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Khanh Do, Jorgelina Muriel Calandria, Nicolas G Bazan; Neuroprotectin D1 (NPD1) modulates amyloid precursor protein (APP) processing in human retinal pigment epithelial cells (RPE). Invest. Ophthalmol. Vis. Sci. 2016;57(12):6032.
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© ARVO (1962-2015); The Authors (2016-present)
Amyloid beta is a major component of drusen in age-related macular degeneration (AMD) (Ratnayaka et al; Eye, 2015). In neuronal cells in culture from human brain expressing APP mutations that cause familial forms of Alzheimer’s disease, NPD1 shifts APP processing from the tandem beta and gamma secretase to alpha secretase (Zhao et al; PLOS One, 2011). We hypothesize that NPD1 deters formation of amyloid-β towards the prosurvival sAPPa, as it occurs in human brain cells. Here we have studied the bioactivity of NPD1 on APP processing in RPE cells.
Plasmid construct containing Swedish double mutant APP protein (APPsw), which dominantly produce sAPPβ (a precursor of amyloid beta), was transfected into human ARPE-19 cells. After 24h, NPD1 was added at 50, 100 and 500 nM concentrations in the oxidative stress-inducing condition (10 ng/mL of tumor necrosis factor alpha (TNFα) plus 600 nM H2O2). The synthesized non-amyloidogenic sAPPα and amyloidogenic sAPPβ were analyzed by western blot after 48h of NPD1 treatment. Non-transfected and non-oxidative stress conditions were used as negative controls. Furthermore, we also investigated the anti-oxidant activity of 2 nM sAPPα protein in preventing oxidative stress-induced apoptosis in ARPE-19 cells. The cell survival ratio was analyzed by Hoechst staining.
At 48h, higher sAPPα production was observed in the increase of NPD1 concentration. The sAPPα expression peaked 1.5 times higher than the non-treated control at 500 nM of NPD1. In contrast, reverse production of sAPPβ was observed, where the highest amount of sAPPβ was detected in the absence of NPD1. An equal amount of holo-APP, regarded as total of all sAPP forms, was detected in all studied condition. Also, we saw that NPD1 decreased the level of amyloid-β secretion to the control level. For anti-oxidant action, our results showed that the presence of 2 nM sAPPα could prevent oxidative stress-induced apoptosis in RPE cells.
We found an NPD1 concentration-dependent increase of non-amyloidogenic sAPPα and decrease of amyloidogenic sAPPβ in RPE cells. These results suggest that NPD1 shifts production of amyloidogenic to the non-amyloidogenic pathway in human RPE cells. Additionally, the protective activity of sAPPα modulated by NPD1 represents a new RPE cell survival route.
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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