Resolving Contributions of Oxygen-Consuming and ROS-Generating Enzymes at the Synapse

Disruption of cellular redox homeostasis is implicated in a wide variety of pathologic conditions and aging. A fundamental factor that dictates such balance is the ratio between mitochondria-mediated complete oxygen reduction into water and incomplete reduction into superoxide radical by mitochondria and NADPH oxidase (NOX) enzymatic activity. Here we determined mitochondrial as well as NOX-dependent rates of oxygen consumption in parallel with H2O2 generation in freshly isolated synaptosomes using high resolution respirometry combined with fluorescence or electrochemical sensory. Our results indicate that although synaptic mitochondria exhibit substantially higher respiratory activities (8–82-fold greater than NOX oxygen consumption depending on mitochondrial respiratory state), NADPH-dependent oxygen consumption is associated with greater H2O2 production (6-7-fold higher NOX-H2O2). We also show that, in terms of the consumed oxygen, while synaptic mitochondria “leaked“ H2O2 during NAD