Autophagy-mediated catabolism of visual transduction proteins prevents retinal degeneration Jingyu Yao1, Lin Jia1, Kecia Feathers1, Chengmao Lin1, Naheed W. Khan1, Daniel J. Klionsky2,3, Thomas A. Ferguson4 and David N. Zacks1,# 1

Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, MI

2

Life Sciences Institute; University of Michigan; Ann Arbor, MI USA;

3

Department of Molecular, Cellular and Developmental Biology; University of Michigan; Ann

Arbor, MI USA; 4

Department of Ophthalmology and Visual Sciences, Washington University School of

Medicine, St Louis, MO, USA #

Corresponding Author: David N Zacks 1000 Wall St. Ann Arbor, MI 48103 Tel: 734-763-7711

Fax: 734-936-2340 Email: [email protected] ABSTRACT Autophagy is a lysosomal degradation pathway critical to preventing the accumulation of cytotoxic proteins. Deletion of the essential autophagy gene Atg5 from the rod photoreceptors of the retina (atg5∆rod mouse) results in the accumulation of the phototransduction protein transducin and the degeneration of these neurons. The purpose of this study is to test the hypothesis that autophagic degradation of visual transduction proteins prevents retinal degeneration. Targeted deletion of both Gnat1 (a gene encoding the alpha subunit of the heterotrimeric G-protein transducin) and Atg5 in the rod photoreceptors resulted in a significantly decreased rate of rod cell degeneration as compared to the atg5∆rod mouse retina, and considerable preservation of photoreceptors. Supporting this we used a novel technique to immunoprecipitate green fluorescent protein (GFP)-tagged autophagosomes from the retinas of the GFP-LC3 mice and demonstrated that the visual transduction proteins transducin and

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ARR/arrestin are associated with autophagosome-specific proteins. Together, this study shows that degradation of phototransduction proteins by autophagy is necessary to prevent retinal degeneration. In addition, we demonstrate a simple and easily reproducible immunoisolation technique for enrichment of autophagosomes from the GFP-LC3 mouse retina, providing a novel application to the study of autophagosome contents across different organs and specific cell types in vivo. Keywords autophagy, photoreceptor, retina, retinal degeneration, transducin

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INTRODUCTION Photoreceptor cells, the light sensing unit within the retina, are highly compartmentalized structures.1 The cell nuclei are confined to the outer nuclear layer (ONL) (Fig. S1), while many of the cells organelles are located in the inner segments (IS) beneath the nuclei. The outer segments (OS) then extends from the IS via a connecting cilium and consists of stacks of membranes containing the light sensing protein RHO (rhodopsin) and the other visual transduction proteins required to convert photons into neuronal signals. Activation of RHO results in membrane potential changes that are transmitted back up through the photoreceptor towards the axonal connections with bipolar cells and eventual transmission of the visual stimulus to the brain through the optic nerve. The photosensitivity of the rods is regulated by the activity of 2 major proteins, transducin and ARR/arrestin. Transducin is a heterotrimeric G-protein that binds activated RHO and propagates the visual stimulus.2 In dark or very low light conditions, the majority of the transducin is localized within the OS, allowing the photoreceptor to maintain high sensitivity in low light conditions (Fig. S1B). In contrast, under bright light conditions, transducin translocates to the IS reducing the sensitivity of the rods. The second photoreceptor protein, ARR, binds the activated RHO and quenches its signaling. Under dark conditions, the majority of ARR is found in the IS, isolated from the stacks of RHO in the OS and thus unable to quench its signaling. Upon switching from dark to light conditions, ARR moves to the OS and quickly binds to and inhibits RHO, thus maintaining the sensitivity of the retina to new visual stimuli. Macroautophagy/autophagy plays important roles in maintaining cellular homeostasis and regulating the response to environmental stressors.3,4 Intracellular accumulation of excess proteins can result in significant disruption of cellular homeostasis and cell death. We have

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previously shown that there is a bimodal peak of autophagy activation within the photoreceptors, the first occurring approximately 4 to 6 h after the shift from dark to light, and the second approximately 4 to 6 h after the shift from light to dark.5 These peaks are driven, in part, by the translocation of transducin or ARR from the outer segment to the inner segment upon the switch from dark to light or light to dark, respectively. It is currently unknown how the translocated proteins are degraded within the inner segment to prevent their accumulation that might result in toxicity. We have previously demonstrated that ATG5 regulates autophagy flux in the retina,6 and it has also been shown by one of the authors (TAF) that the selective knockout of the gene encoding the autophagy protein ATG5 from the rod cells (atg5∆rod mouse) results in an accumulation of transducin and a resultant degeneration of the photoreceptor, suggesting that autophagic mechanisms are involved in regulating the level of this phototransduction protein.7 The purpose of this study is to test the hypothesis that autophagy contributes to the degradation of visual transduction proteins thereby preventing retinal degeneration. RESULTS Knockout of Gnat1 reduces retinal degeneration in the atg5∆rod mouse Our previous study demonstrated that translocation of transducin from the OS to the IS contributes to the dynamic regulation of autophagosome formation in the rod photoreceptors.5 Selective deletion of the Atg5 gene from the rods (the atg5∆rod mouse) results in accumulation of transducin within these cells and a progressive degeneration of the photoreceptors.7 Our hypothesis predicts that if the transducin gene is also knocked out of autophagy-deficient photoreceptors, then the transducin protein cannot accumulate and the rate of degeneration will be reduced. We tested this by crossing the atg5∆rod mouse with the gnat1-/- mouse, which contains a knockout of a gene encoding the alpha subunit of rod transducin (GNAT1/T),8 to

