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Eszter Emri, Xinyi Yang, Janos Kriston-Vizi, Robin Ketteler, Imre Lengyel; The effects of zinc supplementation on autophagy in primary human fetal retinal pigment epithelial cells – new molecular target pathway. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5366.
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© ARVO (1962-2015); The Authors (2016-present)
Autophagy is thought to be a response to increased oxidative stress in age related macular degeneration (AMD) and it has been suggested that modulation of autophagy flux could become a treatment strategy for the disease. The ARED study showed that zinc supplementation can reduce the progression of AMD, raising the possibility, that zinc might be directly involved in regulating cellular processes during disease progression. In this study we tested the hypothesis that zinc supplementation can directly affect autophagy.
Primary human fetal RPE cells (ScienceCell) were cultured on polyester transwells (Corning), coated with Geltrex for 4 and 31 days in the presence or absence of 125 μM externally added ZnSO4. Autophagosome number was induced by Chloroquine or Bafilomycin, visualized using CytoID kit (Enzo) and the Opera high content screening system (PerkinElmer) and quantified by ImageJ. Distribution of autophagosomes were examined by confocal (Zeiss LSM 700) and transmission electron microscopy (Jeol 1010).
There was a small, but significant decrease in autophagosome numbers with increasing differentiation in non-treated cells (1.77±0.31 vs. 0.65±0.15; p<0.05). Treatment with Chloroquine or Bafilomycin increased autophagosome numbers to 7.92±0.45 and 11.25±2.34 respectively, at day 4. The treatment with Bafilomycin did not affect the cells at day 31, while Chloroquine had a small, but significant effect (0.65±0.15 vs 1.17±0.21; p<0.05). Treatment with zinc significantly attenuated the effects of both Chloroquine and Bafilomycin (p<0.05). Examination of the autophagosomes with confocal and transmission electron microscopy showed that autophagosomes were not only present in the cells but also in the sub-RPE space.
Our results support the hypothesis, that zinc supplementation can directly affect autophagy. The observation that autophagosomes are present in the sub-RPE space suggest that they may contribute to deposit formation in AMD. Therefore, the effects observed in the ARED study, at least in part, might be due to a direct influence on autophagy and this could indeed be a target for future treatment strategies for AMD.Aknowledgement: This work was supported by COST TD 1304 ‘The Network for the Biology of Zinc (Zinc-Net)’and EYE-RISK European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No 634479.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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