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Retina  |   November 2015
Early Onset Ultrastructural and Functional Defects in RPE and Photoreceptors of a Stargardt-Like Macular Dystrophy (STGD3) Transgenic Mouse Model
Author Affiliations & Notes
  • Sharee Kuny
    Department of Ophthalmology and Visual Sciences, University of Alberta, Edmonton, Alberta, Canada
  • Woo Jung Cho
    Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada
  • Ioannis S. Dimopoulos
    Department of Ophthalmology and Visual Sciences, University of Alberta, Edmonton, Alberta, Canada
  • Yves Sauvé
    Department of Ophthalmology and Visual Sciences, University of Alberta, Edmonton, Alberta, Canada
    Department of Physiology, University of Alberta, Edmonton, Alberta, Canada
  • Correspondence: Yves Sauvé, Department of Physiology, 7-55 Medical Sciences Building, University of Alberta, Edmonton, AB, Canada, T6G 2H7; [email protected]
Investigative Ophthalmology & Visual Science November 2015, Vol.56, 7109-7121. doi:https://doi.org/10.1167/iovs.15-17567
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      Sharee Kuny, Woo Jung Cho, Ioannis S. Dimopoulos, Yves Sauvé; Early Onset Ultrastructural and Functional Defects in RPE and Photoreceptors of a Stargardt-Like Macular Dystrophy (STGD3) Transgenic Mouse Model. Invest. Ophthalmol. Vis. Sci. 2015;56(12):7109-7121. https://doi.org/10.1167/iovs.15-17567.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: We investigated the interplay between photoreceptors expressing mutant ELOVL4 (responsible for Stargardt-like disease, STGD3) and RPE in the initial stages of retinal degeneration.

Methods: Using electron microscopy and electroretinogram (ERG), we assessed RPE and photoreceptor ultrastructure and function in transgenic ELOVL4 (TG1-2 line; TG) and wild-type (WT) littermates. Experiments were done at P30, 1 month before photoreceptor loss in TG and at P90, a time point with approximately 30% rod loss. To further elucidate the mechanism underlying our ultrastructural and functional results, we undertook Western blotting and immunohistochemistry of key proteins involved in phagocytosis of outer segments by RPE cells.

Results: Firstly, we showed that in TG mouse photoreceptors, endogenous ELOVL4 protein is not mislocalized in the presence of the mutated ELOVL4 protein. Secondly, we found evidence of RPE toxicity at P30, preceding any photoreceptor loss. Pathology in RPE cells was exacerbated at P90. Furthermore, higher proportions of phagosomes remained at the apical side of RPE cells. Subretinal lysosomal deposits were immunopositive for phagocytic proteins. Ultrastructural analysis of photoreceptor (rod) outer segments showed disrupted surface morphology consisting of disc spacing irregularities. Finally, rods and RPE exhibited signs of dysfunction as measured by the ERG a-wave leading edge (P30) and c-wave (P90), respectively.

Conclusions: The presence of human mutant ELOVL4 in transgenic mouse photoreceptors leads to early outer segment disc pathology and RPE cytotoxicity. Defective processing of these abnormal discs by RPE cells ultimately may be responsible for outer segment truncation, photoreceptor death, and vision loss.

