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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

Targeted gene knockout mouse models have helped to identify roles of autophagy in many tissues. Here, we investigated the retinal pigment epithelium (RPE) of Atg7(f/f) Tyr-Cre mice (on a C57BL/6 background), in which Cre recombinase is expressed under the control of the tyrosinase promoter to delete...

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Autores principales: Sukseree, Supawadee, Chen, Ying-Ting, Laggner, Maria, Gruber, Florian, Petit, Valérie, Nagelreiter, Ionela-Mariana, Mlitz, Veronika, Rossiter, Heidemarie, Pollreisz, Andreas, Schmidt-Erfurth, Ursula, Larue, Lionel, Tschachler, Erwin, Eckhart, Leopold
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4990303/
https://www.ncbi.nlm.nih.gov/pubmed/27537685
http://dx.doi.org/10.1371/journal.pone.0161640
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author Sukseree, Supawadee
Chen, Ying-Ting
Laggner, Maria
Gruber, Florian
Petit, Valérie
Nagelreiter, Ionela-Mariana
Mlitz, Veronika
Rossiter, Heidemarie
Pollreisz, Andreas
Schmidt-Erfurth, Ursula
Larue, Lionel
Tschachler, Erwin
Eckhart, Leopold
author_facet Sukseree, Supawadee
Chen, Ying-Ting
Laggner, Maria
Gruber, Florian
Petit, Valérie
Nagelreiter, Ionela-Mariana
Mlitz, Veronika
Rossiter, Heidemarie
Pollreisz, Andreas
Schmidt-Erfurth, Ursula
Larue, Lionel
Tschachler, Erwin
Eckhart, Leopold
author_sort Sukseree, Supawadee
collection PubMed
description Targeted gene knockout mouse models have helped to identify roles of autophagy in many tissues. Here, we investigated the retinal pigment epithelium (RPE) of Atg7(f/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 Atg7(f/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 Atg7(f/f) and Atg7(f/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 Atg7(f/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 Atg7(f/f) mice. Further studies in mice of different genetic backgrounds are necessary to determine the relative contributions of these mechanisms.
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spelling pubmed-49903032016-08-29 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 Sukseree, Supawadee Chen, Ying-Ting Laggner, Maria Gruber, Florian Petit, Valérie Nagelreiter, Ionela-Mariana Mlitz, Veronika Rossiter, Heidemarie Pollreisz, Andreas Schmidt-Erfurth, Ursula Larue, Lionel Tschachler, Erwin Eckhart, Leopold PLoS One Research Article Targeted gene knockout mouse models have helped to identify roles of autophagy in many tissues. Here, we investigated the retinal pigment epithelium (RPE) of Atg7(f/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 Atg7(f/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 Atg7(f/f) and Atg7(f/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 Atg7(f/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 Atg7(f/f) mice. Further studies in mice of different genetic backgrounds are necessary to determine the relative contributions of these mechanisms. Public Library of Science 2016-08-18 /pmc/articles/PMC4990303/ /pubmed/27537685 http://dx.doi.org/10.1371/journal.pone.0161640 Text en © 2016 Sukseree et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Sukseree, Supawadee
Chen, Ying-Ting
Laggner, Maria
Gruber, Florian
Petit, Valérie
Nagelreiter, Ionela-Mariana
Mlitz, Veronika
Rossiter, Heidemarie
Pollreisz, Andreas
Schmidt-Erfurth, Ursula
Larue, Lionel
Tschachler, Erwin
Eckhart, Leopold
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
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
title_full 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
title_fullStr 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
title_full_unstemmed 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
title_short 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
title_sort 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
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4990303/
https://www.ncbi.nlm.nih.gov/pubmed/27537685
http://dx.doi.org/10.1371/journal.pone.0161640
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