Amyloid precursor protein synthesis, processing, and secretion in retinal pigmented epithelial cells: Effects of hypoxia

Abstract

Purpose : Amyloid precursor protein (APP) is a ubiquitously expressed protein that is metabolized to amyloid beta (present in drusen in AMD and in plaques in Alzheimer disease brains) and soluble APP alpha and beta (sAPPα and β). We found that hypoxia dramatically decreased APP expression and secretion in retinal pigmented epithelial cells (RPE). Retromer, a multimeric protein complex that transports APP to the cell surface for secretion, was also decreased by hypoxia. In the current study we examined the mechanisms by which hypoxia affects APP levels and secretion by inducing ER stress and silencing retromer.

Methods : RPE were isolated from canine eyes and grown to confluence. Cells were placed under hypoxic (0.5% O₂) or normoxic (21% O₂) conditions for 2 h (for ³⁵S labeling) or 48 h. Cells were treated with either 1µM tunicamycin or 2.5mM 4-PBA (to induce or inhibit ER stress respectively) or siRNA to knock down retromer. Lysates and cell-conditioned media (CCM) were collected and immunoblotted for APP, sAPPα, and sAPPβ.

Results : As we previously reported, hypoxia significantly decreased APP cellular levels and secretion by 60% in RPE cells. Importantly, inducing ER stress with tunicamycin caused a 77% decrease in APP in RPE lysates and the ER stress inhibitor 4-PBA decreased hypoxia’s effect on APP levels. Previous data showed that with retromer knockdown, APP secretion was significantly decreased in normoxia and hypoxia. In this study retromer knockdown specifically decreased sAPPβ secretion by 57% in normoxia and hypoxia, but had no effect on sAPPα secretion. Preliminary studies with ³⁵S metabolic labeling indicate that hypoxia does not affect APP synthesis.

Conclusions : Hypoxia alters APP expression and secretion in RPE cells. However, preliminary studies indicate that hypoxia may not affect the synthesis of APP. In RPE, ER stress decreased expression of APP by 77%, similar to that seen in hypoxia. This may be the mechanism by which hypoxia caused APP levels to decrease because ER stress inhibition blocked this effect. Interestingly, when retromer was knocked down, sAPPβ but not sAPPα secretion was dramatically lowered. Since formation of sAPPα and β follow different pathways, retromer likely is involved only in sAPPβ processing. Understanding how hypoxia affects APP processing and secretion is vital to our knowledge of RPE pathophysiology.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.