Activity‐Dependent and Compartment‐Specific Neuronal Redox Dynamics

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. However, it has remained technically challenging to quantitatively correlate spatially distinct redox dynamics with subcellular resolution. To address this challenge, we developed red fluorescent redox sensors that can be multiplexed with reduction‐oxidation‐sensitive green fluorescent proteins (roGFP) to image dual 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 roGFP into red emission wavelengths. The construct was then characterized both in solution and through dual compartment imaging in cell lines.ROS increases are ancillary to pathological processes like glutamate excitotoxicity and mitochondrial dysfunction that can propagate neuronal damage. Mitochondria help regulate the metabolic burden through the detoxification of neurotransmitters and the utilization of metabolic substrates, thereby regulating extracellular levels of glutamate. Following preliminary characterization of the sensor in cell lines, we targeted the sensors to mitochondria and cytosol in primary hippocampal neurons and monitored sensor response to oxidizing and reducing agents as well as to neuronal stimulation. Glutamate stimulation of neurons results in an increase in mitochondrial oxidation accompanied by a cytosolic reduction. Pharmacology indicates that the compartment‐specific redox responses are coupled and involve mitochondrial electron transport. Thus, using our sensors we have observed novel redox phenomena in primary neurons.