Early AMD-like defects in the RPE and retinal degeneration in aged mice with RPE-specific deletion of Atg5 or Atg7

Abstract

Purpose: To examine the effects of autophagy deficiency induced by RPE-specific deletion of Atg5 or Atg7 in mice as a function of age.

Methods: Conditional knockout mice with a floxed allele of Atg5 or Atg7 were crossed with inducible VMD2-rtTA/Cre transgenic mice. VMD2-directed RPE-specific Cre recombinase expression was induced with doxycycline feeding in the resulting mice. Cre-mediated deletion of floxed Atg5 or Atg7 resulted in RPE-specific inactivation of the Atg5 or Atg7 gene. Plastic and thin retinal sections were analyzed with light and electron microscopy for histological changes. Photoreceptor outer segment (POS) thickness in plastic sections was measured using the Adobe Photoshop CS4 extended ruler tool. Autophagic adaptor p62/SQSTM1 and markers for oxidatively damaged lipids, proteins, and DNA were examined with immunofluorescence staining of cryosections. Fluorescence signals were quantified using Image J software.

Results: Accumulation of p62/SQSTM1 reflecting autophagy deficiency was observed in the RPE of the Atg5^ΔRPE and Atg7^ΔRPE mice. 3-nitrotyrosine, advanced glycation end products (AGEs), and 8-hydroxy-2'-deoxyguanosine (8-OHdG), markers for oxidatively damaged proteins and DNA, were also found to accumulate in the RPE of these mice. We observed retinal degeneration in 35% of the Atg5^ΔRPE mice and 45% of the Atg7^ΔRPE mice at 8 to 24 months old. Degeneration severity and the number of mice with degeneration increased with age. The mean POS thickness of these mice was 25 µm at 8-12 months, 15 µm at 13-18 months, and 3 µm at 19-24 months, compared to 35 µm, 30 µm, and 24 µm in the wild-type mice, respectively. Early age-related macular degeneration (AMD)-like RPE defects were found in all the Atg5^ΔRPE and Atg7^ΔRPE mice 13 months old or older, including vacuoles, uneven RPE thickness, diminished basal infoldings, RPE hypertrophy/hypotrophy, pigmentary irregularities, and necrosis. The severity of the RPE defects increased with age and in the mice with retinal degeneration. RPE atrophy and choroidal neovascularization (CNV) were occasionally observed in the Atg5^ΔRPE and Atg7^ΔRPE mice with advanced age.

Conclusions: Autophagy deficiency induced by RPE-specific deletion of Atg5 or Atg7 predisposes but does not necessarily drive the development of AMD-like phenotypes or retinal degeneration.

Figure 1

RPE-specific deletion of Atg5 or Atg7 and p62/SQSTM1 accumulation in the RPE of Atg5^ΔRPE and Atg7^ΔRPE mice. A: Reverse transcription polymerase chain reaction (RT-PCR) showed that Atg5 or Atg7 was not expressed in RPE cells isolated from Atg5^ΔRPE or Atg7^ΔRPE mice but was expressed in the neuroretina isolated from these mice. RT–PCR using Efemp1 primers confirmed the integrity of the RNA from all the samples. B: Frozen sections from 8-month-old wild-type, Atg5^ΔRPE, and Atg7^ΔRPE mice were stained with an antibody (green signal) against p62/SQSTM1. Note the bright green staining in the RPE of the Atg5^ΔRPE and Atg7^ΔRPE mice. The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; blue signal). C: The p62 staining in the RPE was quantified using Image J software. n = 3 mice per genotype. Error bars indicate the mean ± standard deviation (SD). WT, wild-type; ONL, outer nuclear layer; IS, photoreceptor inner segment; OS, photoreceptor outer segment.

Figure 2

Increased levels of oxidized proteins and DNA in the RPE of aged Atg5^ΔRPE mice. Frozen sections from 8-month-old wild-type (+/+; A, C, E) and Atg5^ΔRPE (Atg5−/−; B, D, F) mice were stained with antibodies (green signal) against 8-hydroxy-2’-deoxyguanosine (8-OHdG; A and B), 3-nitrotyrosine (C and D), or advanced glycation end products (AGEs; E and F). Note the bright green punctate staining in the RPE of the Atg5^ΔRPE mice. The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; blue signal).

