Degree Name

PhD (Doctor of Philosophy)

Program

Biomedical Sciences

Date of Award

5-2022

Committee Chair or Co-Chairs

Patrick Bradshaw

Committee Members

Alok Agrawal, Victoria Palau, Antonio Rusinol, Douglas Thewke

Abstract

Nearly all multicellular organisms show changes in redox balance with aging leading to oxidative damage of macromolecules. This study investigated the role of the [NADP+]/[NADPH] redox couple in aging. This redox couple plays an important role in maintaining tissue redox balance and becomes slightly more oxidized in aged tissues. NADPH is a major source of reducing equivalents for enzymes that detoxify hydrogen peroxide. However, catalase detoxifies hydrogen peroxide independently of NADPH. But catalase is absent from mitochondria, a major source of hydrogen peroxide, where instead glutathione plays the major role in hydrogen peroxide detoxification in an NADPH-dependent manner. Three major cytoplasmic and three major mitochondrial NADPH generating enzymes were knocked down by the RNAi feeding technique in C. elegans, but little to no significant effects on lifespan were observed. The C. elegans genome contains two predicted, yet uncharacterized, NAD kinase (NADK) genes nadk-1 and nadk-2 to synthesize NADP+. Data suggest that NADK-1 is cytoplasmic, while NADK-2 is mitochondrial. NADK activity assays strongly suggested that both NADK-1 and NADK-2 possess NADK activity. Knockdown of NADKs did not affect the rate of body bending in young nematodes. However, knockdown of nadk-2 in nadk-1 mutant worms slightly decreased lifespan. Deficiency of NADKs increased the [NADP+]/[NADPH] and decreased the [NAD+]/[NADH], similar to redox changes that occur with aging. Unexpectedly, nadk-1 or nadk-2 knockdown resulted in decreased reactive oxygen species (ROS) levels and increased survival in young adult nematodes in the presence of juglone, a superoxide generator. The antioxidant response generated upon NADK knockdown required the transcription factors DAF-16/FOXO, SKN-1/Nrf2, and HSF-1 in nadk-2 knockdown nematodes, but only required HSF-1 in nadk-1 knockdown nematodes. NADK-1 or NADK-2 deficiency led to increased catalase activity and a strong trend for increased cytoplasmic catalase-1 (ctl-1) gene expression. Peroxisomal catalase-2 (ctl-2) mutant worms showed increased SOD activity when either NADK gene was knocked down, while ctl-1 mutant worms showed increased glutathione peroxidase or glutathione reductase activities following NADK knockdown. In summary, NADK knockdown oxidizes the [NADP+]/[NADPH] to compromise the antioxidant system, but young nematodes are able to mount a compensatory broad antioxidant response leading to decreased ROS levels.

Document Type

Dissertation - embargo

Copyright

Copyright by the authors.

Available for download on Thursday, June 15, 2023

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