Tyrosinase-Cre-Mediated Deletion of the Autophagy Gene Atg7 Leads to Accumulation of the RPE65 Variant M450 in the Retinal Pigment Epithelium of C57BL/6 Mice

Abstract

Targeted gene knockout mouse models have helped to identify roles of autophagy in many tissues. Here, we investigated the retinal pigment epithelium (RPE) of Atg7f/f Tyr-Cre mice (on a C57BL/6 background), in which Cre recombinase is expressed under the control of the tyrosinase promoter to delete the autophagy gene Atg7. In line with pigment cell-directed blockade of autophagy, the RPE and the melanocytes of the choroid showed strong accumulation of the autophagy adaptor and substrate, sequestosome 1 (Sqstm1)/p62, relative to the levels in control mice. Immunofluorescence and Western blot analysis demonstrated that the RPE, but not the choroid melanocytes, of Atg7f/f Tyr-Cre mice also had strongly increased levels of retinoid isomerohydrolase RPE65, a pivotal enzyme for the maintenance of visual perception. In contrast to Sqstm1, genes involved in retinal regeneration, i.e. Lrat, Rdh5, Rgr, and Rpe65, were expressed at higher mRNA levels. Sequencing of the Rpe65 gene showed that Atg7f/f and Atg7f/f Tyr-Cre mice carry a point mutation (L450M) that is characteristic for the C57BL/6 mouse strain and reportedly causes enhanced degradation of the RPE65 protein by an as-yet unknown mechanism. These results suggest that the increased abundance of RPE65 M450 in the RPE of Atg7f/f Tyr-Cre mice is, at least partly, mediated by upregulation of Rpe65 transcription; however, our data are also compatible with the hypothesis that the RPE65 M450 protein is degraded by Atg7-dependent autophagy in Atg7f/f mice. Further studies in mice of different genetic backgrounds are necessary to determine the relative contributions of these mechanisms.

Fig 1. Hypothetical model of effects caused by Tyr-Cre-mediated deletion of Atg7.

Expression of the Cre recombinase under the control of the tyrosinase promoter leads to deletion of the floxed Atg7 gene, abrogation of autophagy and accumulation of p62 and other autophagy substrates in a pigment cell-specific manner. In addition to these direct effects, abrogation of autophagy alters cellular homeostasis that manifests in altered gene expression.

Fig 2. RPE65 but not p62 is transcriptionally upregulated in the RPE of Atg7^f/f Tyr-Cre mice.

(A) RNAs from the RPE of Atg7^f/f and Atg7^f/f Tyr-Cre mice were analyzed by quantitative RT-PCR for Atg7, p62 and Rpe65. Expression levels (a.u., arbitrary units) are shown relative to the expression of the house-keeping gene Gapdh. n = 4 mice per genotype. Error bars indicate standard errors of the mean. *p<0.05, considered statistically significant (2-tailed t-test). n.s., not significant. (B) Western blot analysis of RPE lysates from Atg7^f/f and Atg7^f/f Tyr-Cre mice. Protein lysates obtained from freshly isolated RPE sheets of Atg7^f/f and Atg7^f/f Tyr-Cre mice were subjected to Western blot (WB) analysis for microtubule-associated protein light chain 3 (LC3), p62, and RPE65 (retinal pigment epithelium-specific 65 kDa protein). The same protein amounts were run on 3 separate electrophoresis gels. Before exposing the membrane to the primary antibodies, the membranes were stained with Ponceau reagent to visualize the total proteins on the membrane (loading control). A representative Ponceau staining confirming equal loading is shown at the bottom. Furthermore, the intensities of an unspecific band (*) show that there is not more protein loaded in Atg7^f/f Tyr-Cre than in Atg7^f/f lanes. A short and a long exposure of the LC3 Western blot are shown to demonstrate the accumulation of LC3-I, the non-lipidated form of LC3, in Atg7^f/f Tyr-Cre mice (short exposure), and weak bands corresponding to LC3-II, the lipidated form of LC3, in Atg7^f/f mice (long exposure). Note that under the Western blot conditions applied here the levels of p62 and RPE65 were below the detection threshold in RPE lysates from wild-type mice. Positions of protein size markers (kD, kilo-Dalton) are indicated on the right.

Fig 3. Deletion of Atg7 leads to the accumulation of p62 and RPE65 in the RPE.

Double-immunofluorescence labelling of p62 (green) (A, B) and RPE65 (red) (C, D) in Atg7^f/f (A, C, E) and Atg7^f/f Tyr-Cre (B, D, F) eyes. Nuclear DNA was labelled with Hoechst 33258 (blue). Panels E and F show merged images. ONL, outer nuclear layer; PRL, photoreceptor layer; RPE, retinal pigment epithelium; Ch, choroid. Scale bars: 50 μm.

Fig 4. Regulators of the visual cycle are expressed at increased levels in the RPE of Atg7^f/f Tyr-Cre mice.

The expression levels of genes encoding visual cycle regulators (Lrat, Rpe65, Rdh5, Rgr), transcription factors implicated in the homeostasis of the RPE (Sox9, Otx2), and house-keeping genes (Alas1, B2m) were determined by quantitative RT-PCR analysis of RNAs from the RPE of Atg7^f/f (n = 5) and Atg7^f/f Tyr-Cre (n = 4) mice. Expression levels (a.u., arbitrary units) are shown relative to the expression of the house-keeping gene B2m. Error bars indicate standard deviations. *p<0.05, considered statistically significant (two-tailed t-test). n.s., not significant.