Mutations (790-794-delAACTT, 789ΔT+794ΔT, Y270ter) in exon 6 of the ELOVL4 gene are responsible for autosomal dominant Stargardt-like macular dystrophy (STGD3; OMIM 600110), a juvenile form of progressive macular degeneration. All these mutations lead to a premature stop codon, resulting in a truncated ELOVL4 protein lacking its C-terminal endoplasmic reticulum retention signal.1 This truncated form of the protein is enzymatically inactive.2 In vitro, this protein mislocalizes to Golgi membranes and/or perinuclear cytoplasmic aggresomes,3 where it forms complexes with wild-type ELOVL4 and other ELOVL proteins leading to an unfolded protein response (UPR).48 
Intriguingly, the mechanisms responsible for photoreceptor loss in STGD3 are unknown. Unfolded protein response was not observed in vivo, in either a knock-in or a transgenic mouse model of this disease.9,10 In our STGD3 transgenic mouse model (TG1-2 line),11 the only UPR marker transcript found to be elevated (1.45-fold)12 was homocysteine-inducible, endoplasmic reticulum stress–inducible, ubiquitin-like domain member 1 (Herpud1). Protein HERPUD1 has been proposed to delay the degradation of cytosolic proteins.13 In addition to the lack of UPR, the loss of ELOVL4 elongase activity,2 which is essential for the synthesis of very long chain polyunsaturated fatty acids (VLC-PUFAs, of 28–36 carbon chains),14 is not the cause of retinal degeneration in STGD3.10,1520 Finally, there is recent evidence that human mutant ELOVL4 mislocalizes in vivo, in photoreceptor cell bodies of STGD3 transgenic pigs,21 and photoreceptor outer segments of STGD3 transgenic Xenopus laevis.22 Whether such mislocalization occurs in the STGD3 transgenic mouse model and leads to alterations in disc membrane and ultimately to photoreceptor degeneration, however, remains to be deciphered. 
Photoreceptor survival depends on the recycling of their shed outer segments by RPE cells,2328 a process involving the interplay of phagocytosis and autophagy, both important intracellular lysosomal degradative pathways. In RPE cells, phagocytosis coopts the autophagy pathway to increase the efficiency of degradation in a process termed LC3-associated phagocytosis.29 In fact, the major cause of vision loss in the elderly (age-related macular degeneration, AMD) involves RPE phagocytic defects.30,31 Over a 70-year human lifespan, each RPE cell is estimated to have digested a billion discs.32 In mice, a higher photoreceptor density combined with larger RPE cells in the central retina results in a phagocytic load per RPE cell more than 3-fold higher than in the human macula.33 This highly demanding degradative load implies that defects in this sequential process (photoreceptor outer segment binding, internalization, and digestion)24,34 can lead to photoreceptor dysfunction and death.35 Examples include the pathologic accumulation in RPE cells, of photoreceptor outer segments, misfolded protein aggregates, and cytotoxic debris, such as A2E.11,36,37 
In the present study, we sought to determine how expression of human mutant ELOVL4 by photoreceptors might impact their integrity and that of the underlying RPE. Assessment at the ultrastructural level in parallel with functional status provided evidence for photoreceptor outer segment and RPE defects before any cell death. 
Materials and Methods
Animals
The present study was performed on heterozygous transgenic ELOVL4 (TG1-2 mice) and wild-type (WT) littermates from a colony maintained at the University of Alberta. As described previously,38 this line is derived from the TG2 line originally generated by Karan et al.11 but with lower transgene expression levels. Animals were maintained on a 14:10 light–dark cycle, temperature 21.5°C, relative humidity 42%, and supplied ad libitum with water and Laboratory Rodent Diet (5001, LabDiet; PMI Nutrition Intl., Richmond, IN, USA). Experiments were performed in accordance with guidelines of the Institutional Animal Care and Use Committee (University of Alberta) and the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. 
RNA Expression Levels
The expression level of the human mutant ELOVL4 transgene was determined with semiquantitative RT-PCR as described previously.38 Briefly, total RNA was isolated from the neural retina at P30, P60, P90, and P270 (n = 4) using TRIzol (15596-026; Invitrogen, Carlsbad, CA, USA), treated with DNase I (18068-015, Invitrogen), and 0.5 μg was used for cDNA synthesis and PCR (SuperScript One-Step RT-PCR with Platinum Taq, 10928-034; Invitrogen). EcoRI allowed differentiating the human ELOVL4 cDNA, which contains an EcoRI site (384 + 173 bP products) from the mouse Elovl4 cDNA (557 bp product only). 
Transmission Electron Microscopy (TEM)
Following euthanasia and enucleation, corneas were punctured and eyes (minimum of four each from TG and WT animals at P30 and P90) placed in ice-cold TEM-fixative (0.1 M cacodylate buffer, pH 7.4, with 2 mM sucrose, 4% paraformaldehyde [PFA], 2.5% glutaraldehyde [GA]) for 3 hours at 4°C, after which time corneas and lenses were removed. Following 30 minutes of additional fixation, four cuts were made to flatten the posterior eyecup, 1 × 2-mm sample pieces were cut from the central retina 250 to 400 μm from the optic nerve head, and fixation continued for 72 hours at 4°C. 
Sample pieces then were washed for 1 hour in 0.1 M sodium cacodylate buffer, to remove any residual aldehyde, and incubated in 2% osmium tetraoxide (OsO4) in 0.1 M sodium cacodylate buffer for 1 hour to fix lipids. Brief washing with 0.1 M sodium cacodylate buffer (15 minutes), then with 0.1 M sodium acetate buffer, pH 5.2 (5 minutes), was followed by en bloc staining with 2% uranyl acetate in 0.1 M sodium acetate buffer, pH 5.2 for 1 hour to increase contrast. After further washing with 0.1 M sodium acetate buffer, the sample pieces were dehydrated in an ethanol series (50, 70, 80, 90, 95, and 100%), followed by infiltration with Spurr's resin (Sigma-Aldrich Corp., St. Louis, MO, USA). Sample pieces then were placed into flat embedding molds, oriented parallel to photoreceptor outer segments, and the resin thermally polymerized for 24 hours at 65°C. Ultrathin sections (60 nm) were cut with an ultramicrotome (Leica UC7; Leica Microsystems, Inc., Ontario, Canada), and then stained with 2% uranyl acetate and Reinold's lead citrate. The contrasted sections were imaged under a Hitachi H-7650 transmission electron microscope (Hitach High-Technologies Canada, Inc., Rexdale, ON, Canada) at 80 kV equipped with a 16 mega pixel EMCCD camera (XR111; Advanced Microscopy Techniques, MA, Woburn, USA) and imaging software (AMT version 600; Advanced Microscopy Techniques). 
Low Voltage Field Emission Scanning Electron Microscopy (SEM)
Following euthanasia and enucleation, corneas were punctured and eyes (P30 time point only) placed in ice-cold SEM-fixative (0.1 M sodium cacodylate, pH 7.4, with 2.5% GA) for 5 minutes. Retinas were dissected out, fixed for an additional 1 hour, then four cuts were made to flatten the retina, 1 × 2-mm sample pieces were cut from the central retina 250 to 400 μm from the optic nerve head and fixation continued overnight at 4°C. 
After fixation, sample pieces were washed with 0.1 M sodium cacodylate buffer 3 × 15 minutes and repeatedly impregnated with 1% OsO4 and 1% tannic acid to increase bulk electron conductivity as well as to prevent damage and imaging artifacts. Incubation (30 minutes each) in first 1% OsO4 and then 1% tannic acid, repeated two times, was followed with a final 1% OsO4 step. This technique removed the need for sputter coating and allowed for better visualization of close topographic details. Sample pieces then were dehydrated in an ethanol series (30%, 50%, 70%, 80%, 90%, 95%, and 100%) and hexamethyl-disilazane, followed by drying at room temperature. The dried retina specimens were mounted on aluminum stub with conductive paint (colloidal graphite, Product No. 16051; Ted Pella, Inc. Redding, CA, USA), scanned under a Hitachi S-4800 field emission scanning electron microscope at 1 kV, and detected with a back-scattered electron detector. 
Western Blotting
Following euthanasia (systematically within 1.5–2 hours after light onset) and enucleation, retinas or posterior eyecups (retina removed) were dissected out (pooling a minimum of six each from TG and WT animals), homogenized in sample lysis buffer (0.13 M Tris, pH 6.8; 4% SDS; 20% glycerol + HALT Protease inhibitor cocktail, 87786; Thermo Fisher Scientific, Waltham, MA, USA), incubated 30 minutes at 4°C, passed repeatedly through a 26-gauge needle, centrifuged 5 minutes at 4°C to pellet debris, and then frozen in small aliquots at −80°C until use. Protein levels were determined using a Pierce BCA protein assay kit (Cat# PI-23227; Thermo Fisher Scientific). After addition of 2% (vol/vol) 2-mercaptoethanol and 1% (vol/vol) saturated bromophenol blue, samples (30 μg total retina or eyecup protein) were boiled for 5 minutes and resolved by SDS-PAGE on 12% gels. Rhodopsin and α-tubulin blots were run as above but with 1 and 10 μg total protein, respectively. 
Sample lysis buffer was modified for endogenous ELOVL4 Western blots and consisted of 20 mM TrisHCl, pH 7.5; 150 mM NaCl; 1 mM EGTA; 1% Triton X-100 + HALT Protease inhibitor cocktail. Additionally, samples (30 μg total protein from retina, epidermis or liver, positive and negative controls, respectively) were not boiled. 
Samples treated with PNGase F were prepared by diluting 40 μg protein in 0.1 M phosphate buffer, pH 7.2 (with or without 10 U PNGase F, recombinant N-glycosidase F, 1 365 185; Roche, Basel, Switzerland) in a total volume of 20 μL and then incubating overnight at 37°C. After addition of 5 μL ×5 loading dye, 30 μg protein (18.75 μL) was loaded on the gel. 
Proteins were transferred to PVDF membranes, blocked for 1 hour with 5% skim milk powder (or 5% BSA) diluted in TBS-T (20 mM Tris, 137 mM NaCl, 0.1% Tween-20, pH 7.6), and incubated overnight with primary antibodies (see Table) diluted in above blocking solution. The following day membranes were washed 3 × 10 minutes in TBS-T and reacted for 1 hour with anti-mouse, rabbit or goat IgG, HRP-conjugated ECL antibody (NA931, NA934; GE Healthcare, Laurel, MD, USA, and sc-2354; Santa Cruz Biotechnology, Inc., Dallas, TX, USA, respectively; 1:5000 in the blocking solution). After a final extensive washing, protein bands were visualized using ECL reagent (NEL 103, Perkin Elmer, Waltham, MA, USA) on a Carestream Molecular Imaging In-Vivo F PRO with v.5.0.7.22 software. ImageJ v1.48 software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) was used to calculate net intensity of bands (area × mean gray value). Protein levels were normalized for α-tubulin as a loading control. 
Table
 
