June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Amyloid precursor protein synthesis, processing, and secretion in retinal pigmented epithelial cells: Effects of hypoxia
Author Affiliations & Notes
  • Philip Mzyk
    Molecular Biomedical Sciences, North Carolina State University, Raleigh, North Carolina, United States
  • Jill Harned
    Molecular Biomedical Sciences, North Carolina State University, Raleigh, North Carolina, United States
  • Steven Nagar
    Molecular Biomedical Sciences, North Carolina State University, Raleigh, North Carolina, United States
  • Mary Christine McGahan
    Molecular Biomedical Sciences, North Carolina State University, Raleigh, North Carolina, United States
  • Footnotes
    Commercial Relationships   Philip Mzyk, None; Jill Harned, None; Steven Nagar, None; Mary McGahan, None
  • Footnotes
    Support  NIH Grant EY04900 and funds from the State of North Carolina
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 1607. doi:
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      Philip Mzyk, Jill Harned, Steven Nagar, Mary Christine McGahan; Amyloid precursor protein synthesis, processing, and secretion in retinal pigmented epithelial cells: Effects of hypoxia. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1607.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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