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Katherine Jean Davis, Shabnam Pakneshan, Peter Yu Cheng Zhao, Haben Kefella, Ron A Adelman, Lawrence J Rizzolo; Autophagy over the lifespan: using fetal, stem cell, and adult RPE cultures to model the pathogenesis of AMD. Invest. Ophthalmol. Vis. Sci. 2014;55(13):371. doi: https://doi.org/.
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Dysfunctional autophagy in the retinal pigment epithelium (RPE) has been implicated as a potential therapeutic target in age-related macular degeneration (AMD). To explore how autophagy changes over the lifespan and in response to photoreceptor outer segments (POS), we compared induced pluripotent stem cell RPE (iPS-RPE), human fetal RPE (hfRPE), and adult donor RPE (ad-RPE).
RPE was cultured from 16-week human fetuses and cadaveric eyes. Stem cell-derived RPE was prepared from human embryonic stem cells (hESC-RPE) and induced pluripotent stem cells (iPS-RPE). LC3 conversion (immunoblotting) and changes in autophagy-related gene expression (qRT-PCR) were used to monitor autophagy. Relative maturity of RPE cultures was assessed using a panel of signature and maturation genes (qRT-PCR). Autophagy was manipulated with an inhibitor, Spautin-1, and inducer, Rapamycin. iPS-RPE were challenged with porcine POS daily for up to 1 month, and monitored with confocal-immunomicroscopy. The health of RPE cultures was assessed by the transepithelial electrical resistance (TER).
Autophagic flux (LC3 conversion) increased from stem cell to 53-year-old ad-RPE, but was reduced in 90-year-old RPE. Rapamycin stimulated RPE autophagy, but Spautin-1 inhibited autophagy only partially—the strongest effect was on 90-year-old ad-RPE. qRT-PCR revealed quantitative differences in the expression of autophagy- and maturation-related genes. In iPS-RPE, the expression level of most maturation genes was similar to hfRPE, but some were at the level of less mature hESC-RPE. However, iPS-RPE and ad-RPE exhibited substantially higher levels of autophagy-related genes than hfRPE. In iPS-RPE, continuous feeding of POS over three weeks lowered TER to physiologic levels. One-week exposure to POS had little effect on iPS-RPE autophagy gene-expression, but did result in the accumulation of autofluorescent granules.
The characteristics of autophagy depended on the culture model: autophagy gene expression in iPS-RPE more closely resembled ad-RPE than hfRPE. Pending the examination of more donors, partial inhibition by Spautin-1 suggests that a non-canonical autophagy pathway is replaced in old age by the canonical pathway. The accumulation of lipofuscin-like granules induced by POS indicates that complementary RPE cultures will be a valuable aid to explore targets for therapeutic agents for AMD.
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