S or in CHM mouse models [4,7]. Consistent with multiple trafficking defects

S or in CHM mouse models [4,7]. Consistent with multiple trafficking defects in the development of CHM, reduced lysosomal acidification and secretion of cytokines have been detected in monocytes isolated from CHM patients [8] and there is reduced melanosome movement into theAge-Related Changes in RPE of Choroideremia ModelFigure 1. Impaired melanosome distribution in ChmFlox, Tyr-Cre+ mice. Electron micrographs of the RPE of 7-month old ChmFlox (A), ChmFlox, Tyr-Cre+ (B) and ashen (C) mice. Scale bars: 5 mm. The percentage of melanosomes in the apical processes in ChmFlox and ChmFlox, Tyr-Cre+ mice was determined (D). No melanosomes were ever found in the apical processes of ashen mice. Results are mean+/2SEM of 5 to 6 observations. *P,0.05. doi:10.1371/journal.pone.0057769.gapical processes of RPE cells in mouse models of CHM [9]. Furthermore cultured RPE cells acutely depleted of REP1 exhibit reduced lysosome acidification and delayed phagosome degradation [10]. Despite the demonstration of both underprenylated Rabs and trafficking defects in MedChemExpress HIF-2��-IN-1 peripheral cells, the pathological features of CHM are restricted to the eye and characterised by progressive HIV-RT inhibitor 1 custom synthesis degeneration of photoreceptors, RPE and choroid in patients with CHM, leading ultimately to blindness. The particular susceptibility of the eye to loss of REP1 is notcompletely understood. Both the photoreceptors and the RPE are largely postmitotic and have a huge traffic burden from the daily production of outer segments by the photoreceptors and the daily degradation of phagocytosed shed outer segments by the RPE, which may render these cell types particularly susceptible to partial defects in membrane traffic. In this study we use a CHM mouse model previously described where, in addition to the conditional allele of the Chm/Rep1 gene, mice carry the Cre-transgene under the control of the tyrosinaseAge-Related Changes in RPE of Choroideremia Modelpromoter (Tyr-Cre) which leads to a knock-out of the Chm/Rep1 gene only in pigmented cells [9]. These ChmFlox, Tyr-Cre+ mice have enabled us to study the long-term consequences of chronic membrane traffic defects in the RPE.Immuofluorescence AnalysisEyes were fixed in 4 paraformaldehyde in PBS for 2 hours at room temperature or overnight at 4uC, incubated overnight at 4uC in 30 sucrose in PBS and embedded in Tissue Tec. 10-mm thick sections were cut at 220uC and dried at room temperature. Sections were immunolabelled with anti-Rhodopsin (RetP1, from Abcam, UK) antibody followed by secondary antibody conjugated to Alexa488 (from Molecular Probes, UK). Eyecups were cut parallel and adjacent to the optic nerve in order to visualise peripheral and central retina. Samples were analysed by confocal microscopy (Leica TCS-SP2) and the number of phagosomes per length of RPE was counted manually. To eliminate autofluorescence from our analysis, microscope settings were adjusted so that negative controls, where primary antibodies were omitted, were negative. Between 3 and 5 animals were analysed in each group.Materials and Methods MiceAll animals used in this study were treated humanely in accordance with Home Office guidance rules under project licence 70/6176 and 70/7078, adhering to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The conditional knock-out mouse line ChmFlox,Tyr-Cre, that carries the Cre-recombinase transgene under control of the tyrosinase promoter, was generated previously and genotyping of mice was perf.S or in CHM mouse models [4,7]. Consistent with multiple trafficking defects in the development of CHM, reduced lysosomal acidification and secretion of cytokines have been detected in monocytes isolated from CHM patients [8] and there is reduced melanosome movement into theAge-Related Changes in RPE of Choroideremia ModelFigure 1. Impaired melanosome distribution in ChmFlox, Tyr-Cre+ mice. Electron micrographs of the RPE of 7-month old ChmFlox (A), ChmFlox, Tyr-Cre+ (B) and ashen (C) mice. Scale bars: 5 mm. The percentage of melanosomes in the apical processes in ChmFlox and ChmFlox, Tyr-Cre+ mice was determined (D). No melanosomes were ever found in the apical processes of ashen mice. Results are mean+/2SEM of 5 to 6 observations. *P,0.05. doi:10.1371/journal.pone.0057769.gapical processes of RPE cells in mouse models of CHM [9]. Furthermore cultured RPE cells acutely depleted of REP1 exhibit reduced lysosome acidification and delayed phagosome degradation [10]. Despite the demonstration of both underprenylated Rabs and trafficking defects in peripheral cells, the pathological features of CHM are restricted to the eye and characterised by progressive degeneration of photoreceptors, RPE and choroid in patients with CHM, leading ultimately to blindness. The particular susceptibility of the eye to loss of REP1 is notcompletely understood. Both the photoreceptors and the RPE are largely postmitotic and have a huge traffic burden from the daily production of outer segments by the photoreceptors and the daily degradation of phagocytosed shed outer segments by the RPE, which may render these cell types particularly susceptible to partial defects in membrane traffic. In this study we use a CHM mouse model previously described where, in addition to the conditional allele of the Chm/Rep1 gene, mice carry the Cre-transgene under the control of the tyrosinaseAge-Related Changes in RPE of Choroideremia Modelpromoter (Tyr-Cre) which leads to a knock-out of the Chm/Rep1 gene only in pigmented cells [9]. These ChmFlox, Tyr-Cre+ mice have enabled us to study the long-term consequences of chronic membrane traffic defects in the RPE.Immuofluorescence AnalysisEyes were fixed in 4 paraformaldehyde in PBS for 2 hours at room temperature or overnight at 4uC, incubated overnight at 4uC in 30 sucrose in PBS and embedded in Tissue Tec. 10-mm thick sections were cut at 220uC and dried at room temperature. Sections were immunolabelled with anti-Rhodopsin (RetP1, from Abcam, UK) antibody followed by secondary antibody conjugated to Alexa488 (from Molecular Probes, UK). Eyecups were cut parallel and adjacent to the optic nerve in order to visualise peripheral and central retina. Samples were analysed by confocal microscopy (Leica TCS-SP2) and the number of phagosomes per length of RPE was counted manually. To eliminate autofluorescence from our analysis, microscope settings were adjusted so that negative controls, where primary antibodies were omitted, were negative. Between 3 and 5 animals were analysed in each group.Materials and Methods MiceAll animals used in this study were treated humanely in accordance with Home Office guidance rules under project licence 70/6176 and 70/7078, adhering to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The conditional knock-out mouse line ChmFlox,Tyr-Cre, that carries the Cre-recombinase transgene under control of the tyrosinase promoter, was generated previously and genotyping of mice was perf.

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