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create a mouse in which the rod cells are deficient in both gene products (gnat1-/- atg5∆rod ) for comparison to the atg5∆rod (Table 1). Figure 1A demonstrates that in both groups, we detected robust expression of Cre-recombinase and marked reduction of autophagy activation, with significantly reduced levels of ATG5, LC3-I to LC3-II conversion and significant accumulation of SQSTM1 as compared to the (Rho)-Cre-positive-only littermate controls (Fig. 1B to D).9 The small amounts of ATG5 and LC3-II observed are thought to come from other cells in the retina. As expected, there was normal staining for T in the atg5∆rod retinas, but no T was detectable in the gnat1-/- atg5∆rod mouse (Fig. 1B). At age of 2 months, both atg5∆rod mice and gnat1-/- atg5∆rod mice had normal ONL thickness, as measured with the optical coherence tomography (OCT) in vivo retinal imaging system (Fig. 2A), and on histological sections (Fig. 2D). By the age of 10 months, the retina of atg5∆rod mouse showed progressive loss of the photoreceptors, with only 2 or 3 rows of photoreceptor nuclei remaining in the ONL (Fig. 2B) and generalized thinning of the retina (Fig. 2C). In sharp contrast, the retinas of the gnat1-/- atg5∆rod mice had a decreased rate of rod-cell degeneration and less retinal thinning as compared to the Gnat1+/+ atg5∆rod mouse retina (Fig. 2B,C). Preservation of the retina was also confirmed by detecting increased rhodopsin levels in 10 month old gnat1-/- atg5∆rod mice by fluorescent microscopy (Fig. 3A) and western blotting (Fig. 3B). Preservation of cone morphology and function in gnat1-/- atg5∆rod mouse The gnat1-/- mice have no rod function due to the lack of rod transducin, so it is not possible to measure the functional preservation of the rods. However, the loss of rods in the atg5∆rod does also result in a reduction of the cone-mediated visual response.7,10 Therefore, we used cone-mediated responses to light as a measure of overall retinal preservation in the double

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knockout mice. In this study, we tested cone function by performing light-adapted (photopic) single-flash electroretinography (ERG) and 20-Hz flicker response analyses at the age of 2 and 10 months. While significantly reduced amplitudes of the photopic b-wave and 20-Hz flicker response were observed in atg5∆rod mice at the age of 10 months, gnat1-/- atg5∆rod mice displayed significantly higher responses, demonstrating the preservation of cone function (Fig. 4A). Cone-mediated functional vision of atg5∆rod and gnat1-/- atg5∆rod mice was also tested using an optokinetic tracking response system at 2 and 10 months of age.11 At the age of 2 months both groups had relatively normal optokinetic response as compared with (Rho)-Cre controls. By the age of 10 months, although lower than controls, the optokinetic tracking response of the gnat1-/- atg5∆rod mice was significantly higher than that of the atg5∆rod mice (Fig. 4B), further indicating a better preserved cone-mediated visual behavior in gnat1-/- atg5∆rod mice, and by inference, preservation of the rods. In accordance with the visual function results, diminished cone photoreceptor numbers were observed in the atg5∆rod retinas at the age of 10 months, as assessed by immunohistochemical staining for the cone-specific OPN1MW/M-opsin protein, whereas in the gnat1-/- atg5∆rod retinas, the number and the morphology of the cones were preserved (Fig. 4C). Isolation of autophagosomes in vitro—assay development The results described above, as well as our previously published results,5,6 strongly support the hypothesis that autophagy prevents the accumulation of phototransduction proteins in the inner segment of the photoreceptor. One prediction would be that autophagosomes isolated from the retina should contain these proteins on their way to autophagic degradation; thus, we undertook studies to isolate autophagosomes and analyze their content. Previous methods for isolating autophagosomes utilize complex centrifugation protocols to enrich for these

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organelles.12-15 There is one report of a more direct technique based on immunoprecipitation of green fluorescent protein (GFP)-labelled LC3.16 The basic premise of this technique is that since LC3 coats the phagophore and nascent autophagosome, one can then immunoprecipitate the autophagosomes using antibodies against the GFP tag attached to LC3. We applied this technique to the 661W murine cone-photoreceptor cell line17 that was transfected with GFP-LC3 (Fig. 5). Figure 5A shows the fluorescent images of 661W cells expressing GFP-LC3 that were subjected to starvation and treatment with the fusion inhibitor bafilomycin A1 to increase autophagy and visualize autophagosomes.18 Green puncta consistent with the formation of autophagosomes are readily observed in the treated cells. We then used an anti-GFP antibody to immunoisolate the autophagosomes, and performed western blot analyses. We found marked accumulation of the autophagosome-associated proteins GFP-LC3 and SQSTM1within the immunoprecipitate (Fig. 5B), strongly suggesting that we had enriched for the autophagosome fraction of the cells. We did detect significant levels of endogenous LC3-II as well (Fig. 5B), consistent with autophagosome enrichment. The ATG12–ATG5 complex interacts with the phagophore and assists in the lipidation of LC3 for autophagosome formation. We found only small amounts of the ATG12–ATG5 complex in our immunoprecipitation fraction (Fig. 5B), suggesting we were enriching for organelles more advanced along the maturation pathway to the autophagosome. Immunoisolation of retinal autophagosomes in vivo To directly test the hypothesis that autophagosomes sequester the visual transduction proteins transducin and ARR within the photoreceptor we applied this technique to the retinas of the GFP-LC3 transgenic mouse. This mouse contains the GFP protein bound to LC3 in all cells.19 Retinas were harvested 7 h after light onset, as this is the time point when the formation

