Redox Biology Core

PROJECT SUMMARY – GEROSCIENCE REDOX BIOLOGY CORE (GRBC) The goal of the Geroscience Redox Biology (GRB) Core is to provide investigators with a coordinated and comprehensive evaluation of the molecules, pathways, and systems that contribute to oxidative metabolism and oxidative stress using specialized analyses of samples. The state-of-the-art analyses the Core provides allow an integration of measures of redox and energy balance, oxidative damage, and mitochondrial function that are all critical players in aging and age-related diseases and are a key factor in geroscience. The Core leaders, Drs. Van Remmen and Humphries are experts in redox biology and mitochondrial function in aging. The Specific Aims for this Core are the following: Specific Aim 1.) To perform accurate and comprehensive analyses of redox and energetic status, oxidative stress and damage markers and antioxidant protein expression. This includes: GSH/GSSG, and ATP, ADP, and AMP in cells/tissues measured by HPLC; oxidative damage (F2- isoprostanes (lipid peroxidation) and protein carbonyls (protein oxidation) in cells/tissues, and expression of mitochondrial, antioxidant and TCA cycle related proteins in collaboration with the MPAC. Redox and oxidative damage endpoints are present in limited amounts and may be unstable. The expertise in the Core allows for these analyses to be performed with the highest possible rigor, sensitivity and accuracy. In addition, the analyses we provide are especially appropriate for a Core because they require costly equipment and significant technologic expertise that can limit these types of analyses as routine measures in individual laboratories. Finally, because we are analyzing levels of highly specific biological compounds, our redox and oxidative damage assays can be applied to tissues or cells from any living organism. Specific Aim 2.) To perform quantitative high precision measurements of nicotinamide adenine dinucleotides (NADPH/NADP+, NADH/NAD+) and additional markers of redox status by LC/QTOF. NADPH/NADP+ and NADH/NAD+ will be measured in cell/tissue lysates or isolated mitochondria. Additional small molecule markers of redox status and TCA cycle intermediates can be quantified to complement analyses performed in Aim 1. Specific Aim 3.) To provide assays of mitochondrial function using permeabilized tissue, isolated mitochondria, and cell culture models. Mitochondrial function (respiration, ETC activities, oxidant generation) will be measured in isolated mitochondria or fresh tissue using the O2K Oroboros respirometer. Mitochondrial function in cells is measured using the Seahorse Extracellular Flux Analyzer. NADH oxidase, an overall measure of ETC activity and an indicator of mitochondrial function and quantity will be measured spectrophotometrically in frozen tissue. Overall, the Geroscience Redox Biology Core will provide a comprehensive panel of endpoint markers of redox balance and oxidative stress as well as measures of contributing factors such as mitochondrial function and free radical generation that can impact many aspects of aging, healthspan and age-related disease.