List and Details of Antibodies
Table
 
List and Details of Antibodies
Immunohistochemistry
Wild-type and TG 20 μm retina cryosections were blocked for 1 hour in PBS + 0.3% Triton X-100 + 10% normal goat serum and then reacted overnight in a humid container with primary antibodies diluted as appropriate (see Table) in a 1:10 dilution of the above blocking solution. The following day, sections were washed extensively in PBS and reacted for 1 hour with species appropriate Alexa fluorescent dyes (1:1000; Molecular Probes, Eugene, OR, USA). After further extensive washing in PBS, slides were coated with a 4′,6-diamidino-2-phenylendole (DAPI)–containing antifade reagent (P36931; Life Technologies, Carlsbad, CA, USA) and coverslipped. Images were captured from the center of the retina (400 ± 200 μm from the optic disc) using a confocal microscope (Zeiss LSM710, with a Plan-Neofluar ×40/1.3 oil objective; Carl Zeiss Meditec, Jena, Germany). Brightness and contrast levels were adjusted if necessary (Adobe Photoshop CC; Adobe Systems, San Jose, CA, USA). 
RPE Flat Mounts
Flat mounts of RPE were prepared as described by Narimatsu et al.39 Briefly, after careful removal of cornea, lens, and retina, the posterior eyecups were fixed in 4% PFA for 30 minutes. After making four radial cuts to flatten and 30 minutes further fixation in PFA (or methanol for β-catenin), eyecups were washed 3 × 10 minutes with PBS. All steps were performed at room temperature unless otherwise noted. For detection of β-catenin, eyecups were treated with L.A.B. Solution (24310, Polysciences, Inc., Warrington, PA, USA) for 20 minutes, blocked for 1 hour in PBS + 0.1% Tween-20 + 0.3% Triton X-100 + 5% normal goat serum and then incubated overnight at 4°C with β-catenin antibody (1:50, see Table) diluted in PBS + 0.3% Triton X-100. The following day, eyecups were washed as before and then incubated with goat anti-rabbit Alexa 488 (1:1000 dilution in 1:10 block) + 10 μg/mL Hoechst (Bisbenzimide, H33258, Sigma-Aldrich Corp.) for 1 hour with shaking. For detection of F-actin, eyecups were incubated with A488 Phalloidin (A12379, 1:40 dilution in 1:10 block; Molecular Probes) for 30 minutes, then 10 μg/mL Hoechst in PBS with shaking for 1 hour. After extensive washing, all eyecups were mounted with Vectashield (H-1000; Vector Laboratories, Burlingame, CA, USA) mounting media and coverslipped. Images were captured from the central retina (∼400 μm from the optic disk) with a Leica DM6000B fluorescence microscope and DFC360FX camera, using a HCX PL APO 40.0 ×1.25 oil objective, and LAS AF v2.2.0 software (Leica Microsystems, Inc.). Brightness and contrast levels were adjusted if necessary (Adobe Photoshop CC; Adobe Systems). 
Electroretinogram (ERG) Functional Studies
Dark- and light-adapted ERG responses were recorded in WT (n = 9) and TG (n = 11) mice at P30 and P90 as described previously.38 In brief, mice were dark-adapted for 1 hour before preparation for simultaneous bilateral recording, under dim red light. Light stimulation, signal amplification (0.3–300 Hz bandpass), and data acquisition were provided by a full-field ERG system (Espion E2 system; Diagnosys LLC, Littleton, MA, USA). For all ERG tests, only the eye corresponding to the highest maximal dark- adapted a-wave amplitude was selected for statistical analysis. 
Under dark adaptation, single white flashes of light were presented in 19 steps at increasing intensities from −5.22 to 2.86 log cds/m2. Dark-adapted a-waves elicited by the four brightest stimulus intensities (1.37, 1.89, 2.39, and 2.86 log cds/m2) were fitted with the Hood and Birch equation40 of the Lamb and Pugh phototransduction activation model,41 which describes the response (R) as a function of flash intensity (I), and time (t): R(I,t) = {1 − exp[−I · S · (ttd)2] · Rmp3}. 
The main parameters derived by this model are the amplitude of the saturated rod response (Rmp3; μV) and the sensitivity parameter (S; m2 cd−1 s−3); td is a brief delay before the onset of the a-wave. Best fitting values of Rmp3 and S were estimated using a χ2 minimization curve-fitting method in Igor Pro (Wavemetrics, Inc., Lake Oswego, OR, USA). Fitting was restricted to the leading edge of the a-wave response. 
Light-adapted responses were recorded after 10 minutes of light adaptation to a background illumination of 30 cds/m2. Intensity response functions were generated in response to incremental flashes ranging in intensity from −1.62 to 2.86 log cds/m2
Function of the RPE was assessed by recording the c-wave, a positive ERG deflection generated in response to a light-induced decline in subretinal [K+]. To avoid rod bleaching, we relied on a single flash (1.36 log cds/m2) as the visual stimulus (as opposed to a sustained stimulus), repeated 6 times at 20-msec intervals to provide signal averaging. C-waves elicited in this fashion achieve maximal amplitudes of approximately 800 msec as reported previously.42,43 The c-wave amplitude was measured as the difference between the dark-adapted ERG apex voltage value at 800 ms post stimulus and the voltage value at the tail of the PIII component.42,43 This approach precluded having to make statements on multiple positive-going peaks elicited from TG mice for which no clear c-wave was apparent between the tail of PIII and up to 2000 msec post flash presentation. 
Statistics
Statistical significance between groups was assessed with repeated-measures ANOVA using the Greenhouse-Geisser correction for sphericity. Post hoc analyses were done between the individual groups and at individual stimulus strengths or time points using the Bonferroni technique for multiple comparisons. GraphPad Prism was used for 1-way ANOVA, linear regression, correlation analyses, and Mann Whitney U test for single variable comparison (GraphPad Prism 5; Graphpad Software, Inc., La Jolla CA, USA). We used SPSS for repeated-measures ANOVA (SPSS, Inc., Chicago, IL, USA). For all comparisons done in this study, significance was set at P < 0.050. 
Results
Transgene Versus Endogenous Elovl4 Ratios Are Stable in our TG Mouse Colony
Ratios of human ELOVL4 transgene (384 bp + 173 bp products) over mouse endogenous Elovl4 levels (557 bp) were 0.74, 0.89, 1.12, and 1.26 at P30, P60, P90, and P270, respectively demonstrating an increasing proportion of ELOVL4 transgene over Elovl4 levels with age and extent of retinal degeneration (Fig. 1A). The ratio of 1.26 at P270 is in agreement with that previously published for this line (ratio = 1.34)38 indicating stable TG expression in our colony over more than 5 years (∼10 generations). 
Figure 1
 
Endogenous ELOVL4 protein not mislocalized in the presence of mutant protein; ELOVL4/Elovl4 ratios stable. (A) Ratios of human ELOVL4 TG over mouse endogenous Elovl4 levels were 0.74, 0.89, 1.12, and 1.26 at P30, P60, P90, and P270, respectively. (B) Western blot with C-terminal antibody recognizing endogenous ELOVL4 protein only. Two bands are seen in retina corresponding to active, glycosylated ELOVL4 at ≈35 kDa (arrowhead) and the inactive, nonglycosylated form at ≈32 kDa. A nonspecific band is detected at ≈17 kDa. Lower, α-tubulin loading control. Right demonstrates downward shift from ≈35 kDa (arrowhead) to ≈30 kDa (arrow) with PNGase F treatment. E, epidermis, positive control; L, liver, negative control. (C) Graphic representation of endogenous ELOVL4 levels. Initially, 22% lower in TG at P30 but not significantly different at P60 or P90. (D) Immunohistochemistry with same antibody showing that in TG, endogenous ELOVL4 is not mislocalized in photoreceptors and, therefore, remains strictly confined to inner segments and perinuclear cytoplasm. OS, outer segments; IS, inner segments. Scale bars: 10 μm.
Figure 1
 