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of green puncta within the photoreceptors corresponds to the peak of autophagy activation.5 As previously described,5 the puncta localized to the inner segment and the perinuclear area of the ONL (Fig. 6A). Fluorescence microscopy of the enriched autophagosome fraction (Fig. 6B) showed vacuole-shaped structures coated with GFP. Transmission electron microscopy (TEM) of these structures confirmed that they were double-membrane structures, consistent with them being autophagosomes (Fig. 6C). The major portion of the isolation product was abundant vesicular structures that looked like mature double-membrane autophagosomes containing electron-dense material, likely representing engulfed cellular material. We observed some LC3positive tubules and some double-membrane cistern-shaped structures (Fig. 6D), which might represent preclosure structures (i.e., phagohpores) along the course of autophagosome maturation or ruptured autophagosomes. These findings provided strong morphological confirmation that our immunoisolation technique was enriching for autophagosomes from GFP-LC3 mouse retina. Further confirmation was provided by western blot analysis, which identified multiple autophagosome-related molecules in the immunoisolated autophagosomes, including GFP-LC3, SQSTM1/p62, and WDFY3/ALFY (Fig. 7A). Importantly there was exclusion of proteins not expected within the autophagosome fraction, such as the ATG12–ATG5 complex. As with the in vitro preparation, we detected much higher levels of endogenous LC3-II in the immunoisolated fraction, as compared to LC3-I, consistent with autophagosome enrichment. Our hypothesis predicts that the autophagosomes would contain more transducin than ARR when harvested from light-adapted retinas, when the transducin is localized to the inner segment, and that the opposite would be true for autophagosomes harvested from dark-adapted retinas, when ARR is primarily localized to the inner segment. Our findings confirm this prediction, with the transducin and ARR levels within the enriched autophagosome fraction varying depending on dark- or light-

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adaptation (Fig. 7B). This last finding would be consistent with our previous results that the light- and dark-induced peaks in autophagy are caused, at least in part, by the translocation of these visual transduction proteins from the outer segment to the inner segment. The protein profile of the enriched autophagosomes from the GFP-LC3 retinas was also assessed by proteomics analysis. Consistent with the results of western blot, the presence of the phototransduction proteins transducin and ARR, along with other autophagy-associated proteins, was confirmed by proteomics analysis. In total, over 200 unique proteins were identified in the enriched autophagosome fraction representing all aspects of cellular function and localization that would be expected to be found in an autophagosome (Fig. 7C). Importantly, proteins not expected to be found in the autophagosome were not detected in the autophagosome-enriched fraction. Examples include lysosomal proteins such as LAMP1, LAMP2, CTSB and CTSD. In addition, we did not detect any single-membrane vesicle structures by TEM. Altogether, our data demonstrated that we are enriching for autophagosomes. DISCUSSION Previous work shows that deletion of the essential autophagy gene Atg5 in rod photoreceptors leads to their degeneration. Degeneration is accompanied by the accumulation of the phototransduction protein, transducin, suggesting that one role for autophagy is to regulate the level of this protein. In addition these studies suggest that the increased levels of transducin might be involved in retinal degeneration as this protein can be toxic to cells 7. In this work, we show that by removing the stress of transducin accumulation in the ATG5-deficient atg5∆rod mouse, we greatly reduce the rate of photoreceptor degeneration, further reinforcing the hypothesis that the degradation of phototransduction proteins by autophagy is critical to retinal homeostasis.

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The removal of excess and damaged proteins by mechanisms such as autophagy is critical to the long term survival of many cell types. Autophagy is particularly important in postmitotic cells where it functions to remove damaged components that would otherwise accumulate and harm the cell. 20,21 This concept has been borne out in numerous model systems, and dysfunctional autophagy has been implicated in neurodegenerative diseases such as Alzheimer and Huntington diseases.22-24 In rod photoreceptors the light-/dark-induced translocation of phototransduction proteins is thought to regulate light sensitivity whereby the concentration of transducin and ARR/arrestin in the photoreceptor OS is the critical factor. In bright light the translocation of transducin from the OS reduces sensitivity and the movement of ARR/arrestin into the OS quenches rhodopsin activation. Our results directly confirm that autophagy also serves as a critical pathway for preventing the excessive accumulation by degrading these translocating proteins. This key process reduces the potential for toxicity of accumulating proteins. Although our studies demonstrate that autophagic regulation of phototransduction proteins prevent retinal degeneration we sought additional evidence for a role of the autophagy pathway. We did this by directly showing that ARR/arrestin and transducin could be recovered from autophagic vacuoles isolated from the retina. Typically, autophagosome purification has been a tedious process requiring high-speed centrifugation of cellular organelles across density gradients. In these protocols there is also the requirement for large amounts of starting material, as the yields tend to be low. For example, purification of autophagosomes from a single rat liver, which contains approximately 3.5 g of hepatocytes, will typically yield approximately 5 mg of autophagosome product.12-14 Due to the small size of the mouse eye (average 15 to 20 mg) and retina (< 2 mg),25 centrifugation protocols would require extensive amounts of eyes and would

