Elated to the observed phenotypes in our cells, we analyzed the PGC-1 expression. Our data showed lower PGC-1 expression in AMD iPSC-RPE cells compared to normal iPSC-RPE in accordance with the phenotypic disease state of the cells. We further analyzed SIRT1 expression in AMD iPSCRPE and normal iPSC-RPE. Accordingly, SIRT1 protein levels were reduced in the AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE as compared to normal RPE-iPSCRPE. SIRT1 is known to directly affect PGC-1 activity through phosphorylation and deacetylation [23]. It is also know that AMPK activation increases PGC-1 expression, and activates PGC-1 by direct phosphorylation [68]. AMPK activation also induces SIRT1-mediated PGC-1 deacetylation [69]. AMPK activation is triggered by increases in cellular AMP/ATP ratio [61]. Inactive AMPK results in lower autophagy dynamics causing accumulation of lipids and unwanted materials in the cells. Inactive AMPK also induces PGC-1 inactivation and results in lower mitochondrial biogenesis and turnover affecting mitochondrial activity. Increased total ATP caused by glycolysis that we observed in the AMD iPSCRPE cells could result in AMPK inactivation and consequently lower the PGC-1 expression and activation. Based on our observations we hypothesize that repressed AMPK / SIRT1 / PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28893839 PGC-1 pathway could affect mitochondrial biogenesis and remodeling, causing increased ROS production, leading to RPE and retinal degeneration in AMD. Figure 7, summarizes our hypothesis. Our data suggest involvement of SIRT1/PGC-1 pathway in the pathophysiology of AMD and open new avenues for development of novel and targeted drugs for treatment of AMD.Fig. 7 The role of SIRT1/PGC-1 repression in the pathophysiology of AMD. SIRT1 deacetylates and activates PGC-1. AMPK increases SIRT1 activity and directly phosphorylates and activates PGC-1. The reduction in SIRT1 activity would reduce deacetylation rate of PGC-1. Hyperacetylation decreases PGC-1 activity which translates to lower mitochondrial content and activity, lowered mitochondrial respiratory capacity, lowered ROS detoxification and increased ROS production, contributing to the pathophysiology of AMD. Ac acetylation, p phosphorylationConclusions Our study identified SIS3 msds morphological and functional differences between normal iPSC-RPE and AMD iPSCRPE generated from RPE of healthy donors and RPE of donors with AMD. We also observed that the iPSC-RPE generated from skin biopsy of a dry AMD patient exhibit the same disease cellular phenotypes and therefore can be used for in vitro disease modeling. The morphological changes observed in AMD RPE-iPSC-RPE and Skin-iPSC-RPE consist of disintegrated mitochondria, increased numbers of autophagosomes, and lipid droplets. We further performed functional analyses of AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE and observed increased susceptibility to oxidative stress, higher ROS production under oxidative stress, decreased mitochondrial activity, inability to increase SOD2 expression underoxidative stress conditions, and higher cytoplasmic glycogen as compared to normal RPE-iPSC-RPE. In addition, we observed lower SIRT1 levels and lower PGC-1 expression in AMD iPSC-RPE as opposed to normal iPSC-RPE. In summary, our study suggests that repressed SIRT1/ PGC-1 causing impaired mitochondrial activity in RPE as responsible mechanisms for the disease cellular phenotypes that we observed in AMD RPE. Our data provide new insight in molecular mechanisms of AMD and could be used for develop.
