Abstract
Purpose :
Claudin-19 regulates many of the proteins that define the phenotype of the retinal pigment epithelium (RPE). Mutations of claudin-19 affect the visual cycle, and the development of the retina. This study asks whether claudin-19 might affect phagocytosis.
Methods :
RPE was generated from human induced pluripotent stem cells. Claudin-19 was knocked-down using siRNA. The transepithelial electrical resistance (TER) was used to monitor the effectiveness of the knockdown. Photoreceptor outer segments (POS) were fed to the RPE for 2 hr, followed by washing with EDTA to remove non-internalized POS. The degradation of internalized rhodopsin was followed for 4 days. Micro-arrays and PCR were used to assess the effects on gene expression. Immunoblotting was used to examine the steady-state levels of key proteins for autophagy, LC3-associated phagocytosis (LAP), and stimulation of the AMPK pathway. Cell lysates were assayed for the activity of superoxide dismutase.
Results :
Knock-down of claudin-19 substantially reduced the TER without disassembling the apical junctional complex. There was no discernable effect on the uptake of POS, but the degradation of rhodopsin was retarded. The steady-state levels increased for LC3 and the cargo-adapter, SQSTM1, but not for beclin-1 or ATG5. There was little or no increase for the phosphorylation of AMPK and no detectable effect on the phosphorylation of the downstream kinases ULK1, raptor, or beclin-1. The major effect on the transcriptome was a 2-10x increase in expression for various metallothioneins. The activity of SOD increased 2.4x.
Conclusions :
The absence of claudin-19 caused RPE to mount a defense against oxidative stress. This included induction of enzymatic and non-enzymatic mechanisms that remove oxygen-reactive species. The response included a reduction in phagocytosis, itself a contributor to oxidative stress.
This is a 2020 ARVO Annual Meeting abstract.