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be very challenging. In comparison, an immunopurification-based autophagosome enrichment scheme by immunoprecipitation of GFP-tagged LC3B requires less protein resources, and has been used to imunoisolate LC3-positive membrane compartments from GFP-LC3 transfected cell lines.16 The technique we employed in this study is derived from this immunoprecipitation technique. We confirmed this technique first in vitro, and then applied it to the GFP-LC3 mouse. The typical yield from 2 mouse retinas was approximately 50 g, which was sufficient to allow for most protein work such as western blot analysis. In addition, the technique we describe can be performed very rapidly, with typical enrichment time less than 3 h. This increased efficiency is expected to greatly facilitate future studies of the autophagosome and its content. The electron micrographs showing that the structures we harvest are double membrane serve to strongly support the conclusion that we have a highly enriched population of autophagosomes. An argument against this conclusion is that LC3 is typically cleaved off of the surface of the autophagosome by ATG4, although the precise timing or mechanism of regulation of the deconjugation event are not known.26 Thus, the population of vesicles that we collect could represent a phagophore (autophagosome precursor), which we are able to capture just prior to closure of the vesicle and cleavage of the LC3 off its surface. Additionally, there is the possibility that the presence of the GFP tag on the LC3 protein alters the kinetics of the cleavage by ATG4 sufficiently to allow for persistence of LC3-coated autophagosomes for enough time to enable the immunoprecipitation. The likelihood of these structures representing autophagosomes is also supported by the relative absence of the ATG12–ATG5(-ATG16L1) complex, which would be present on phagophores, but not on autophagosomes. Altogether, this study demonstrates that autophagy plays a critical role in maintaining photoreceptor homeostasis by degrading excess phototransduction proteins, preventing their

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accumulation in the inner segment that would otherwise cause retinal degeneration. In addition, our results provide strong confirmation that our immunoisolation technique was sufficient for enrichment of autophagosomes from the GFP-LC3 mouse retina, validating a novel technique for the study of the autophagosome and its contents across different organs and specific cell types in vivo. MATERIALS AND METHODS Animals The gnat1-/- mouse contains a knockout of the gene encoding the alpha subunit of rod transducin (Table 1).7 The atg5∆rod mouse contains a rod-specific deletion of the Atg5 gene, and was originally generated by crossing the Atg5flox/flox mouse with a (Rho)-Cre mouse.7,9 These 2 strains were bred together to generate mice whose rod cells were deficient in both the Gnat1 and Atg5 genes [gnat1-/-; Atg5flox/flox; (Rho)-Cre] and control [gnat1+/+; Atg5flox/flox; (Rho)-Cre] littermates. Mice were genotyped by PCR analysis of genomic DNA isolated from tail tips. The (Rho)-Cre-only mouse had no retinal degeneration9 and will not be discussed further. The GFPLc3 mice19 (Riken Laboratories, Tsukuba, Japan) were used for purification of autophagosomes in the retina. Mice were bred and housed under standard 12-h light/12-h dark conditions in the University of Michigan, Kellogg Eye Center animal facility. All animal experiments were conducted in accordance with the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research and approved by the University Committee on the Use and Care of Animals of the University of Michigan. All mice were genotyped and only those negative for mutation in the Crb1/Rd8 gene27 were used.

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Tissue collection Retinal tissue was carefully dissected at various ages as previously described.5 Due to the bimodal temporal variation in autophagy levels in the photoreceptor, all tissue collections were performed at the same time of day, 1:00 pm. Briefly, mice were euthanized and eyes were immediately enucleated and placed in a dish. The connective tissue, muscle, and optic nerve were removed from the back of the eye, and the cornea and lens were removed to form an eye cup. The retina was then carefully dissected off of the retinal pigment epithelium. To collect dark-adapted retinas, mice were dark-adapted overnight and eyes were enucleated and dissected under dim red light. Electroretinography Electroretinography (ERG) was performed using the Espion e2 recording system (Diagnosys, Lowell, MA, USA) as described previously.28 Mice were anesthetized with an intraperitoneal injection of ketamine at 80 mg/kg (Hopira, 0409-2051-05) and xylazine at 10 mg/kg ( NAND, 139-236). Body temperature was maintained at 37°C with a heating pad. After pupil dilation with topical phenylephrine (2.5%) and tropicamide (1.0%), corneal ERGs were recorded from both eyes using gold wire loops mounted in a contact lens electrode (Mayo Corporation, Japan) and a drop of 2% methylcellulose for corneal hydration. A gold wire loop placed in the mouth was used as reference, and a ground electrode was on the tail. Light adaptation was performed by exposure to a white 32 cd.m-2 light, rod-suppressing background. The light adapted ERGs were recorded at 1.09 log cd.s.m-2. Ten to 25 responses were recorded at 3 to 60 s depending upon the stimulus intensity intervals. B-wave amplitude of the averaged response was measured from baseline to the positive peak of the waveform. The flicker response was taken with 20-Hz light flickers.