Endogenous ELOVL4 protein not mislocalized in the presence of mutant protein; ELOVL4/Elovl4 ratios stable. (A) Ratios of human ELOVL4 TG over mouse endogenous Elovl4 levels were 0.74, 0.89, 1.12, and 1.26 at P30, P60, P90, and P270, respectively. (B) Western blot with C-terminal antibody recognizing endogenous ELOVL4 protein only. Two bands are seen in retina corresponding to active, glycosylated ELOVL4 at ≈35 kDa (arrowhead) and the inactive, nonglycosylated form at ≈32 kDa. A nonspecific band is detected at ≈17 kDa. Lower, α-tubulin loading control. Right demonstrates downward shift from ≈35 kDa (arrowhead) to ≈30 kDa (arrow) with PNGase F treatment. E, epidermis, positive control; L, liver, negative control. (C) Graphic representation of endogenous ELOVL4 levels. Initially, 22% lower in TG at P30 but not significantly different at P60 or P90. (D) Immunohistochemistry with same antibody showing that in TG, endogenous ELOVL4 is not mislocalized in photoreceptors and, therefore, remains strictly confined to inner segments and perinuclear cytoplasm. OS, outer segments; IS, inner segments. Scale bars: 10 μm.
Endogenous ELOVL4 Protein Levels and Localization are Unaffected in TG
Limited by the absence of an available antibody specific for human mutant ELOVL4 protein, only endogenous protein could be examined by Western blotting and immunohistochemistry. Protein ELOVL4 is known to be glycosylated by a simple oligosaccharide complex at the N-terminus of the protein.7,44 Using a C-terminal antibody that recognizes only endogenous ELOVL4 protein, a product at ≈35 kDa corresponding to glycosylated, active ELOVL4, was observed as well as two additional bands; one band running just below the ELOVL4 band at ≈32 kDa that could be an inactive form of the protein, as well as a band of 17 kDa also present in the negative control tissue (liver) and, therefore, likely nonspecific (Fig. 1B). The ≈32 kDa band has been previously seen in cell culture (Agbaga MP, personal communication, 2014) and inconsistencies in appearance could be due to experimental variations in sample preparation or loading. Digestion with PNGase F to remove N-glycosylation (Fig. 1B, right) led to a small shift in the 35 kDa ELOVL4 band so that it ran below the 32 kDa band. A 35 kDa band was seen in the positive control (epidermis) at ≈×5 retina levels, as well as an additional higher molecular weight band of ≈55 kDa. Wild-type retinas demonstrated a 29% reduction in glycosylated ELOVL4 protein from P30 to P60 after which protein levels remained stable. In TG, endogenous ELOVL4 levels were initially 22% lower (P30) but resembled WT levels at P60 and P90 (Fig. 1C). 
Using the same C-terminal ELOVL4 antibody, we confirmed that in TG photoreceptors, endogenous ELOVL4 protein is not mislocalized in the presence of mutant ELOVL4 protein; as such, the endogenous ELOVL4 protein remains in its normal locations, that is, inner segments and perinuclear cytoplasm (Fig. 1D). 
RPE Monolayer Integrity is Preserved in TG
No disruptions in cell–cell (adherens) junctions were observed in RPE flat mounts immunostained for β-catenin (Fig. 2) at a time point (P60) corresponding to the beginning of rod photoreceptor death in TG.12 Similarly, no notable changes were detected in actin cytoskeleton (phalloidin-stained). 
Figure 2
 
RPE monolayer integrity preserved. RPE flat mount staining at P60 showed no disruptions using β-catenin for cell-cell junctions, phalloidin for actin cytoskeleton, or Hoechst for nuclei. Scale bars: 25 μm.
Figure 2
 
RPE monolayer integrity preserved. RPE flat mount staining at P60 showed no disruptions using β-catenin for cell-cell junctions, phalloidin for actin cytoskeleton, or Hoechst for nuclei. Scale bars: 25 μm.
Earliest Anatomical Defects in TG Are Observed at the Ultrastructural Level
Although outer segment truncation and rod photoreceptor death could not be detected at P30,12 several early signs of pathology in outer segments and RPE cells were already evident at this age. Ultrastructural examination of the outer segment surface with SEM, as well as analysis of cross-sections with TEM, revealed abnormal morphology (Fig. 3). Outer segments in TG had a rough surface appearance compared to those in WT. In contrast to the tightly packed discs of homogeneous form in WT, discs in TG demonstrated an irregular packing pattern with large gaps (Fig. 3, asterisks). Early pathologic changes in RPE cells included vacuolization, distension of basal infoldings, disorganization of apical villi, and abnormal appearance of RPE nuclei as well as other organelles (Figs. 4B–D). 
Figure 3
 
Early photoreceptor defects. Outer segment discs demonstrate a high degree of variability in appearance in TG at P30 with large gaps (asterisks) between discs. SEM, upper panel. Scale bar: 2 μm; 1 μm for close-ups. TEM, lower panel. Scale bar: 500 nm.
Figure 3
 
Early photoreceptor defects. Outer segment discs demonstrate a high degree of variability in appearance in TG at P30 with large gaps (asterisks) between discs. SEM, upper panel. Scale bar: 2 μm; 1 μm for close-ups. TEM, lower panel. Scale bar: 500 nm.
Figure 4
 
Early RPE defects. At P30, RPE appearance in TG ranges from normal to exhibiting features of early pathology (WT, [A]; TG, [BD]). Vacuolization is exacerbated at P90 (WT, [E]; TG, [FH]). Scale bar: 2 μm.
Figure 4
 
Early RPE defects. At P30, RPE appearance in TG ranges from normal to exhibiting features of early pathology (WT, [A]; TG, [BD]). Vacuolization is exacerbated at P90 (WT, [E]; TG, [FH]). Scale bar: 2 μm.
By P90, vacuolization was exacerbated (Figs. 4F–H) and in the most extreme case, large vacuoles filled almost the entire RPE cell. Abnormalities could be seen in other RPE cell organelles, including a less ordered rough endoplasmic reticulum (RER), mitochondria with swollen intercristal spaces (Fig. 5, TG, arrow and asterisks, respectively), and abnormal melanosomes (Fig. 5, TG, arrowhead). Also evident, were lipid and large subretinal lysosomal deposits containing undigested outer segments (Fig. 6; arrow and asterisks in inset, respectively). Retinal pigment epithelium thickening did not reach significance at P90 (TG, 8.71 ± 1.72 μm, n = 4; WT, 6.49 ± 1.51 μm, n = 4). 
Figure 5
 
Other RPE organelles affected. Mitochondria with swollen intercristal spaces (asterisks), a more fragmented RER (arrow), and an example of an abnormal melanosome (arrowhead) in TG at P90. Scale bars: 500 nm.
Figure 5
 
Other RPE organelles affected. Mitochondria with swollen intercristal spaces (asterisks), a more fragmented RER (arrow), and an example of an abnormal melanosome (arrowhead) in TG at P90. Scale bars: 500 nm.
Figure 6
 
Debris accumulated. Lipid (arrow) and subretinal lysosomal deposits, with distinguishable photoreceptor outer segments inside (asterisks in inset), are observed in TG at P90. Scale bars: 2 μm; 500 nm (inset).
Figure 6
 
Debris accumulated. Lipid (arrow) and subretinal lysosomal deposits, with distinguishable photoreceptor outer segments inside (asterisks in inset), are observed in TG at P90. Scale bars: 2 μm; 500 nm (inset).
Finally, although numbers of phagosomes 1.5 hours after light onset were not different at P90 (TG, 4.33 ± 1.56, n = 4; WT, 5.04 ± 1.11, n = 4; per ∼30 μm), TG demonstrated significant (P = 0.040, Mann-Whitney U test, 1-tailed, confidence interval 95%) delays in phagosome movement through the RPE cell (Fig. 7, arrows) with approximately 2-fold more phagosomes still located in the apical half of the RPE when compared to WT (58.33% in TG versus 30.77% in WT). 
Figure 7
 
Phagosome movement delayed. At P90, phagosome movement is delayed (arrows) in TG with approximately 2-fold more phagosomes still found in the apical half of the RPE. Scale bar: 2 μm.
Figure 7
 
