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.

Filamentous Aggregation of Sequestosome-1/p62 in Brain Neurons and Neuroepithelial Cells upon Tyr-Cre-Mediated Deletion of the Autophagy Gene Atg7

“…Outside of the brain, p62 accumulations were detected in skin melanocytes [ 18 ], choroid melanocytes of the eye, and retinal pigment epithelial cells [ 20 ]. However, the strongest accumulation of p62, as judged from immunohistochemical and immunofluorescence analysis, was found in the epithelial cells of the ciliary body of Atg7 f/f Tyr-Cre mice (Fig.…”

“…The insoluble debris was removed by centrifugation, and the protein concentration of the supernatant was measured by the bicinchoninic acid (BCA) method (Pierce, Rockford, IL). Western blot analysis was performed as described previously [ 20 ]. Twenty microgram protein was loaded per lane on SDS polyacrylamide gels (ExcelGel SDS, gradient 8–18, Amersham Biosciences) on a horizontal electrophoresis system (Amersham Biosciences) and thereafter blotted onto a nitrocellulose membrane.…”

“…In previous studies, we have generated Atg7 f/f Tyr-Cre mice for the investigation of the role of autophagy in pigment cells [ 18 – 20 ]. The Tyr-Cre gene utilizes promoter and enhancer elements from tyrosinase ( Tyr ), a gene encoding the enzyme that converts tyrosine to melanin via tyrosine hydroxylase and dopa oxidase catalytic activities in pigment cells.…”

“…Atg7 f/f Tyr-Cre mice show mild hypopigmentation of hair and tail skin [ 18 ] but otherwise appear phenotypically normal. Autophagy is also suppressed in the retinal pigment epithelium of Atg7 f/f Tyr-Cre mice leading to the accumulation of p62 and an increase in the abundance of a degradation-prone variant of retinal pigment epithelium-specific 65 kDa protein (RPE65) [ 20 ].…”

IL-10 inhibits retinal pigment epithelium cell proliferation and migration through regulation of VEGF in rhegmatogenous retinal detachment

“…RRD prevents the outer retina layer from obtaining nutrition from choroid, which may lead to atrophy of the detached retina and its subsequent degeneration (8,9). Untreated RRD may lead to impairment or loss of vision, and recovery is unlikely (10). The human eye is made up of millions of non-renewable RPE cells organized into a single-cell layer (11).…”