, lower right panel), Ulk1 (Figure 4D, lower right panel), or Atg13 (Figure 4E, lower right panel) had no effect on co-localization of LC3 with engulfed POS (arrowheads), nor on POS-induced LC3-II formation (Figure S2B). In support of this process as phagocytosis and not autophagy, TEM analysis of RPE-J cells that had phagocytosed POS revealed only single membrane phagosomes (Figure 4F, black arrows).

Figure 4. Components of the autophagy pathway promote LC3 translocation to the outer segment containing phagosome.

Figure 4

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GFP-LC3+ RPE-J cells were transfected with scrambled (Scr) (Panels A–E) or Atg5, Beclin1, Fip200, Ulk1, or Atg13 (panels A, B, C, D, E, respectively) siRNA oligonucleotides and fed labeled POS (red) or treated with rapamycin. Representative images are shown. Inset images in lower panels indicate individual phagosomes denoted by arrows or arrowheads. The percentage (%) of GFP-LC3+ phagosomes (arrows and arrowheads) was quantified following POS feeding from time-lapse captured images (Bar graphs). The number of GFP-LC3+ punctae/cell was quantified 24 h following rapamycin treatment (bar graphs). *** = p< .05. F. Representative electron micrographic image of a phagosome in an RPE-J cell taken 6 h following POS feeding demonstrating a single membrane structure (black arrows). Scale bars = 10 μm. See also Figure S2.

One function of LAP in macrophages (Martinez et al., 2011; Sanjuan et al., 2007) and in epithelial cells (Florey et al., 2011) is to accelerate the maturation of the phagosome as it fuses to lysosomes. Indeed, we observed that in RPE-J cells engulfed POS co-localized with LAMP1, LAMP2, and cathepsin D (Figure 5A, left panels, arrows) indicating that POS degradation was proceeding (Bosch et al., 1993; Rakoczy et al., 1997; Ramkumar et al., 2010). Silencing of Atg5 prevented co-localization of these molecules involved in phagosome maturation and cargo degradation (Figure 5B, arrows). Further, the accumulation of LAMP1 and the maturation of cathepsin D, both induced by exposure of RPE-J cells to POS, were diminished in cells lacking Atg5 (Figure 5C) (single channel fluorescent images are provided in Figure S3A). This observation was supported by in vivo evidence when we examined RPE cells from the Atg5ΔRPE mouse where Lamp1 expression was reduced following RPE phagocytosis as was the availability of mature cathepsin D (Figure S3B).

Figure 5. LC3 associated phagocytosis facilitates phagosome maturation and degradation of engulfed POS by RPE cells.

Figure 5

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GFP-LC3+ RPE-J cells were transfected with A. scrambled (Scr) or B. Atg5 siRNA oligonucleotides and fed labeled POS (red). Cells were stained for Lamp1, Lamp2, or Cathepsin D and co-localization was assessed by confocal microscopy. Representative images (of >25 cells visualized) are shown (taken at 16 h post-POS feeding). C. RPE-J cells were transfected with scrambled (Scr) or Atg5 siRNA oligonucleotides and fed POS. Cells were harvested at 1, 2, 6 h and western blotting was performed for Lamp1, Lamp2, and Cathepsin D. D. RPE-J cells were transfected with scrambled (Scr) or Atg5 siRNA oligonucleotides and fed POS. At 1, 3 and 6 h post feeding confocal images were captured; z- stack images were reconstructed and rotated 90°. In the “No POS” group a nuclear stain (Draq5) was used to visualize nuclei (green). Images at 1, 3 and 6 h do not show the nuclear stain but the position of the nuclei in the cells is indicated by the dotted line oval. The basal surface of the RPE is denoted (dotted straight line). Scale bars = 10μm. See also Figure S3.

As POS were engulfed by RPE-J cells, we noted that they rapidly moved through the epithelial layer past the nucleus (Figure 5D, dotted ovals) toward the basal surface of the cell (Figure 5D, left, dotted line). This movement of POS was not apparent in cells lacking Atg5 (Figure 5D, right) as the POS were located more apically at comparable time points to controls similar to what we observed in the Atg5ΔRPE mouse (Figure 3). Thus, while binding and engulfment were independent of Atg5 (Figure S2C) their ability to move through the RPE cells and localize to the phagosome for digestion is impaired when LAP is disrupted.