Phagosome movement delayed. At P90, phagosome movement is delayed (arrows) in TG with approximately 2-fold more phagosomes still found in the apical half of the RPE. Scale bar: 2 μm.
Subretinal Deposits in TG Immunopositive for Phagocytic Markers
Figure 8 demonstrates the levels and cellular localization of key phagocytosis proteins assessed at P90. These proteins exhibited mild elevation in some animals, ranging from WT to approximately 10% to 20% increased levels. Integrin β5 (involved in photoreceptor outer segment binding and synchronization of phagocytic rhythm) and late stage lysosomal enzyme, cathepsin D (involved in photoreceptor outer segment degradation) demonstrated small increases when compared to WT. Interestingly, these two proteins intensely stained subretinal deposits. Early autophagy marker, vacuole membrane protein 1 (VMP1), involved in the regulation of the initial steps of autophagy through interaction with beclin 1,45 also showed some variability. Levels of the autophagosomal marker LC3B, a lipidated form of microtubule-associated protein 1 light-chain 3 (LC3), and the only protein found on the inner membrane of the autophagosome,46 were likewise affected. With the exception of subretinal deposit staining, no changes were observed in the distribution of these proteins in TG. 
Figure 8
 
Subretinal deposits in TG immuno-positive for phagocytic markers. (A) Retinal pigment epithelium and outer retinal staining at P90. Subretinal deposits were intensely stained for integrin β5 and cathepsin D. With the exception of this subretinal deposit staining, no changes were observed in the distribution of these proteins in TG. OS, outer segments; IS, inner segments; ONL, outer nuclear layer. Scale bars: 20 μm; 5 μm (insets). (B) Western blots with the same antibodies. Protein levels are represented graphically to the right. Retinal pigment epithelium–enriched eyecups and retinas from n = 9 TG, n = 8 WT for the first blot (open red circles, actual blot shown on left) and n = 7 TG, n = 9 WT for the second blot (open black triangles, blot not shown). Transgenic levels are represented as relative to WT and are corrected for α-tubulin loading control. Dotted line indicates ratio of 1.00 relative to WT. Proteins ranged from WT levels to mildly elevated. E, eyecup with neural retina removed; R, retina; i, immature; m, mature. Asterisk in LC3B denotes possible dimer.
Figure 8
 
Subretinal deposits in TG immuno-positive for phagocytic markers. (A) Retinal pigment epithelium and outer retinal staining at P90. Subretinal deposits were intensely stained for integrin β5 and cathepsin D. With the exception of this subretinal deposit staining, no changes were observed in the distribution of these proteins in TG. OS, outer segments; IS, inner segments; ONL, outer nuclear layer. Scale bars: 20 μm; 5 μm (insets). (B) Western blots with the same antibodies. Protein levels are represented graphically to the right. Retinal pigment epithelium–enriched eyecups and retinas from n = 9 TG, n = 8 WT for the first blot (open red circles, actual blot shown on left) and n = 7 TG, n = 9 WT for the second blot (open black triangles, blot not shown). Transgenic levels are represented as relative to WT and are corrected for α-tubulin loading control. Dotted line indicates ratio of 1.00 relative to WT. Proteins ranged from WT levels to mildly elevated. E, eyecup with neural retina removed; R, retina; i, immature; m, mature. Asterisk in LC3B denotes possible dimer.
Early Functional Deficits in TG Are Detected in Rods and RPE
Figure 9 summarizes findings on the functional impact of the pathophysiological changes reported above on photoreceptors and RPE. Figure 9A shows representative ERG traces recorded under dark adaptation, with emphasis on stimulus strengths eliciting a-waves (photoreceptoral function). Figure 9B shows the corresponding intensity-response curves (for all intensities tested) for the dark-adapted mixed a-wave (top graph) and b-wave (middle graph). Age-matched comparisons showed reductions in TG versus WT starting at P30 for a-wave but not until P90 for b-wave. The lack of a-wave amplitude differences under photopic adaptation (purely cone-driven photoreceptor function) at P30 and P90 (Fig. 9B, lower graph) supports that mixed a-wave amplitude reductions reflect rod dysfunction already occurring at P30. 
Figure 9
 
Early functional deficits in rods and RPE. (A) Representative examples of dark-adapted ERG traces at P30 and P90. (B) Electroretinogram intensity-response functions for WT and TG at P30 and P90: dark-adapted a- and b-wave amplitudes (top and middle panel); light-adapted a-wave amplitudes (bottom panel). (C) Ensemble fit of the P3 model to the leading edges of the dark-adapted ERG a-waves. Representative ERG recordings (solid lines) and best-fit curves (dashed lines) elicited by white flash stimuli of 0.88 to 2.86 scot log cd s/m2 (top to bottom). (D) Phototransduction activation parameters. Average maximum response amplitude (Rmp3, top) and sensitivity (S) parameters (bottom). (E) Rhodopsin protein levels in retina. Levels in TG match WT at P30 and P60; at P90 they are ≈80% of WT levels. Antibody detects monomeric ≈35 kDa and multimeric (bracket) forms of rhodopsin (α-tubulin loading control). (F) Electroretinogram c-wave amplitudes as a functional correlate of RPE integrity. Left: Representative examples of WT (top) and TG (bottom) at P90; c-wave amplitudes = 182 and 121 μV, for WT and TG, respectively, corresponding to the amplitude at 800 ms minus that at the tail of PIII, which consistently occurred between 200 to 300 ms. Right: Average c-wave amplitudes for WT and TG at P30 and P90. Triangles indicate PIII component negative peaks. Asterisks indicate statistical significance between WT and TG (P < 0.050). Error bars: SEM.
Figure 9
 
