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(Delta RPE) and Atg7(Delta RP)E 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(Delta RPE) mice and 45% of the Atg7.RPE 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 mu m at 8-12 months, 15 mu m at 13-18 months, and 3 mu m at 19-24 months, compared to 35 mu m, 30 mu m, and 24 aem in the wildtype mice, respectively. Early age-related macular degeneration (AMD)-like RPE defects were found in all the Atg5(Delta RPE) and Atg7.RPE 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(Delta RPE) and Atg7(Delta RPE) 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.