Silencing Ulk1 in RPE cells had no effect on POS induced LC3-I to LC3-II conversion but prevented conventional autophagy activated by rapamycin (Figure S2B). When we examined RPE cells from Ulk1−/− mice in the morning (8:00 AM) when phagocytosis is maximal we detected LC3-II (Figure 6A); however, we also observed the accumulation of p62/SQSTM [a hallmark of defective autophagy (Klionsky et al., 2012; Mizushima et al., 2010; Takamura et al., 2011)] in the Ulk1−/− RPE indicating that the enzymatic production of LC3-II was likely due to POS induced LAP as opposed to classical autophagy. Autophagy- and LAP- defective Atg5ΔRPE RPE cells also had increased p62/SQSTM1 as expected. Further, unlike Atg5ΔRPE mice (Figure 2), the ERG responses in Ulk1−/− animals showed no defects (Figure 6B) and the association of Atg5 with engulfed POS in RPE from Ulk1−/− mice was indistinguishable from that in the RPE of Ulk1+− control animals (Fig 6C). This supports the idea that POS phagocytosis promotes a non-canonical form of autophagy (i.e. LAP) that is dependent on LC3 and Atg5 but is not classical autophagy which requires Ulk1, Fip200, and Atg13 (Martinez et al., 2011; Sanjuan et al., 2007; Sanjuan et al., 2009).

Figure 6. Autophagy deficient Ulk1−/− mice have normal vision and demonstrate association of Atg5 with phagocytosed POS.

Figure 6

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A. Western blots for LC3 and p62 in the RPE were performed on 16 weeks Ulk1−/− and Ulk1+/− mice. B. ERG recording on 16 week old Ulk1−/− and Ulk1+/− mice demonstrating that scotopic and photopic responses were normal. C–D. Association of Atg5 and POS 16 week old Ulk1−/− and Ulk1+/− mice as detected by co-stains for Atg5 and rhodopsin on RPE flat mounts. E–F. Reconstructed confocal Z-stack images were rotated 90° to visualize Atg5/rhodopsin association in Ulk1−/− and Ulk1+/− mice.

Non-canonical autophagy in the RPE contributes to the retinoid supply for the photoreceptors

Although we did not observe obvious decreases in rod and cone photoreceptor numbers, disruption of LAP in the RPE reduced ERG responses (see Figure 2). This suggests that LAP may be linked to other functions of the RPE cells that support visual function. An interesting possibility was that phagocytosis of POS is linked to the visual cycle, a process that recycles photo-bleached retinoids from the photoreceptors back to the retina for proper visual function (Tang et al., 2013). Initially we examined the quantities of RPE proteins known to be involved in this multi-step enzymatic pathway and found no difference between control and Atg5ΔRPE RPE cells (Figure S4) suggesting that the machinery for intrinsic chromophore synthesis was in place. Therefore, we sought to determine if the decrease in rod and cone function observed in Atg5ΔRPE mice was the result of a deficit in intrinsic chromophore synthesis by the visual cycle. To test this hypothesis, ERG recordings were performed on Atg5ΔRPE and littermate control mice (Figure 7A) and the mice were allowed to recover for 1 week in a normal light cycle environment. Mice then received an intraperitoneal injection of 0.25 mg/animal of the 9-cis RAL, a functional analog of the visual chromophore 11-cis RAL. The animals were then dark-adapted for 24 h prior to scotopic and photopic ERG recordings. While pre-treatment results confirmed the difference between Atg5ΔRPE and control, post-treatment results indicated no significant difference in rod and cone function between the two groups (Figure 7B). This single treatment provided only short-term functional rescue as previously shown (Tang et al., 2010). To confirm that retinoid metabolism was directly modulated by the disruption of non-canonical autophagy, retinoids were extracted from eyecups of 20-week old Atg5ΔRPE and littermate control Atg5f/f mice following a 24 h period of dark-adaptation. The results, quantitated according to previously published protocol (Tang et al., 2011), show that the amounts of 11-cis, all-trans, and 13-cis RAL were significantly reduced in Atg5ΔRPE mice compared to littermate controls (Figure 7C). All-trans retinyl-esters (RE) were not significantly different between Atg5ΔRPE and a control, indicating that vitamin A delivery through the blood to the RPE was unaffected. These results are consistent with the idea that the deficit in visual function in Atg5ΔRPE mice can be attributed to an inadequate recovery of retinoids due to disruption of the non-canonical pathway of autophagy. Significant quantities of ROL (any isomeric form) were not detected.