Early functional deficits in rods and RPE. (A) Representative examples of dark-adapted ERG traces at P30 and P90. (B) Electroretinogram intensity-response functions for WT and TG at P30 and P90: dark-adapted a- and b-wave amplitudes (top and middle panel); light-adapted a-wave amplitudes (bottom panel). (C) Ensemble fit of the P3 model to the leading edges of the dark-adapted ERG a-waves. Representative ERG recordings (solid lines) and best-fit curves (dashed lines) elicited by white flash stimuli of 0.88 to 2.86 scot log cd s/m2 (top to bottom). (D) Phototransduction activation parameters. Average maximum response amplitude (Rmp3, top) and sensitivity (S) parameters (bottom). (E) Rhodopsin protein levels in retina. Levels in TG match WT at P30 and P60; at P90 they are ≈80% of WT levels. Antibody detects monomeric ≈35 kDa and multimeric (bracket) forms of rhodopsin (α-tubulin loading control). (F) Electroretinogram c-wave amplitudes as a functional correlate of RPE integrity. Left: Representative examples of WT (top) and TG (bottom) at P90; c-wave amplitudes = 182 and 121 μV, for WT and TG, respectively, corresponding to the amplitude at 800 ms minus that at the tail of PIII, which consistently occurred between 200 to 300 ms. Right: Average c-wave amplitudes for WT and TG at P30 and P90. Triangles indicate PIII component negative peaks. Asterisks indicate statistical significance between WT and TG (P < 0.050). Error bars: SEM.
To further characterize photoreceptor function, we quantified activation parameters of rod phototransduction (Rmp3 and S) by fitting dark-adapted a-wave leading edges.40 Figure 9C shows representative examples of leading edge curve fitting. At P30, the maximum saturated rod photoresponse (Rmp3) was reduced in TG (−264.2 ± 48.3 μV) compared to WT (−347.6 ± 70.4 μV), while the sensitivity parameter (S) was unchanged (25.8 ± 5.5 and 25.7 ± 3.2 scot cd−1s−1m2 for TG and WT, respectively; Fig. 9D). Similarly, ERG recordings at P90 revealed reduced Rmp3 amplitude in TG (−210.1 ± 45.6 μV) compared to WT (−267.0 ± 68.4 μV), while sensitivity was unchanged (24.2 ± 5.3 and 23.5 ± 4.9 scot cd−1 s−1 m2, for TG and WT, respectively; Fig. 9D). 
Since rhodopsin is a key player in rod phototransduction activation, we undertook Western blotting to assess retina protein levels (Fig. 9E). Rhodopsin monoclonal antibody 4D2 recognized both monomeric approximately 35 kDa and multimeric forms (Fig. 9E, bracket). Levels of rhodopsin in TG retina at P30 and P60 were similar to those of age-matched WT (Fig. 9E, graph on right). In TG retinas at P90, rhodopsin levels were approximately 80% of those in WT, in agreement with a photoreceptor population in TG reduced to ≈70% the WT value at this age.20 
The functional correlate of RPE integrity was obtained by recording the c-wave component of the ERG (Fig. 9F, representative c-wave traces are shown on the left). Significant c-wave amplitude reductions were observed at P90 in TG (104.1 ± 32.3 μV) compared to WT (151.3 ± 53.4 μV). 
Discussion
Before photoreceptor death, this study identified pathology in rod photoreceptors and RPE. The rationale for choosing P30 and P90 as time points was based on our previous work12 demonstrating that anatomical structure and fatty acid composition are comparable to WT at P30. Rod loss begins at P60, exhibiting a central to peripheral gradient, and by P90, approximately 30% loss is observed in the central retina. 
Stable ratios of mutant ELOVL4 to endogenous Elovl4 over more than 10 generations of breeding allowed us to make objective correlations between former12,20,38 and present work. The proportion of mutant ELOVL4 over endogenous Elovl4 increases as retinal degeneration progresses in TG. These findings are in agreement with those of Karan et al.,11 which showed decreasing amounts of endogenous Elovl4 in the presence of increasing transgene levels (inducing larger degeneration patterns) in lines TG1 through TG3. However, no significant differences were found in endogenous ELOVL4 protein levels in these TG mice. Endogenous protein levels in TG were initially (P30) 22% lower than normal but stabilized close to WT values at the start of degeneration (P60).12 Mandal et al.47 also reported no reductions in endogenous ELOVL4 levels of similarly aged TG2 retinas. Together, these data suggest that only the expression of mutant ELOVL4 transcript and related protein increases as degeneration progresses in TG. It is conceivable that this altered retinal environment may progressively affect the activity of the IRBP (human interphotoreceptor retinoid-binding protein) gene promoter that drives transgene expression in our model.11 Of note, methylation state and external culture conditions have been found to modulate the expression of the IRBP promoter in vitro.48 
While in principle, human STGD3 could be modeled using a knock-in approach, mice generated in this manner49 displayed a phenotype incompatible with STGD3: primary defect specific to s-opsin containing cones accompanied by protracted cell death (10 months) selective to far peripheral retina. Other models, such as Elovl4 conditional knockouts (cKO)10,17 with almost complete depletion of VLC-PUFAs, displayed no distinctive phenotype, which suggests that retinal degeneration in our TG model has to be due to abnormal accumulation of the mutant protein. A direct example of pathologic accumulation of proteins underlying photoreceptor cell death occurs in Bardet-Biedl syndrome, a case in which nonouter segment proteins accumulate in outer segments due to mutations preventing their export (Seo, et al. IOVS 2010;56:ARVO E-Abstract 4644). Recent work in transgenic X. laevis expressing murine hemagglutinin-tagged mutant ELOVL422 demonstrated a similar phenomenon. A large fraction of the mutant protein was misrouted to outer segments, whereas the endogenous protein remained in inner segments. Accordingly, ELOVL4 endogenous protein was not mislocalized in the presence of human mutant protein in TG mouse photoreceptors. Difficulties in generating an antibody against the mutant protein specifically have to date hindered abilities to determine if mutant protein is misrouted to outer segments in vivo. New methodologies, such as phage display libraries (used by our lab) generated two novel antibodies, both of which failed specificity tests for the human mutant ELOVL4 protein. 
Although introduced in all photoreceptors, the presence of the human mutant ELOVL4 protein impacts cones much later in degeneration (as in human STGD3), when at least half of the rod complement is lost.20 The distinctive vulnerability of rods (comprising ∼98% of the mouse retina, compared with an average close to 95% in humans) is due, at least in part, to a precarious combination of high disc DHA content (the most oxidizable fatty acid; required for disc membrane fluidity) and extremely high oxygen levels (required for passive diffusion in an avascular environment),50 both of which are modulated by the constant turnover of their outer segment discs. Rod function (as opposed to cone) was first affected in TG (P30). According to earlier studies,51,52 a decline in the rod a-wave amplitude and in saturated response Rmp3, with no change in either outer segment length or cell number, points to a dysfunction in the biochemical phototransduction activation steps leading to the closure of cGMP gated ion channels in outer segments. Specific aspects of these steps can be quantified by mathematically fitting the a-wave leading edge (elicited by a single high energy white flash) with equations that take into account the kinetics of these distinct biochemical steps.53 Thus, our results of reduced Rmp3 values are indicative of defective biochemical processes in photoreceptors, before their death. 
Electron microscopic examination of the photoreceptor outer segment surface in TG revealed large gaps in disc spacing at P30. Previous work by Karan et al.11 described ultrastructural disorganization of outer segment disk membranes at 2 months of age in TG3 with the highest levels of transgene expression, by which time more than half of the photoreceptor complement had already been lost. Raz-Prag et al.54 also reported “unusual gaps” in their Elovl4+/− knockout mouse model, albeit at a much later age (16 to 22 months of age). Similarly, in homozygous transgenic STGD3 mice completely lacking C28-C36 acyl phosphatidylcholine lipids, Kedzierski et al. (IOVS 2010;51:ARVO E-Abstract 2939) demonstrated disruptions in the organization of distal disks affecting rod and cone function. 
Dysfunction of RPE (indicated by dark-adapted c-wave amplitude) was not detected until P90. Pathologic changes in the apical RPE have been shown to result in reduced ERG c-wave amplitudes.55 Kim et al.56 reported RPE apical microvilli defects and reduced ERG c-wave amplitudes as early as 1 month of age in Ahr/− (aryl hydrocarbon receptor, AHR) mice. Aryl hydrocarbon receptor serves a protective role essential for toxin metabolism and cellular debris clearance. Reduced in aging RPE, its deficiency in mice causes an AMD-like pathology.57 
While RPE monolayer integrity was preserved, ultrastructural examination revealed early signs of RPE cell toxicity in TG; including vacuolization, distension of basal infoldings, disorganized apical villi, and dysmorphic organelles (E. Nandrot, personal communication, 2014). Examples of other models displaying these features include Elovl4 5-bp deletion knock-in mice (but at advanced age; 22 months),49 RPE-specific Sod2 deletion mice,58 NUC1 mutant rats (with a spontaneous Cryba1 mutation),59 and Cryba1 cKO mice.60 Zigler et al.59 demonstrated that not only is Cryba1 expressed in the RPE but also that CRYBA1/βA3/A1-crystallin has an essential role in the degradation of outer segment discs that have been internalized in phagosomes. This protein regulates endolysosomal acidification and Cryba1 cKO mice showed decreased V-ATPase activity, increased lysosomal pH, and decreased cathepsin D activity.60 With lysosomal dysregulation impairing autophagy and phagocytosis, Cryba1 cKO mice not only share ultrastructural findings with TG, but interestingly, fundus images from Cryba1 cKO mice also closely resemble those of TG.20 
Mitochondrial damage in TG was manifested as a focal loss of cristae, a feature of aging, exacerbated in AMD.61 Using human AMD samples, Terluk et al.62 found that RPE mitochondria suffer early insults in AMD and proposed that these could be attributed to homeostasis defects, including autophagy, which would be responsible for the degradation of damaged mitochondria. In vitro studies using human RPE cells showed that impairment of autophagy, with inhibitor 3-methyladenine, in the presence of A2E also led to an increased number of abnormal mitochondria displaying loss of cristae.63 Preet et al.64 reported mitochondrial swelling and RER reductions in the bipolar cells of alloxin-induced diabetic rat retinas. Rough endoplasmic reticulum fragmentation, observed in the present study, could be due to ER stress, although as discussed previously only one ER stress–induced gene was found to be slightly upregulated in TG eyecups (with retina) at a later stage of degeneration (9 months).12 We cannot rule out the possibility that more changes may have been detected with RPE alone and/or at different ages. 
The presence of lipid deposits in TG at P90 is in agreement with our previous findings that all-trans A2E and iso-A2E accumulate in TG and are 2.2-fold that of WT levels at this same age.38 In a model for the role of autophagy in AMD, it was proposed that normally, damaged proteins trigger autophagy, leading to lysosomal degradation for recycling. However, in the disease state this self-renewal is less efficient and these proteins, as well as reactive metabolites, such as A2E, accumulate.11,36,37,65 More recently, it has been demonstrated that bisretinoids trap cholesterol and bis(monoacylglycerol) phosphate within the RPE, ultimately leading to tubulin acetylation and inhibition of autophagosome motility.66 Whether A2E-induced inhibition of autophagosome trafficking has a role in STGD3 remains to be elucidated. 
The approximately 2-fold higher proportion of phagosomes retained in the apical half of the RPE 1.5 hours after light onset do support that trafficking delays may have a role in pathology. Using the TG2 ELOVL4 mouse line11 Esteve-Rudd et al.67 observed increased amounts of undigested photoreceptor outer segments in the cytoplasm of RPE cells 2.5 hours after light onset. When RPE cells in primary culture were presented with the mutant photoreceptor outer segments, binding and ingestion were normal; however, rates of degradation were impaired. In our study, the presence of undigested photoreceptor outer segments in subretinal deposits (immunopositive for phagocytic proteins) in vivo also might be suggestive of degradation impediments. 
In summary, taken together all of the above-mentioned RPE ultrastructural changes, phagosome retention in the apical RPE, outer segment–containing subretinal deposits, along with rod outer segment trunctation,12 are suggestive of a photoreceptor-induced RPE phagocytic defect. Dysregulated autophagy has been involved in the pathology of various retinopathies including AMD, retinitis pigmentosa, and Leber's congenital amaurosis (for a review on autophagy see the report by Bo et al.28). In mouse photoreceptors, the presence of mutant ELOVL4 protein affects outer segment structure (whether mislocalized to this location or not), which may lead to dysregulated autophagy and impeded disc recycling. 
Conclusions
Stargardt's disease (STGD), a recessive form of early onset macular degeneration caused by mutations in the ABCR gene,68 has been defined primarily as a defect of the RPE.69 Rim protein (RmP) is an ABC transporter present exclusively in outer segments70 and yet, mutations in RmP ultimately lead to what is described by Weng et al.71 as “rod-mediated ‘poisoning' of the macular RPE.” Our results indicated that Stargardt-like disease (STGD3), an autosomal dominant form of juvenile maculopathy caused by mutations in the ELOVL4 gene, also involves rod-mediated RPE cytotoxicity. Exactly how mutant ELOVL4 leads to alterations in disc membrane ultrastructure and/or biochemistry has yet to be determined. 
Acknowledgments
The authors thank Xuejun Sun and Geraldine Barron at the Cross Cancer Institute Cell Imaging Facility for technical assistance with the Zeiss LSM710 confocal microscope, and Emeline Nandrot for her comments on transmission electron microscopy findings. The authors also acknowledge Mandy Hong for ERG recordings on TG and age-matched WT littermates, and Frederic Gaillard and Camille Dejos for proofreading. 
Supported by Canadian Institutes of Health Research (CIHR) Grant (#79278); Alberta Innovates Health Solution Collaborative Research Innovation Opportunity (AIHS-CRIO; #201200139); The Olive Young Foundation; an AIHS Graduate Student Research Assistantship (#201201795; ID); Senior Scholarship of the Alberta Heritage Foundation for Medical Research (#200800242; YS). 
Disclosure: S. Kuny, None; W.J. Cho, None; I.S. Dimopoulos, None; Y. Sauvé, None 
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Figure 1
 