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Optokinetic tracking responses The optokinetic response was measured in (Rho)-Cre, atg5rod∆, and gnat1-/- atg5rod∆ mice (n = 5 each), using an OptoMotry system (Cerebral Mechanics, Lethbridge, AB Canada), following previously described methods.29 Briefly, an awake and freely moving mouse was placed onto the elevated platform at the center of the OptoMotry device, surrounded by 4 computer screens arranged in a quadrangle around the platform. The computer screens created a virtual image of a rotating cylinder with vertical sine-wave grating of varying contrast. A camera was place on the top to monitor the movement of the mouse. The tracking of the rotating gratings by the mouse was scored by the movements of its head and neck (i.e., the optokinetic reflex response). The maximum spatial frequency (in cycles/degree [c/d]) in 100% background contrast that generated a tracking movement of the animal was recorded for each eye by a genotypemasked observer. Cell culture and transfection The 661W photoreceptor cell line17 was maintained in DMEM containing 10% fetal bovine serum, 300 mg/L glutamine, 32 mg/L putrescine (Sigma, P-7505), 40 µL/L of βmercaptoethanol (Sigma, M-6250), and 40 µg/L of both hydrocortisone 21-hemisuccinate (Sigma, H-2270) and progesterone (Sigma, P-7505). The media also contained penicillin (90 U/mL) and streptomycin (0.09 mg/mL). Cells were grown at 37°C in a humidified atmosphere of 5% CO2 and 95% air. GFP-LC3 was subcloned from pBABEpuro-GFP-LC3 (addgene, 22405; deposited by Dr. J. Debnath) into the AgeI and SbfI sites of the pGW1-CMV vector, a gift from Jason Miller (Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, MI). Inserts were verified by DNA sequencing. 661W cells were transfected using ViaFect transfection reagent (Promega, E4981) according to the manufacturer’s instruction.

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Autophagosome immunoisolation To prepare cell lysates of GFP-LC3-transfected 661W cells stably expressing GFP-LC3 for immunoprecipitation, cells were washed three times with Hanks balanced salt solution (Gibco, 14025092), and starved by incubating in Hanks balanced salt solution containing 100 nM bafilomycin A1 (Invitrogen, tlrl-baf1) for 2 h. Cells were then washed with cold PBS and homogenized in 1 ml of cold lysis buffer containing protease inhibitor (250 mM sucrose (SigmaAldrich, S0389), 1mM EDTA [Thermo Fisher Scientific, AM9262], 10 mM HEPES [SigmaAldrich, H3375], pH 7.4, complete protease inhibitor tablet [Roche, 11697498001]) by passing 15 times through a 22-gauge needle on ice. To prepare retina lysates, 2-month-old GFP-LC3 mice were given an intraperitoneal injection of 50 mg/kg body weight chloroquine (SigmaAldrich, C6628). Five h after the treatment, retinas were dissected, and homogenized using a pellet pestle motor (Kontes, 749540-0000). The lysate was then passed 10 times through a 26gauge needle and 5 times through a 30-gauge needle. GFP-based immunoisolation was performed using µMACS GFP Isolation Kit (Miltinyl Biotec, 130-091-125) with modifications. The lysate of cell lines and retinas was centrifuged at 1,000 x g for 10 min at 4°C. The postnuclear supernatant fraction was centrifuged at 17,000 x g for 20 min and the supernatant fraction was discarded to remove residual cytosolic GFP-LC3. The pellet fraction was resuspended in 1 mL lysis buffer and was incubated with µMACS antiGFP magnetic microbeads (supplied in the kit) for 1 h on ice with mixing. Lysate-bead mixture was applied to a LS column (MACS Miltenyi Biotec, 130-042-701), which was placed in a magnetic µMACS Separator (MACS Miltenyi Biotec, 130-042-602). The column was then washed and eluted by preheated elution buffer. For transmission electron microscopy or fluorescence microscopy, the immunoprecipitated material was dissociated from the column by

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pH shift using trimethylamine (pH 11.8; Sigma-Aldrich, 243205) then neutralizing with 1 M MES (Sigma-Aldrich, M5287). Western blot analysis Proteins were separated by 4-15% SDS-PAGE (Tris-HCl Ready Gels; Bio-Rad Laboratories) and transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories, 1620177). The membranes were incubated overnight with primary antibodies. A full list of antibodies is shown in Table 2. Secondary polyclonal goat anti-immunoglobulins antibodies were from Dako (P0488 or P0447). Detection was by SuperSignal West Dura Substrate (Thermo Scientific, 34075) according to the manufacturer’s protocols. Quantitative densitometry of the immunoblots was performed using ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html ), and expressed as the mean density (± SD) from replicate experimental groups. All experiments were performed a minimum of 3 times. Transmission electron microscopy Immunoisolated autophagosomes were prepared and immediately sent for transmission electron microscopy. Five µl of the sample was deposited on 200-mesh formvar-coated grids (Polysciences, Inc., 24915). After 20 sec, the excess liquid was removed with a Whatman filter paper. The remaining sample was allowed to dry at room temperature. Five µl of 1% uranyl acetate was then deposited on the sample and the excess was removed after several seconds. Once the grid was dry, samples were observed with a JEOL transmission electron microscope. Images were recorded digitally using a Hamamatsu ORCA-HR digital camera system operated using AMT software (Advanced Microscopy Techniques Corp., Danvers, MA, USA).