Figure 7. Delivery of retinoids restores visual function.

Figure 7

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A. ERG recordings were performed on Atg5ΔRPE (n=8) and littermate control mice (n=8). B. After 1 week of recovery in a normal light cycle environment each mouse received a single intraperitoneal injection of 0.25 mg of 9-cis RAL, a functional analog of 11-cis RAL. The animals were then dark-adapted for 24 h followed by scotopic and photopic ERG recordings. C. Retinoids were extracted from eyecups of 20- week old Atg5ΔRPE (n=4, gray bar) and Atg5f/f littermate control (n=5, black bar) mice dark- adapted for 24 h prior to the experiments. RAL = retinal; RE = retinyl-esters. See also Figure S4.

DISCUSSION

The RPE is essential to the health and longevity of the retina by performing a number of functions to support vision. Two of its essential processes are phagocytosis of POS, which removes the most distal tips of the photoreceptors to support disk renewal, and the visual cycle, which maintains the supply of chromophore for regeneration of photo-bleached visual pigments (Strauss, 2005; Tang et al., 2013). Disruptions in either of these recycling processes can have serious consequences for the long term health of the retina (Duncan et al., 2003; Fan et al., 2008; Nandrot et al., 2006; Ruiz et al., 2007; Travis et al., 2007; Znoiko et al., 2005). Studies presented here show that the phagocytic function of the RPE and the visual cycle are linked via a non-canonical form of autophagy. We demonstrate that the lipidated form of the autophagy protein LC3 associates with the phagosome in a manner dependent on Atg5 and Beclin1, but independent of the autophagy pre-initiation complex consisting of Ulk1/Atg13/Fip200. This process, termed LAP, was necessary for the degradation of the phagocytosed outer segments. Importantly, our studies show that this non-canonical form of autophagy supported optimal visual function by supplying a portion of the retinoids required for chromophore synthesis. Thus, we have established a link between RPE phagocytosis, autophagy, and the visual cycle.

Cells utilize phagocytosis to ingest extracellular particles by invaginations of the plasma membrane following engagement of specific cell surface receptors while autophagy isolates portions of the cytoplasm in double membrane structures to target cytoplasmic proteins, organelles, and lipids for degradation. Recent studies from several laboratories (DeSelm et al., 2011; Florey et al., 2011; Henault et al., 2012; Martinez et al., 2011) have shown that there is a functional convergence of autophagy and phagocytosis where components of the autophagy pathway have been co-opted by phagocytosis to increase the efficient degradation of the phagocytosed cargo by aiding in lysosome/phagosome fusion as well as the maturation of the phagolysosome. In macrophages degradation of apoptotic corpses utilizes LAP as a mechanism to degrade apoptotic bodies and inhibit the immune response. Disruption of this process prevented effective degradation and led to the release of pro-inflammatory cytokines (Martinez et al., 2011). Similarly, host defense against microbial infection involves the recruitment of LC3 to phagosomes, promoting their maturation and microbial killing (Henault et al., 2012; Huang et al., 2009). In a similar manner fusion of the lysosome to the phagosome in dendritic cells required Atg5 and LC3 for efficient antigen degradation and presentation to T cells (Lee et al., 2010). In osteoclasts, proteins essential for autophagy, including Atg5, Atg7, Atg4B, and LC3, were shown to be important for generating the osteoclast ruffled border, the secretory function of osteoclasts (lysosome-membrane fusion), and bone resorption (DeSelm et al., 2011). Herein we show that degradation of outer segment tips following RPE phagocytosis is also dependent on a non-canonical form of autophagy and that its disruption not only prevented POS degradation but had a significant impact on vision.

We addressed the importance of LAP in the RPE by deleting the essential autophagy gene Atg5 specifically in this cell layer. In normal RPE cells POS were contained in single membrane phagosomes as reported (Bosch et al., 1993; Gordiyenko et al., 2010; Strauss, 2005). In the absence of Atg5 degenerating phagosomes containing undigested POS material were prevalent. In addition, without Atg5 POS were unable to penetrate into the RPE in the same manner as in control cells. These findings, along with the observations that there was no difference in the levels of known phagocytic receptors on the RPE and that uptake of POS was not impaired without Atg5, suggest that phagosomes require Atg5 to move through the RPE and enter the lysosomal compartment for degradation. This was confirmed by our findings that the lysosomal proteins Lamp1 and Lamp2 and the degradative enzyme cathepsin D were not able to associate with the phagocytosed POS without Atg5.

