Neuron Activity Dependent Redox Compartmentation Revealed with a Second Generation Red-Shifted Ratiometric Sensor

Area of Science:

• Neuroscience
• Cell Biology
• Biochemistry

Background:

• Oxidative stress is implicated in neurological disorders, but understanding subcellular redox dynamics during neuronal activity is challenging.
• Existing redox probes lack the multicolor capabilities needed for compartment-specific analysis in single cells, often masked by population averaging.

Purpose of the Study:

• To develop a second-generation, genetically encoded, excitation ratiometric redox-sensitive fluorescent protein sensor (rogRFP2) with improved red emission for quantitative live-cell imaging.
• To utilize rogRFP2 to investigate activity-dependent redox changes in individual neurons and their subcellular compartments.

Main Methods:

• Engineering of a second-generation red-shifted redox-sensitive fluorescent protein sensor (rogRFP2) using a Förster resonance energy transfer relay strategy.
• Quantitative live-cell imaging of cultured neurons using rogRFP2 to measure compartment-specific redox dynamics.
• Ratiometric one- and two-photon redox imaging in rat brain slices and Drosophila retinas.

Main Results:

• Observed an anticorrelation between mitochondrial oxidation and cytosolic reduction in response to neuronal activity.
• Demonstrated that this redox behavior is dependent on Complex I activity in the mitochondrial electron transport chain.
• Showed that cocultured astrocytes can modulate these activity-dependent redox changes.
• Validated rogRFP2 for ratiometric redox imaging in brain slices and fly retinas.

Conclusions:

• The novel rogRFP2 sensor enables quantitative, compartment-specific redox imaging in live cells, brain slices, and fly retinas.
• This tool provides new insights into the relationship between neuronal activity and subcellular redox changes.
• rogRFP2 is a powerful tool for advancing redox biology research in vitro and in vivo across model organisms.