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" 0.71% ± 0.12 H₂O₂ during NAD⁺-linked resting, 0.21% ± 0.04 during NAD⁺-linked active respiration, and 0.07% ± 0.02 during FAD⁺-linked active respiration, NOX converted 38% ± 13 of O₂ into H₂O₂. Our results indicate that NOX rather than mitochondria is the major source of synaptic H₂O₂. The present approach may assist in the identification of redox-modulating synaptic factors that underlie a variety of physiological and pathological processes in neurons.