Abstract
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.
Highlights
Substantial evidence indicates that the synapse is a center stage for brain physiology and pathology [1]
It is likely that the initial product of all NADPH oxidases (NOX) enzymes is O2∙−, which spontaneously dismutates to H2O2 via superoxide dismutase (SOD), it is clear that H2O2 is predominantly produced by several NOX isoforms, NOX4, Duox1, and Oxidative Medicine and Cellular Longevity
Since HRP has been shown to catalyze the oxidation of NADPH with subsequent generation of H2O2 [16], we compared NOX-dependent oxygen consumption and hydrogen peroxide production obtained by using the AR/HRP system and the HPO sensor
Summary
Substantial evidence indicates that the synapse is a center stage for brain physiology and pathology [1]. Mitochondria are one source of cellular ROS. Recent data indicate that NADPH oxidases (NOX), the enzyme family known to generate ROS as their only and primary function, are widely expressed in the CNS where they considerably contribute to ROS generation [7, 8]. NOX2 and NOX4 have been characterized in the neurons of adult mouse nervous system, potentially contributing to wide range of physiologic functions and to several neurological disorders [9]. It is likely that the initial product of all NOX enzymes is O2∙−, which spontaneously dismutates to H2O2 via superoxide dismutase (SOD), it is clear that H2O2 is predominantly produced by several NOX isoforms, NOX4, Duox, and Oxidative Medicine and Cellular Longevity
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