Real-Time Imaging of NADPH Oxidase Activity in Living Cell by using Novel Bio-Sensor

Abstract

Production of reactive oxygen species (ROS) has been implicated as a causal agent in sporadic variants of many pathological conditions conditions, including cardiovascular, inflammatory and neurodegenerative diseases, aging, muscular dystrophy, and muscle fatigue. NADPH oxidases (Nox) have recently gained attention as an important source of ROS involved in redox signaling. However, our knowledge of the source of ROS has been limited by the relatively impoverished array of tools available to study them and the limitations of all imaging probes to provide meaningful spatial resolution. We have developed a redox sensitive protein to specifically assess Nox activity by linking redox-sensitive GFP (roGFP) to the Nox activating protein, p47phox. Our Nox redox probe allowed for dynamic live cell imaging of Nox activity associated with respiratory burst activity and skeletal muscle contraction. Grx1-roGFP has previously been reported as an efficient sensor in live imaging of the glutathione redox potential in different cellular compartments. The dynamic and specific analyses of redox-based assays under different physiological and pathological conditions are greatly attributed by these biosensors. using Grx1-roGFP and our novel p47phox –roGFP sensor, we were able to measure intra-cellular ROS production and glutathione redox potential in a mouse model of Duchenne Muscular Dystrophy with dynamic live cell imaging. Our studies suggest that NADPH oxidase plays a major role in both extra- and intracellular ROS generation in muscular dystrophy. As Nox has been linked to pathological redox signaling, our newly developed Nox biosensor will allow for development of therapeutic Nox inhibitors.