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Histology and immunohistochemistry The superior region of each eye was marked by a burner (GEMINI Cautery System, GEM5917) before enucleation for orientation. To obtain cryosections, enucleated eyes were fixed in freshly prepared 4% paraformaldehyde overnight and subsequently processed as previously described.5 Briefly, the cornea and lens were removed and the eyecup was rinsed 3 times in phosphate-buffered saline (PBS; Sigma Life Science, P4417), transferred to 10% and then 20% sucrose in PBS for 2 h each, before embedding in OCT (Tissue Tek; Sakura Finetek, 4583) mixed in a ratio of 1:1 with 20% sucrose. A cryostat was used to obtain serial sections of 10-µm thickness. Eyes to be used for paraffin sections were prepared using a fixation method involving freeze substitution. Briefly, each enucleated eye was snap frozen in dry ice-cooled liquid propane for 30 sec, and then transferred to dry ice-cooled methanol containing 3% glacial acetic acid. Eyes immersed in this fixative were kept at -80°C for 48 h, followed by overnight at -20°C. After eyes were warmed to room temperature, the cornea and lens were removed in 100% ethanol and left for 1 to 2 h in 100% ethanol, 2×30 min in 100% ethanol, 2×30 min in citrate solvent (Fisher, 22143975), and 3×40 min in paraffin. Eyes were embedded in paraffin in an orientation-specific manner. Sections were cut using a microtome (Shandon AS325, Thermo Scientific, USA) at 6 μm. Representative sagittal sections crossing the optic nerve from each eye were used for immunohistochemistry, and hematoxylin (Fisher Scientific, 3536-16) and eosin (Fisher Scientific, E511) staining; for the latter, 3 representative thin sections crossing the optic nerve were used from each eye and images were captured with a Leica DM6000 microscope (Leica Crop., Wetzlar, Germany). Sections were deparaffinized in xylene, hydrated through gradient alcohols, and washed in PBS. Antigen retrieval was performed at 98°C using 1 mM EDTA in

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0.05% Tween-20 (Sigma, P7949) for 30 min and left at room temperature for 30 min before washing with PBS. For immunohistochemistry, sections were blocked with 10% goat serum (Sigma Life Science, G9023) in PBS for 1 h, and incubated with primary antibodies in PBS containing 0.2% Triton X-100 (Sigma Life Science, 9002-93-1) in a humidity chamber overnight at 4ºC. After washing and incubation for 1 h at room temperature with secondary antibodies, sections were counterstained with ProLong Gold with DAPI (Invitrogen, P36941) to reveal cell nuclei. Images were obtained using a confocal microscope (Leica SP5, Leica Corp., Mannheim, Germany) and were taken at the comparable area of sections. All images in each individual experiment were acquired with a fixed detection gain. For cultured GFP-LC3 transfected cell lines, confluent cultures on chamber slides were fixed in 4% paraformaldehyde at room temperature. Slides were washed in PBS, and were counterstained with ProLong Gold with DAPI. To observe immunoisolated autophagosomes from cultured cells, 10 µl of the eluted immunoprecipitate was transferred to a glass slide, covered and observed under a confocal microscope. Spectral domain optical coherence tomography Mice were anesthetized with an intraperitoneal injection of ketamine at 80 mg/kg (Hopira, 0409-2051-05) and xylazine at 10 mg/kg (NAND, 139-236). Pupils were dilated with topical phenylephrine (2.5%, Paragon BioTek, inc., 42702-102-15) and tropicamide (1.0%, AKORN, 17478-101-12). Systane lubricant eye drops (Alcon, 9004494-0109) were given for corneal hydration. Optical coherence tomography (OCT) was performed with a spectral domain optical coherence tomography system (Bioptigen Inc., Durham, NC, USA). A volume analysis centered on the optic nerve head was performed, using 100 horizontal, raster, and consecutive B

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scan lines, each one composed of 1200 A-scans. The volume size was 1.6 x 1.6 mm. Outer nuclear layer thickness was assessed at areas of superior, inferior, nasal and temporal with distances of 500 µm from the optic nerve head. Generation of the retina heat map and the grid of the calculated thicknesses of the retina areas were performed using InVivoVueTM Diver 2.4 software (Bioptigen Inc., Durham, NC). Mass spectroscopy Immunoisolated autophagosomes were prepared and sent to MS Bioworks (Ann Arbor, MI, USA) for mass spectroscopy analysis. Briefly, the isolated autophagosome sample was processed by SDS-PAGE using a 10% Bis-Tris NuPAGE gel (ThermoFisher Scientific, NP0301) with the MES buffer system. The digest sample was analyzed by nano LC-MS/MS with a Waters NanoAcquity HPLC system (Waters Corp., Milford, MA, USA) interfaced to a ThermoFisher Q Exactive (ThermoFisher Scientific, Waltham, MA, USA). The mass spectrometer was operated in data-dependent mode, with the Orbitrap (ThermoFisher Scientific, Waltham, MA, USA) operating at 60,000 FWHM and 17,500 FWHM for MS and MS/MS, respectively. The 15 most abundant ions were selected for MS/MS. Data were searched using a local copy of Mascot with the following parameters: Enzyme: Trypsin/P; database: SwissProt Mouse; fixed modification: Carbamidomethyl (C); variable modifications: Oxidation (M), acetyl (N-term), pyro-Glu (N-term Q), deamidation (N/Q); mass values: Monoisotopic; peptide mass tolerance: 10 ppm; fragment mass tolerance: 0.02 Da; max missed cleavages: 2. Mascot DAT files were then parsed into the Scaffold software for validation, and filtering. Quantitative data are reported as spectral counts (SpC) and normalized spectral abundance factors (NSAFs).

