September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Hypoxia alters the expression, movement, and secretion of APP in retinal pigmented epithelial (RPE) cells
Author Affiliations & Notes
  • Philip Mzyk
    Ophthalmology, North Carolina State University, Raleigh, North Carolina, United States
  • Mary Christine McGahan
    Ophthalmology, North Carolina State University, Raleigh, North Carolina, United States
  • Footnotes
    Commercial Relationships   Philip Mzyk, None; Mary McGahan, None
  • Footnotes
    Support  NIH Grant EY04900 and funds from the State of North Carolina
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 84. doi:
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      Philip Mzyk, Mary Christine McGahan; Hypoxia alters the expression, movement, and secretion of APP in retinal pigmented epithelial (RPE) cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):84.

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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 processed into both amyloid beta (present in drusen in AMD and in plaques in Alzheimer disease brains) and soluble APP (sAPP) which is an iron regulatory protein. Significantly, iron levels are increased in AMD retinas. Hypoxia contributes to numerous retinal diseases and our findings indicated that hypoxia dramatically lowered polarized APP secretion from retinal pigmented epithelial (RPE) cells. In this study we determined the effects of hypoxia on the regulation of APP levels in RPE cell lysates.

Methods : RPE were isolated from canine eyes and grown to confluence. Cells were then dispersed on 6 well tissue culture plates or on cell culture inserts and grown to confluent polarized monolayers. Cells were placed under hypoxic (0.5% O2) or normoxic (21% O2) conditions for 24 or 48 hours. Lysates and cell-conditioned media (CCM) were collected and analyzed by immunoblotting for APP.

Results : As previously reported, hypoxia dramatically decreased APP secretion (26% and 55% of control at 24 and 48h, respectively). Importantly, there was a very large decrease (47%) in intracellular APP present in polarized RPE cells at 48 hours which may explain the decrease in APP secretion. In polarized cells there was approximately a 50% decrease in basolateral APP secretion at 48 hours, which closely matched the decrease in the amount of APP in the lysates. However, there was a dramatic 84% decrease in apical APP secretion which did not match the change seen in the cell lysates.

Conclusions : Hypoxia alters intracellular APP levels in RPE, and therefore the amount available for secretion. Importantly, while the hypoxia-induced reduction in APP matches the amount secreted in the basolateral direction, the amount secreted in the apical direction is reduced more significantly. This indicates the possibility that there are dynamic changes in polarized movement of APP in RPE caused by hypoxia. Other preliminary data indicate that this could be due to changes in the level or function of the intracellular protein transporter, retromer. Significantly, a reduction in APP levels in cells could cause a deleterious increase in iron levels because of APP’s role in regulation of iron efflux from cells. Understanding how hypoxia affects protein processing, localization, and secretion is vital to our knowledge of RPE pathophysiology.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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