Mechanism of Action of Nicotinamide Phosphoribosyltransferase Mediated Signaling in Oxidative Stress
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Worldwide there are an estimated 1 billion people that are affected by a form of neurodegenerative disease. The most common forms of neurodegenerative disease are Alzheimer’s disease and Parkinson’s disease, along with diseases that are attributed to damage of the retina and optic nerve, such as macular degeneration and glaucoma. Nearly five million people have been diagnosed with Alzheimer’s disease in the United States of America alone (1). Another disease that effects 4 million people in the US is glaucoma, and it is the second most common form of blindness after age-related macular degeneration (AMD) (2). Alzheimer’s disease, AMD, and glaucoma are all neurodegenerative diseases that are caused by oxidative stress. Neuroprotection and protection of cells supporting the function of neurons could provide novel therapeutic approaches to treating and preventing neurodegenerative diseases when therapies do not exist or existing therapies fail (3). Nicotinamide phosphoribosyltransferase (Nampt) is an enzyme critical for cellular energy metabolism, and it also functions as a proinflammatory cytokine and growth factor with neuroprotective properties and in these contexts is referred to as Pre-B-Cell Colony Enhancing Factor (PBEF) and Visfatin, respectively. Nampt/PBEF/Visfatin has been implicated in a number of human diseases, including acute lung injury, rheumatoid arthritis, vascular disorders, and diabetes. Based on previous evidence suggesting a protective role of Nampt/PBEF/Visfatin in ischemia, the hypothesis was tested that Nampt/PBEF/Visfatin exerts protective effects against oxidative stress in established in vitro models of neurodegeneration and of cellular degeneration related to neurodegenerative diseases. The PBEF gene was cloned into a prokaryotic expression vector, recombinantly expressed as a Glutathione-S-transferase-fusion protein, and purified by affinity chromatography. Optimization of a fluorescent enzyme activity assay was performed in order to quantify the Nampt/PBEF/Visfatin-mediated conversion of nicotinamide to nicotinamide mononucleotide, confirming enzymatic activity of recombinant Nampt/PBEF/Visfatin in vitro. Testing of neuroprotective properties of recombinant Nampt/PBEF/Visfatin was conducted in the human neuroblastoma cell line SH-SY5Y using the calcein-acetomethoxy ester uptake assay, which measures cellular viability. Testing of siRNA transfection mediated knockdown of endogenous Nampt was performed in the retinal pigment epithelial cell line ARPE-19 and primary isolated optic nerve head astrocytes as models for cells supporting the viability and function of retinal neurons. The use of siRNA in both ARPE-19 and ONHA cell lines demonstrated how the loss of endogenous Nampt affected each cell line when treated with tBHP. Visual cell counts were performed after knockdown of Nampt and treatment of tBHP, and rNampt was used as a neuronal protective in order to produce cellular recovery. Recombinant Nampt/PBEF/Visfatin was enzymatically active and applied to each cell type extracellularly in the pre-treatment stage when fresh media was added to the cells. This addition of rNampt reduced cell death resulting from chemically induced oxidative stress in vitro by approximately 25% in SH-SY5Y cell line. Similar results were seen in both ARPE 19 cells and ONHA when oxidative stress was induced by ROS. Removal of endogenous Nampt by pharmacological inhibition or siRNA-mediated knockdown had no effect on general cell viability but cell lines ARPE-19 and ONHA became more sensitive to oxidative stress and cells entered apoptosis earlier than WT. After knockdown of endogenous Nampt cells were pre-treated with rNampt and shown to have increased cell survival from neuronal protection from oxidative stress. An improved understanding of the mechanisms of action underlying Nampt/PBEF/Visfatin-mediated neuro- and cellular protection has the potential to contribute to the development of new therapies to treat neurodegenerative diseases.
Table of Contents
Introduction -- Materials and methods -- Results -- Discussion -- Future directions
Ph.D. (Doctor of Philosophy)