Endogenous ELOVL4 protein not mislocalized in the presence of mutant protein; ELOVL4/Elovl4 ratios stable. (A) Ratios of human ELOVL4 TG over mouse endogenous Elovl4 levels were 0.74, 0.89, 1.12, and 1.26 at P30, P60, P90, and P270, respectively. (B) Western blot with C-terminal antibody recognizing endogenous ELOVL4 protein only. Two bands are seen in retina corresponding to active, glycosylated ELOVL4 at ≈35 kDa (arrowhead) and the inactive, nonglycosylated form at ≈32 kDa. A nonspecific band is detected at ≈17 kDa. Lower, α-tubulin loading control. Right demonstrates downward shift from ≈35 kDa (arrowhead) to ≈30 kDa (arrow) with PNGase F treatment. E, epidermis, positive control; L, liver, negative control. (C) Graphic representation of endogenous ELOVL4 levels. Initially, 22% lower in TG at P30 but not significantly different at P60 or P90. (D) Immunohistochemistry with same antibody showing that in TG, endogenous ELOVL4 is not mislocalized in photoreceptors and, therefore, remains strictly confined to inner segments and perinuclear cytoplasm. OS, outer segments; IS, inner segments. Scale bars: 10 μm.
Figure 1
 
Endogenous ELOVL4 protein not mislocalized in the presence of mutant protein; ELOVL4/Elovl4 ratios stable. (A) Ratios of human ELOVL4 TG over mouse endogenous Elovl4 levels were 0.74, 0.89, 1.12, and 1.26 at P30, P60, P90, and P270, respectively. (B) Western blot with C-terminal antibody recognizing endogenous ELOVL4 protein only. Two bands are seen in retina corresponding to active, glycosylated ELOVL4 at ≈35 kDa (arrowhead) and the inactive, nonglycosylated form at ≈32 kDa. A nonspecific band is detected at ≈17 kDa. Lower, α-tubulin loading control. Right demonstrates downward shift from ≈35 kDa (arrowhead) to ≈30 kDa (arrow) with PNGase F treatment. E, epidermis, positive control; L, liver, negative control. (C) Graphic representation of endogenous ELOVL4 levels. Initially, 22% lower in TG at P30 but not significantly different at P60 or P90. (D) Immunohistochemistry with same antibody showing that in TG, endogenous ELOVL4 is not mislocalized in photoreceptors and, therefore, remains strictly confined to inner segments and perinuclear cytoplasm. OS, outer segments; IS, inner segments. Scale bars: 10 μm.
Figure 2
 
RPE monolayer integrity preserved. RPE flat mount staining at P60 showed no disruptions using β-catenin for cell-cell junctions, phalloidin for actin cytoskeleton, or Hoechst for nuclei. Scale bars: 25 μm.
Figure 2
 
RPE monolayer integrity preserved. RPE flat mount staining at P60 showed no disruptions using β-catenin for cell-cell junctions, phalloidin for actin cytoskeleton, or Hoechst for nuclei. Scale bars: 25 μm.
Figure 3
 
