Date of Award

January 2015

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Lawrence J. Rizzolo

Subject Area(s)

Medicine, Cellular biology, Pathology

Abstract

Purpose: Dysfunctional autophagy in the retinal pigment epithelium (RPE) has been implicated as a therapeutic target in age-related macular degeneration (AMD). To explore how RPE autophagy changes over the lifespan and in response to phagocytosis of photoreceptor outer segments (POS), we compared stem cell-derived RPE (hESC-RPE, iPS-RPE), human fetal RPE (hfRPE), the ARPE-19 cell line, and adult RPE (ad-RPE).

Methods: 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 (qRT2-PCR) were used to monitor autophagy. Relative maturity of RPE cultures was assessed using a panel of signature and maturation genes (qRT2-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 monitored by the transepithelial electrical resistance (TER).

Results: Autophagic flux (immunoblot) increased from stem cell to a peak in 78-year-old RPE, but was reduced in 91-year-old RPE. Spautin-1 inhibited autophagy only partially—the strongest effect was on ARPE-19 and 91-year-old ad-RPE. qRT2-PCR revealed quantitative differences in the expression of autophagy- and maturation-related genes. In iPS-RPE, the expression level of most maturation genes was most similar to hfRPE. However, iPS-RPE and ad-RPE exhibited substantially higher levels of autophagy-related genes than hfRPE. Continuous feeding of POS to iPS-RPE for three weeks lowered TER to physiologic levels. In iPS-RPE, three-weeks of exposure to POS had little effect on autophagy or signature gene-expression, but did result in the accumulation of autofluorescent granules. Continuous feeding of POS to hfRPE for one week increased the expression of autophagy genes to levels observed in iPS- and ad-RPE.

Conclusions: The characteristics of autophagy depended on the culture model: autophagy gene expression in iPS-RPE more closely resembled adult RPE than hfRPE. Partial inhibition by Spautin-1 suggests the presence of a non-canonical RPE autophagy pathway that is lost in old age. 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.

Comments

This is an Open Access Thesis.

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