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Statistical analysis Experimental results are expressed as mean ± SD. Values of SD-OCT were analyzed by two-tailed Student t tests. The nonparametric Kruskal-Wallis (K-W) test was used for western blotting analysis. Differences were considered significant at P < 0.05. Abbreviations ARR , arrestin; GFP

, green fluorescent protein;

GNAT1

, guanine nucleotide binding protein (G-protein) alpha transducing activity

polypeptide 1 (also known as the α-subunit of transducin or Tα); IS

, inner segment;

OCT , optical coherence tomography; ONL , outer nuclear layer; OS

, outer segments;

RHO , rhodopsin Commercial Interest None Support: National Eye Institute R01-EY020823 (DNZ), Beckman Initiative for Macular Research (DNZ), Foundation Fighting Blindness (DNZ), Research to Prevent Blindness, Inc. (DNZ), University of Michigan Core Center for Vision Research (NEI-EY007003); Washington University Department of Ophthalmology and Visual Sciences Core Grant (NEI-EY02687), Washington University Department of Ophthalmology and Visual Science unrestricted grant from Research to Prevent Blindness, Inc. DJK was supported by NIH grant GM053396.

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REFERENCES 1. Mustafi D, Engel AH, Palczewski K. Structure of cone photoreceptors. Prog Ret Eye Res. 2009;28:289-302 2. Calvert PD, Strissel KJ, Schiesser WE, Pugh EN Jr, Arshavsky VY. Light-driven translocation of signaling proteins in vertebrate photoreceptors. Trends Cell Biol. 2006;16:560-568 3. Yang Z, Klionsky DJ. Mammalian autophagy: core molecular machinery and signaling regulation. Curr Opin Cell Biol. 2010;22:124–131 4. Yoshimori, T. Autophagy: a regulated bulk degradation process inside cells. Biochem. Biophys. Res. Commun. 2004;313:453–458 5. Yao J, Jia L, Shelby SJ, Ganios AM, Feathers K, Thompson DA, Zacks DN. Circadian and noncircadian modulation of autophagy in photoreceptors and retinal pigment epithelium. Invest Ophthalmol Vis Sci. 2014;55:3237-3246 6. Chinskey ND, Zheng QD, Zacks DN. Control of photoreceptor autophagy after retinal detachment: the switch from survival to death. Invest Ophthalmol Vis Sci. 2014;55:688-695 7. Zhou Z, Doggett TA, Sene A, Apte RS, Ferguson TA. Autophagy supports survival and phototransduction protein levels in rod photoreceptors. Cell Death Differ. 2015; 22:488-498 8. Calvert PD, Krasnoperova NV, Lyubarsky AL, et al. Phototransduction in transgenic mice after targeted deletion of the rod transducin alpha-subunit. Proc Nat Acad Sci USA. 2000;97:13913–13918 9. Li S, Chen D, Sauvé Y, McCandless J, Chen YJ, Chen CK. Rhodopsin-iCre transgenic mouse line for Cre-mediated rod-specific gene targeting. Genesis. 2005;41:73-80.

21

10. Yang Y. Mohand-Said S, Danan A et al. Functional cone rescue by RdCVF protein in a dominant model of retinitis pigmentosa. Mol Ther. 2009;17:787-795. 11. Pinto LH, Enroth-Cugell C. Tests of the mouse visual system. Mamm Genome. 2000;11:531536 12. Marzella L, Ahlberg J, Glaumann H. Isolation of autophagic vacuoles from rat liver: morphological and biochemical characterization. J Cell Biol 1982; 93:144-54 13. Strømhaug PE, Berg TO, Fengsrud M, Seglen PO. Purification and characterization of autophagosomes from rat hepatocytes. Biochem J 1998; 335:217-24 14. Seglen PO, Brinchmann MF. Purification of autophagosomes from rat hepatocytes. Autophagy. 2010;6:542-547 15. Chen X1, Li LJ, Zheng XY, Shen HQ, Shang SQ. Isolation of autophagosome subpopulations after induction of autophagy by calcium. Biochem Cell Biol. 2015;93:180184 16. Gao W, Kang JH, Liao Y, Ding WX, Gambotto AA, Watkins SC, Liu YJ, Stolz DB, Yin XM. Biochemical isolation and characterization of the tubulovesicular LC3-positive autophagosomal compartment. J Biol Chem. 2010;285:1371-1383 17. al-Ubaidi MR, Font RL, Quiambao AB, Keener MJ, Liou GI, et al. Bilateral retinal and brain tumors in transgenic mice expressing simian virus 40 large T antigen under control of the human interphotoreceptor retinoid-binding protein promoter. The Journal of cell biology 1992;119: 1681–1687. 18. Klionsky DJ, Abdelmohsen K, Abe A, et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy. 2016;12:1-222

