Multiplex Imaging of Activity‐Dependent Changes in Neuronal Redox Dynamics Using Compartment‐Specific Redox Probes

Abstract

Reactive oxygen species (ROS) are integral components of redox signaling in cells and mediate physiologically essential signaling across compartments. Under pathological conditions, excessive presence of ROS can lead to oxidative stress that damages cellular components and leads to cellular dysfunction and death. To understand dynamic redox changes and identify the mechanisms that disrupt redox homeostasis, we need high‐precision tools that can be used to define redox processes in live cells under physiological and pathophysiological conditions.Genetically encoded fluorescent sensors that measure cellular redox status can be targeted to specific subcellular compartments to study the redox changes in intracellular compartments during live‐cell imaging. Reduction‐oxidation‐sensitive green fluorescent proteins (roGFPs) are redox sensors designed by modifying a green fluorescent protein to encode two cysteines engineered proximal to the chromophore, and a change in green fluorescence is observed upon reduction of the disulfide bond formed by the cysteines. However, it has remained technically challenging to quantitatively correlate spatially distinct redox dynamics with subcellular resolution.To address this challenge, we developed genetically‐encoded sensors that can be used along with roGFP to visualize redox changes in multiple compartments within the same cell. Our approach utilized Förster‐type resonance energy transfer (FRET) in a spectral relay strategy that extends the fluorescence emission of the redox‐sensitive green fluorescent protein (roGFP) into red emission wavelengths. We targeted the sensors to mitochondria and cytosol in primary hippocampal neurons and monitored sensor response to oxidizing and reducing agents. Upon neuronal stimulation of the neurons with glutamate, we observed an increase in mitochondrial oxidation accompanied by a cytosolic reduction. Pre‐treatment with antioxidants like MitoTEMPO and N‐Acetyl Cysteine attenuated both the neuron‐activity dependent cytosolic reduction and mitochondrial oxidation implying that these redox changes were coupled. In a mixed culture system, astrocytes are known to have a neuroprotective effect from oxidative insults and glutamate excitotoxicity. We targeted the redox sensors in a neuron‐astrocyte co‐culture system to observe the neuroprotective effect of astrocytes on neuronal redox dynamics. Using this sensor in combination with the roGFP, we can conduct live‐cell imaging with spatio‐temporal accuracy to delineate sub‐cellular changes in redox dynamics that result in pathological oxidative stress.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.