Modulation of NAD+/NADH Levels in ARPE-19 Cells Leads to Functional Changes in Oxidative Phosphorylation and Glycolysis
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CitationWill-Orrego, Adrian. 2020. Modulation of NAD+/NADH Levels in ARPE-19 Cells Leads to Functional Changes in Oxidative Phosphorylation and Glycolysis. Master's thesis, Harvard Extension School.
AbstractDysregulation of the retinal pigmented epithelial cell (RPE) metabolism through mitochondrial dysfunction leads to photoreceptor degradation, implicating the RPE cell in retinal degeneration (Zhao et al., 2011). Aging decreases bioavailability of NAD+ (Goody & Henry, 2018; Liu et al., 2012) , increasing likelihood of dysregulation in a host of NAD+-regulated pathways, like the sirtuin family of proteins which regulate cellular metabolism, DNA repair, and other crucial functions (Avalos, Bever, & Wolberger, 2005; Gaur et al., 2017). NAMPT is the rate-limiting enzyme of the salvage pathway, responsible for recycling NAM and setting up NAD+ biosynthesis. We hypothesize disruption of NAD+ biosynthesis by inhibiting NAMPT will imbalance the NAD+/NAM ratio and downregulate genes controlled by the Sirtuin family leading to functional changes in metabolism and cellular health.
Our experiments demonstrate intracellular levels of NAD+/NADH are crucial to ARPE-19 cell function. Decreasing available NAD+/NADH downregulates expression of genes associated with RPE activity and differentiation. Dysfunctional RPE cells generate less mitochondrial sourced ATP becoming heavily reliant on glycolysis for energy. Supplementing NAMPT inhibited ARPE-19 cells with NMN (a NAD precursor) reversed SeaHorse assay metabolic dysfunction phenotype and qPCR molecular signature potentially reverting ARPE-19 function to baseline. Further characterization of NAD+/NADH dysregulation in human retina may yield insights into macular degeneration.
Citable link to this pagehttps://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37365016