22

19. Mizushima N, Kuma A. Autophagosomes in GFP-LC3 transgenic mice. Meth Mol Biol. 2008;445:119-124 20. Díaz-Villanueva JF, Díaz-Molina R, García-González V. Protein Folding and Mechanisms of Proteostasis. Int J Mol Sci. 2015;16:17193-17230. 21. Lee WS, Yoo WH, Chae HJ. ER Stress and Autophagy. Curr Mol Med. 2015;15:735-745. 22. Navone F, Genevini P, Borgese N. Autophagy and Neurodegeneration: Insights from a Cultured Cell Model of ALS. Cells. 2015;4:354-386 23. Milisav I, Šuput D, Ribarič S. Unfolded Protein Response and Macroautophagy in Alzheimer's, Parkinson's and Prion Diseases. Molecules. 2015;20:22718-22756 24. González-Polo RA, Pizarro-Estrella E, Yakhine-Diop SM, Rodríguez-Arribas M, GómezSánchez R, Pedro JM, Fuentes JM. Is the Modulation of Autophagy the Future in the Treatment of Neurodegenerative Diseases? Curr Top Med Chem. 2015;15:2152-2174. 25. Wisard J, Chrenek MA, Wright C, Dalal N, Pardue MT, Boatright JH, Nickerson JM. Noncontact measurement of linear external dimensions of the mouse eye. J Neurosci Methods. 2010;187:156-166 26. Tanida I, Sou YS, Ezaki J, Minematsu-Ikeguchi N, Ueno T, Kominami E. HsAtg4B/HsApg4B/autophagin-1 cleaves the carboxyl termini of three human Atg8 homologues and delipidates microtubule-associated protein light chain 3- and GABAA receptor-associated protein-phospholipid conjugates. J Biol Chem. 2004;279:36268-36276 27. Mattapallil MJ, Wawrousek EF, Chan CC, Zhao H, Roychoudhury J, Ferguson TA, Caspi RR. The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes. Invest Ophthalmol Vis Sci. 2012;53:2921-2927

23

28. Yao J, Jia L, Khan N et al. Deletion of autophagy inducer RB1CC1 results in degeneration of the retinal pigment epithelium. Autophagy. 2015;11:939-953 29. Douglas RM, Alam NM, Silver BD, McGill TJ, Tschetter WW, Prusky GT. Independent visual threshold measurements in the two eyes of freely moving rats and mice using a virtualreality optokinetic system. Vis Neurosci. 2005;22:677–684

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Table 1. Description of the different mouse strains Genotyping

Mouse strain

Biological effects

Gnat1 –/–

Atg5

(Rho)-Cre

wt/wt

–

flox/flox

wt/wt

flox/flox

–

(Rho)-Cre

wt/wt

wt/wt

+

atg5∆rod

wt/wt

flox/flox

+

gnat1-/-

Rod photoreceptors are deficient in the protein encoded by Gnat1- the α-subunit of

transducin (Tα). Atg5

-/-

gnat1 atg5

∆rod

–/–

flox/flox

Atg5 gene is flanked by two loxP sequences, no functional change. Cre-unfloxed control. Cre expressed only in the rod photoreceptors. Tissue-specific knockout. Atg5 knockout in the rod photoreceptors. Created by crossing the

Atg5flox/flox with the (Rho)-Cre. Tissue-specific knockout. Both Gnat1 and

+

Atg5 are knocked out in the rod photoreceptors. Created by crossing the atg5∆rod with the

gnat1-/-.

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Table 2. List of antibodies used in this study. Antibody

Host

Immunoblota

Immunostaininga

Supplier

LC3

rabbit

1:1000

-

Cell Signaling Technology, 4108

ATG5

rabbit

1:1000

-

Novus Biologicals, NB100-53818S

SQSTM1/p62

rabbit

1:1000

-

Novus Biologicals, NBP1-48320S

WDFY3

rabbit

1:250

-

Cre-recombinase

mouse

-

1:1000

Aviva Systems Biology, APR50850_P050 Millipore, MAB3120

Cre-recombinase

rabbit

1:10000

1:1000

Novagen, 6905-3

RHO/Rhodopsin (4d2)

mouse

1:1000

1:1000

Novus Biologicals, NBP1-48334

GNAT1

rabbit

1: 500

1:1000

GeneTex, GTX87657

ARR/Visual Arrestin OPN1MW/MOpsin GAPDH

rabbit

1:4000

-

Thermo Scientific, PA1-731

rabbit

-

1:75

mouse

1:60,000

-

A kind gift from Debra Thompson (University of Michigan) Ambion Applied Biosystems, AM4300

a

Ratios listed are the dilution used for each particular experiment.

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Figure 1. Crossing of the atg5∆rod mouse with the gnat1-/- mouse resulted in a mouse in which the rod photoreceptors both lacked the ability to activate autophagy as well as the -subunit of the transducin molecule. (A) Immunohistochemical staining confirms that the atg5∆rod mouse contains the GNAT1 protein (T). Cre-recombinase is detected in the outer nuclear layer, as expected, due to the derivation of the atg5∆rod mouse from the cross between the Atg5-floxed mouse and a mouse with Rho-promoter-driven expression of CRE (the [Rho]-Cre mouse). (B,C) Western blot analysis confirms that the atg5∆rod and the gnat1-/- atg5∆rod mouse strains both lack ATG5, whereas only the gnat1-/- atg5∆rod mouse lacks T. Both strains show markedly diminished autophagy activation, as demonstrated by the significant reduction in LC3 lipidation

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and increased accumulation of SQSTM1, as compared to the (Rho)-Cre mouse. (D) Densitometry analysis of the western blot bands confirms the accumulation of SQSTM1 and reduction in LC3-II formation. Normalization for each of the measures was to the value for the (Rho)-Cre retina. The asterisk signifies statistical significance at a P