Another important function of the RPE is the visual cycle, which involves metabolism of retinoids for phototransduction. Alterations in essential proteins of process such as LRAT, RPE65, or CRALBP have been shown to lead to severe visual dysfunction (Ruiz et al., 2007; Travis et al., 2007; Zhang et al., 2011). We did not detect significant changes in the levels of these key visual cycle markers, nevertheless, the Atg5-deficient RPE was unable to support optimal visual function as evidenced by a consistent loss of ERG amplitudes with age. This loss of retinal function can have multiple underlying causes, the most obvious would be the loss of photoreceptor cells; however, the photoreceptor cell count in the Atg5-deficient animals did not significantly differ from that of normal mice, indicating that the deficiency in these animals is more subtle. Indeed, Atg5ΔRPE mice had decreased 11-cis RAL compared to normal mice and the loss of visual function was restored by exogenous supplementation with the chromophore analog, 9-cis RAL. This provides a direct link between deficiencies in POS degradation and the visual cycle. Although the exact relationship between Atg5 function and the visual cycle is not completely understood, Atg5 mediated phagocytic processes in the RPE must be critical to the recycling of optimal amounts of retinoids.

Recent studies have suggested that the decline in autophagy with age contributes to a number of age-related diseases (Cuervo et al., 2005; Rubinsztein et al., 2011) as cells with diminished degradative processes have difficulty clearing damaged organelles, proteins, and lipids. The deterioration of the visual cycle with age is also well-documented (Gaffney et al., 2012; Kolesnikov et al., 2010) and one hypothesis suggests that it is mediated by retinoid deficiency reflected in a reduction in available 11-cis RAL chromophore (Jackson et al., 2002). This may be a reason why elderly individuals have difficulties with night vision and supplementation with nutritional vitamin A can improve this condition (Owsley et al., 2006). Thus, proteins of the visual cycle are in place but retinoid recycling might be impaired with age. In light of our results showing that we can restore vision in LAP-deficient mice with retinoid supplementation suggests that many of these symptoms are consistent with a loss of LAP function and phagocytic activity with age. However, further studies are required to uncover all the molecular details of the coupling between phagocytosis and the visual cycle.

There is a convergence of the visual cycle and RPE phagocytosis (Graphical Abstract). The RPE performs two vital functions that support vision. Phagocytosis of the POS tips (left), which are shed in the early morning, supports photoreceptor disk renewal. This is mediated by a non-canonical form of autophagy termed LAP. As depicted, once engulfed the shed outer segments enter the phagosome and the Atg12-Atg5-Atg16L complex is recruited, leading to the lipidation of the cytosolic form of LC3 (LC3-I) by PE and its recruitment to the phagosome. Only then does the lysosome fuse to the phagosome forming the phagolysosome leading to maturation and degradation of the ingested POS cargo. Degradation products are then removed from the cell by transport to the choroid while some materials are recycled to the photoreceptors to replenish necessary components.

The second critical process performed by the RPE is the classic visual cycle (right) in which the chromophore 11-cis RAL is generated from all-trans ROL (vitamin A) for use by the photoreceptors in vision. The visual cycle begins following the absorption of light by visual pigments containing 11-cis RAL, which isomerizes to the all-trans configuration. All-trans RAL is released from opsin and reduced to all-trans ROL by retinoid dehydrogenases (RDH). All-trans ROL is transported to the RPE (presumably via IRBP) where it is esterified by lecithin retinol acyltransferase (LRAT) to form all-trans RE. The isomerohydrolase RPE65 converts all-trans RE to 11-cis ROL, which is then oxidized to 11-cis RAL and transported to photoreceptors to complete the visual cycle. Our studies show that these two critical pathways converge as the recovery of all-trans ROL for 11-cis RAL synthesis is aided by the POS phagocytosis and degradation pathway that is dependent on the autophagy proteins LC3, Atg5 and Beclin1, but not on the autophagy pre-initiation complex composed of ULK1, FIP200, and ATG13 and therefore consistent with a role for LAP in this process.

