Reactive oxygen species (ROS) such as H2O2 can damage cellular components and are formed as a byproduct of mitochondrial oxidative metabolism. Studies using the nematode C. elegans have found that increasing ROS during development or early adulthood can extend lifespan, while increasing ROS during later adulthood normally decreases lifespan. NADPH provides the reducing power for several cellular antioxidants and is synthesized in a two-step reaction from NAD+ with the first step being catalyzed by NAD kinase (NADK). In this study, it questioned the effects of knocking down C. elegans cytoplasmic NADK, nadk-1 globally or in a neuron or intestine-specific manner starting from early development on oxygen consumption and ROS levels. It was hypothesized that knocking down the cytoplasmic NADK in C. elegans alters NADP+/NADPH ratios. This led to the prediction that ROS and O2 consumption levels would be decreased, and thioredoxin reductase activity levels will be increased in global and tissue-specific knockdowns. Three different strains of nematodes were used for this study: N2 wild type, intestinal RNAi sensitive strain VP303, and neuronal RNAi sensitive strain TU3401. The nadk-1 RNAi was introduced to the nematodes through their food source. To measure ROS levels, nematodes were treated with a tert-butylhydroperoxide insult to induce ROS generation, along with H2DCFDA dye to fluoresce when oxidized by ROS, pipetted into a 96-well plate, and ran through a microplate reader. For the oxygen consumption measurements, the nematodes were run through a Clark O2 electrode. Finally, Thioredoxin Reductase activity levels were measured using an assay kit that would inhibit testing groups thioredoxin reductase to only measure glutathione reductase levels and subtract it from uninhibited testing groups. The nematodes were pipetted into a 96-well plate and ran through a microplate reader. All experiments were normalized to average protein levels of C. elegans and analyzed using a t-test. Whole body knockdown of nadk-1 to decrease cytoplasmic NADPH levels decreased oxygen consumption and tert-butyl hydroperoxide-stimulated ROS levels, which was phenocopied by intestine-specific or neuron-specific knockdown. Thioredoxin reductase measurements following nadk-1 knockdown showed a trend toward increased activity. These results establish that nadk-1 does have antioxidant properties but does not extend lifespan as is the case of other NADPH generating enzyme knockdowns.
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| Identifer | oai:union.ndltd.org:ETSU/oai:dc.etsu.edu:asrf-2141 |
| Date | 25 April 2023 |
| Creators | Regan, Jake, Bradshaw, Patrick |
| Publisher | Digital Commons @ East Tennessee State University |
| Source Sets | East Tennessee State University |
| Detected Language | English |
| Type | text |
| Source | Appalachian Student Research Forum |
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