Early photoreceptor defects. Outer segment discs demonstrate a high degree of variability in appearance in TG at P30 with large gaps (asterisks) between discs. SEM, upper panel. Scale bar: 2 μm; 1 μm for close-ups. TEM, lower panel. Scale bar: 500 nm.
Figure 3
 
Early photoreceptor defects. Outer segment discs demonstrate a high degree of variability in appearance in TG at P30 with large gaps (asterisks) between discs. SEM, upper panel. Scale bar: 2 μm; 1 μm for close-ups. TEM, lower panel. Scale bar: 500 nm.
Figure 4
 
Early RPE defects. At P30, RPE appearance in TG ranges from normal to exhibiting features of early pathology (WT, [A]; TG, [BD]). Vacuolization is exacerbated at P90 (WT, [E]; TG, [FH]). Scale bar: 2 μm.
Figure 4
 
Early RPE defects. At P30, RPE appearance in TG ranges from normal to exhibiting features of early pathology (WT, [A]; TG, [BD]). Vacuolization is exacerbated at P90 (WT, [E]; TG, [FH]). Scale bar: 2 μm.
Figure 5
 
Other RPE organelles affected. Mitochondria with swollen intercristal spaces (asterisks), a more fragmented RER (arrow), and an example of an abnormal melanosome (arrowhead) in TG at P90. Scale bars: 500 nm.
Figure 5
 
Other RPE organelles affected. Mitochondria with swollen intercristal spaces (asterisks), a more fragmented RER (arrow), and an example of an abnormal melanosome (arrowhead) in TG at P90. Scale bars: 500 nm.
Figure 6
 
Debris accumulated. Lipid (arrow) and subretinal lysosomal deposits, with distinguishable photoreceptor outer segments inside (asterisks in inset), are observed in TG at P90. Scale bars: 2 μm; 500 nm (inset).
Figure 6
 
Debris accumulated. Lipid (arrow) and subretinal lysosomal deposits, with distinguishable photoreceptor outer segments inside (asterisks in inset), are observed in TG at P90. Scale bars: 2 μm; 500 nm (inset).
Figure 7
 
Phagosome movement delayed. At P90, phagosome movement is delayed (arrows) in TG with approximately 2-fold more phagosomes still found in the apical half of the RPE. Scale bar: 2 μm.
Figure 7
 
Phagosome movement delayed. At P90, phagosome movement is delayed (arrows) in TG with approximately 2-fold more phagosomes still found in the apical half of the RPE. Scale bar: 2 μm.
Figure 8
 
Subretinal deposits in TG immuno-positive for phagocytic markers. (A) Retinal pigment epithelium and outer retinal staining at P90. Subretinal deposits were intensely stained for integrin β5 and cathepsin D. With the exception of this subretinal deposit staining, no changes were observed in the distribution of these proteins in TG. OS, outer segments; IS, inner segments; ONL, outer nuclear layer. Scale bars: 20 μm; 5 μm (insets). (B) Western blots with the same antibodies. Protein levels are represented graphically to the right. Retinal pigment epithelium–enriched eyecups and retinas from n = 9 TG, n = 8 WT for the first blot (open red circles, actual blot shown on left) and n = 7 TG, n = 9 WT for the second blot (open black triangles, blot not shown). Transgenic levels are represented as relative to WT and are corrected for α-tubulin loading control. Dotted line indicates ratio of 1.00 relative to WT. Proteins ranged from WT levels to mildly elevated. E, eyecup with neural retina removed; R, retina; i, immature; m, mature. Asterisk in LC3B denotes possible dimer.
Figure 8
 
Subretinal deposits in TG immuno-positive for phagocytic markers. (A) Retinal pigment epithelium and outer retinal staining at P90. Subretinal deposits were intensely stained for integrin β5 and cathepsin D. With the exception of this subretinal deposit staining, no changes were observed in the distribution of these proteins in TG. OS, outer segments; IS, inner segments; ONL, outer nuclear layer. Scale bars: 20 μm; 5 μm (insets). (B) Western blots with the same antibodies. Protein levels are represented graphically to the right. Retinal pigment epithelium–enriched eyecups and retinas from n = 9 TG, n = 8 WT for the first blot (open red circles, actual blot shown on left) and n = 7 TG, n = 9 WT for the second blot (open black triangles, blot not shown). Transgenic levels are represented as relative to WT and are corrected for α-tubulin loading control. Dotted line indicates ratio of 1.00 relative to WT. Proteins ranged from WT levels to mildly elevated. E, eyecup with neural retina removed; R, retina; i, immature; m, mature. Asterisk in LC3B denotes possible dimer.
Figure 9
 
Early functional deficits in rods and RPE. (A) Representative examples of dark-adapted ERG traces at P30 and P90. (B) Electroretinogram intensity-response functions for WT and TG at P30 and P90: dark-adapted a- and b-wave amplitudes (top and middle panel); light-adapted a-wave amplitudes (bottom panel). (C) Ensemble fit of the P3 model to the leading edges of the dark-adapted ERG a-waves. Representative ERG recordings (solid lines) and best-fit curves (dashed lines) elicited by white flash stimuli of 0.88 to 2.86 scot log cd s/m2 (top to bottom). (D) Phototransduction activation parameters. Average maximum response amplitude (Rmp3, top) and sensitivity (S) parameters (bottom). (E) Rhodopsin protein levels in retina. Levels in TG match WT at P30 and P60; at P90 they are ≈80% of WT levels. Antibody detects monomeric ≈35 kDa and multimeric (bracket) forms of rhodopsin (α-tubulin loading control). (F) Electroretinogram c-wave amplitudes as a functional correlate of RPE integrity. Left: Representative examples of WT (top) and TG (bottom) at P90; c-wave amplitudes = 182 and 121 μV, for WT and TG, respectively, corresponding to the amplitude at 800 ms minus that at the tail of PIII, which consistently occurred between 200 to 300 ms. Right: Average c-wave amplitudes for WT and TG at P30 and P90. Triangles indicate PIII component negative peaks. Asterisks indicate statistical significance between WT and TG (P < 0.050). Error bars: SEM.
Figure 9
 
Early functional deficits in rods and RPE. (A) Representative examples of dark-adapted ERG traces at P30 and P90. (B) Electroretinogram intensity-response functions for WT and TG at P30 and P90: dark-adapted a- and b-wave amplitudes (top and middle panel); light-adapted a-wave amplitudes (bottom panel). (C) Ensemble fit of the P3 model to the leading edges of the dark-adapted ERG a-waves. Representative ERG recordings (solid lines) and best-fit curves (dashed lines) elicited by white flash stimuli of 0.88 to 2.86 scot log cd s/m2 (top to bottom). (D) Phototransduction activation parameters. Average maximum response amplitude (Rmp3, top) and sensitivity (S) parameters (bottom). (E) Rhodopsin protein levels in retina. Levels in TG match WT at P30 and P60; at P90 they are ≈80% of WT levels. Antibody detects monomeric ≈35 kDa and multimeric (bracket) forms of rhodopsin (α-tubulin loading control). (F) Electroretinogram c-wave amplitudes as a functional correlate of RPE integrity. Left: Representative examples of WT (top) and TG (bottom) at P90; c-wave amplitudes = 182 and 121 μV, for WT and TG, respectively, corresponding to the amplitude at 800 ms minus that at the tail of PIII, which consistently occurred between 200 to 300 ms. Right: Average c-wave amplitudes for WT and TG at P30 and P90. Triangles indicate PIII component negative peaks. Asterisks indicate statistical significance between WT and TG (P < 0.050). Error bars: SEM.
Table
 
List and Details of Antibodies
Table
 
List and Details of Antibodies
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