title: 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.
authors.affiliation: Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna, Austria.
authors.forename: Supawadee
authors.initials: S
authors.lastname: Sukseree
authors.affiliation: Department of Ophthalmology, Medical University of Vienna, Vienna, Austria.
authors.forename: Ying-Ting
authors.initials: YT
authors.lastname: Chen
authors.affiliation: Department of Ophthalmology, Medical University of Vienna, Vienna, Austria.
authors.forename: Maria
authors.initials: M
authors.lastname: Laggner
authors.affiliation: Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna, Austria.|Christian Doppler Laboratory for Biotechnology of Skin Aging, Vienna, Austria.
authors.forename: Florian
authors.initials: F
authors.lastname: Gruber
authors.affiliation: Institut Curie, PSL Research University, INSERM U1021, CNRS UMR3347, Normal and Pathological Development of Melanocytes, Orsay, France.|INSERM, Orsay, France.|Equipe labellisée - Ligue Nationale contre le Cancer, Université Paris 11, Orsay, France.
authors.forename: Valérie
authors.initials: V
authors.lastname: Petit
authors.affiliation: Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna, Austria.|Christian Doppler Laboratory for Biotechnology of Skin Aging, Vienna, Austria.
authors.forename: Ionela-Mariana
authors.initials: IM
authors.lastname: Nagelreiter
authors.affiliation: Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna, Austria.
authors.forename: Veronika
authors.initials: V
authors.lastname: Mlitz
authors.affiliation: Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna, Austria.
authors.forename: Heidemarie
authors.initials: H
authors.lastname: Rossiter
authors.affiliation: Department of Ophthalmology, Medical University of Vienna, Vienna, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Pollreisz
authors.affiliation: Department of Ophthalmology, Medical University of Vienna, Vienna, Austria.
authors.forename: Ursula
authors.initials: U
authors.lastname: Schmidt-Erfurth
authors.affiliation: Institut Curie, PSL Research University, INSERM U1021, CNRS UMR3347, Normal and Pathological Development of Melanocytes, Orsay, France.|INSERM, Orsay, France.|Equipe labellisée - Ligue Nationale contre le Cancer, Université Paris 11, Orsay, France.
authors.forename: Lionel
authors.initials: L
authors.lastname: Larue
authors.affiliation: Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna, Austria.
authors.forename: Erwin
authors.initials: E
authors.lastname: Tschachler
authors.affiliation: Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna, Austria.
authors.forename: Leopold
authors.identifier: http://orcid.org/0000-0002-5645-2036
authors.initials: L
authors.lastname: Eckhart
mesh_terms: D000818:Animals; D001343:Autophagy / Q000235:genetics / Q000502:physiology; D000071193:Autophagy-Related Protein 7 / Q000235:genetics / Q000502:physiology*; D015153:Blotting, Western; D005260:Female; D005455:Fluorescent Antibody Technique; D017353:Gene Deletion; D019426:Integrases / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D014442:Monophenol Monooxygenase / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D019745:cis-trans-Isomerases / Q000378:metabolism*; D000099178:Retinoid Isomerohydrolase
chemical_list: D000071193:Autophagy-Related Protein 7; D019426:Integrases; D014442:Monophenol Monooxygenase; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C502661:Atg7 protein, mouse; C045073:Cre recombinase