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Nady Golestaneh, Nisi Jiang, Olga Postnikova, Amrita Cheema; Repressed AMP- activated protein kinase, Sirtuin1 and peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha, overactive mammalian target of rapamycin, and dysregulated metabolites in RPE of AMD donor eyes.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):365. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Age-related macular degeneration (AMD) is a major cause of blindness. Recently we showed that autophagy dynamic and flux are reduced in AMD RPE. However, the underlying mechanisms inducing these defective metabolic homeostases leading to AMD remain elusive. We hypothesize that repressed AMPK/SIRT1/PGC-1α pathway and overactive mTOR induce autophagic dysregulation, lipid accumulation, mitochondrial dysfunction, resulting in drusen formation, RPE and photoreceptor degeneration in AMD.
RPE from AMD and normal donor eyes were isolated, purified with MACS, and cultured on transwells to generate RPE monolayers. Electron microscopy (EM) was performed. Next generation RNA sequencing followed by Real Time PCR were used. Western blot and Immunoprecipitation were used to measure levels of total proteins, acetylated or phosphorylated levels of target proteins. Metabolomic analysis was performed to identify any metabolite dysregulation.
EM revealed distinct phenotypes including mitophagosome accumulation and disintegrated mitochondria in AMD RPE. AMD RPE exhibited increased PARP2 and differential expression of mitochondrial genes and genes regulating mitochondrial functions. Reduced SIRT1 and PGC-1α protein levels and increased acetylated PGC-1α (inactive form) levels were observed in AMD RPE compared to normal RPE. AMPK activity was repressed in AMD-RPE compared to normal RPE as shown by phospho-AMPK, phospho acetyl-CoA carboxylase, and phospho-raptor (Ser792) levels in the presence of nutrients or after starvation followed by IGF-1 incubation. mTOR pathway analysis revealed that normal RPE rapidly induced phosphorylation of p70S6K (Thr389) after starvation and incubation with IGF-1, followed by decreased phosphorylation after 30 min and 1h. Conversely, AMD RPE showed basal levels of p70S6K phosphorylation during starvation in the absence of IGF-1, and sustained phosphorylation levels of p70S6K (Thr389) after 1h incubation with IGF-1. A number of metabolites including glycosphingolipid, Anandamide, Phosphoglycerifdes were upregulated in AMD RPE.
Our data support our hypothesis that repressed AMPK/SIRT1/PGC-1α and overactive mTOR contribute to AMD pathophysiology and induce AMD-like phenotypes in RPE of AMD donor eyes.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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