EXPERIMENTAL PROCEDURES

Mice

BEST1(VMD2)-cre transgenic mice, in which cre recombinase is controlled by a fragment of the human VMD2 promoter (Esumi et al., 2004), were crossed to the Atg5flox/flox (generously provided by Dr. Noboru Mizushima, Tokyo Medicine and Dental University, Tokyo, Japan) (Hara et al., 2006) to generate the Atg5ΔRPE mouse strain. Ulk1−/− and Ulk1+/− mice were kindly provided by Dr. Mondira Kundu (St. Jude Children’s research Hospital, Memphis, TN). In all experiments littermate control animals were used (Atg5f/+;cre+ or Atg5f/f). All experiments contained at least three mice per group and were repeated a minimum of three times. Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Washington University School of Medicine. All experiments contained at least three mice per group and were repeated a minimum of three times. See Extended Experimental Procedures for details.

Cell Lines

The rat RPE-J cell line (ATCC, Manassas, VA) was cultured at 33°C and 5% CO2 in DMEM with 4% FBS for undifferentiated colonies. For phagocytosis assay, cells were grown in the presence of 10 nM retinoic acid (Sigma, St. Louis, MO) for six days to acquire a differentiated polarized RPE phenotype. See Extended Experimental Procedures for details.

Transfection assays

RPE-J cells were transfected with siRNA oligonucleotides against rat Atg5, Beclin1, Fip200, ULK1 and Atg13 purchased from Dharmacon (Lafayette, CO).

Bovine retinal outer segment isolation and fluorescent labeling

Fresh bovine eyes were used for POS isolation and labeling. See Extended Experimental Procedures for details.

POS phagocytosis assay

Unlabeled or fluorescent labeled POS were resuspended in the cell culture media and added to the apical surface of differentiated RPE-J at a ratio of 10:1 (POS:cell). Phagocytosis was allowed for various lengths of time and excessive POS were washed extensively with PBS. Phagocytosis was quantified by confocal microscopy. See Extended Experimental Procedures for details.

Time-lapse imaging of phagocytosis

Time lapse imaging was performed on Zeiss LSM 510 (Carl Zeiss Microscopy, Thornwood, NY) inverted confocal microscope equipped with a 488 nm argon ion laser, and 543 nm and 633 nm HeNe lasers. See Extended Experimental Procedures for details.

RPE flat mounts

Posterior cups of eye were prepared from enucleated eyes were isolated and stained with indicated antibodies and appropriate secondary antibodies. See Extended Experimental Procedures for details.

Electroretinography (ERG)

Electroretinograms were recorded using a Tucker–Davis System 3 Complete ABR/OAE Workstation (Tucker–Davis Technologies, Gainesville, FL, USA). See Extended Experimental Procedures for details.

Retinoid treatments

9-cis RAL (Sigma- Aldrich, St. Louis, MO; 0.25 mg/animal) was dissolved in 20 μl of 100% ethanol. For intraperitoneal (IP) delivery, 9-cis RAL was dissolved in 200 μl vehicle (10% ethanol/10% bovine serum albumin in 0.9% NaCl) and injected into the peritoneal cavity. Retinoid was handled under dim red light.

Retinoid analysis

All procedures were performed under dim red light on dark-adapted animals using methods modified from those previously described (Tang et al., 2011). For each sample, 2 eyecups from 20 week Atg5ΔRPE and Atg5f/f littermate control were pooled after hemi-section and the removal of the anterior segment. Data were then averaged for each group Atg5ΔRPE (n=4) and Atg5f/f (n=5). See Extended Experimental Procedures for details.

Statistical Analyses

All data were analyzed using two-tailed unpaired Student’s t test. Statistically significance was considered as P values <0.05 indicated by asterisk. Error bars represent standard error.

Details for Histology, Transmission Electron Microscopy, Reagents and Antibodies are available in the Extended Experimental Procedures.

Supplementary Material

01

HIGHLIGHTS.

  • RPE cells utilize autophagy proteins LC3, Atg5, and Beclin1 for phagosome formation.

  • LC3 is recruited to phagosomes in RPE cells.

  • RPE specific deletion of Atg5 in mice decreases vision.

  • Retinoids are recovered through phagocytosis of photoreceptor outer segments.

Acknowledgments

This work was supported by a National Institutes of Health Grants EY015570 (TAF), AI44848 (DRG), AI40646 (DRG) and the Department of Ophthalmology and Visual Sciences core grant (EY02687). Support was also received from Research to Prevent Blindness (New York, N.Y., USA) and the BrightFocus Foundation (Clarksburg, MD). The authors would like to thank Wandy Beatty for performing the TEM and Jayoung Choi for assistance with the Figure layouts.

Footnotes

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