Figure 3

Increased levels of oxidized proteins and DNA in the RPE of aged Atg7^ΔRPE mice. Frozen sections from 8-month-old wild-type (+/+; A, C, E) and Atg7^ΔRPE (Atg7−/−; B, D, F) mice were stained with antibodies (green signals) against 8-hydroxy-2’-deoxyguanosine (8-OHdG; A and B), 3-nitrotyrosine (C and D), or advanced glycation end products (AGEs; E and F). Note the bright green staining in the RPE of Atg7^ΔRPE mice. The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; blue signal).

Figure 4

Quantification of 8-OHdG, 3-nitrotyrosine, and AGEs in the RPE of Atg5^ΔRPE and Atg7^ΔRPE mice. The immunofluorescence staining of 8-OHdG, 3-nitrotyrosine, and advanced glycation end products (AGEs) in the RPE was quantified using Image J software. n = 3 mice per genotype. Error bars indicate the mean ± standard deviation (SD). WT, wild-type control.

Figure 5

Retinal degeneration in aged Atg5^ΔRPE and Atg7^ΔRPE mice. Representative images of toluidine blue–stained sections showing the retina layers of 17-month-old wild-type, Atg5^ΔRPE, and Atg7^ΔRPE mice. Note the retina layers of the Atg5^ΔRPE and Atg7^ΔRPE mice without retinal degeneration (no rd) were similar to those of the wild-type mice but the outer plexiform layer (OPL), ONL, IS, and OS were diminished in the Atg5^ΔRPE and Atg7^ΔRPE mice with retinal degeneration (rd). The image for the Atg7^ΔRPE mice without retinal degeneration was from a pigmented background, and the other images were from albino mice. Arrows indicate engorged RPE cells. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer.

Figure 6

POS thickness in Atg5^ΔRPE and Atg7^ΔRPE mice with retinal degeneration. The thickness of the photoreceptor outer segment (POS) was measured in the retinas of the mice at 8–12, 13–18, and 19–24 months of age. Each graph represents data from 1 µm toluidine blue–stained sections of three mice (n = 3) per genotype and age group. On each section, nine data points with 250 µm intervals starting from the optic nerve head were measured, and each data point was the mean of three measurements. The value represents the mean of the data points from three mice ± standard deviation (SD). wt, wild-type control mice.

Figure 7

Thinned RPE in aged Atg5^ΔRPE and Atg7^ΔRPE mice. Representative electronic micrographs showing the RPE region of the 17-month-old wild-type (+/+), Atg5^ΔRPE (Atg5−/−), and Atg7^ΔRPE (Atg7−/−) mice. Note that the RPE thicknesses of the Atg5^ΔRPE and Atg7^ΔRPE mice are approximately one third to one half that of the wild-type mice. The wild-type and Atg5^ΔRPE mice shown were pigmented, and the Atg7^ΔRPE mouse was albino. Arrow indicates a hypotrophic RPE cell. Scale bar = 10 µm.

Figure 8

Diminished RPE basal infoldings in aged Atg5^ΔRPE and Atg7^ΔRPE mice. Representative electronic micrographs showing the RPE basal infolding area of 17-month-old wild-type (+/+), Atg5^ΔRPE (Atg5−/−), and Atg7^ΔRPE (Atg7−/−) mice. Note the scarce basal infoldings (arrow) in the Atg5^ΔRPE and Atg7^ΔRPE mice. Scale bar = 2 µm.

Figure 9

CNV in aged Atg5^ΔRPE and Atg7^ΔRPE mice. Representative electronic micrographs showing the RPE area of 17-month-old wild-type (+/+), Atg5^ΔRPE (Atg5−/−), and Atg7^ΔRPE (Atg7−/−) mice. Note the blood vessels (thin arrows) in the RPE layer of the Atg5^ΔRPE or Atg7^ΔRPE mice. Thick arrow indicates a capillary endothelial cell (Atg5−/−) or a platelet (Atg7−/−). Scale bar = 5 µm.