Measuring Mitochondrial Hydrogen Peroxide Levels and Glutathione Redox Equilibrium in Drosophila Neuron Subtypes Using Redox-Sensitive Fluorophores and 3D Imaging

Abstract

Disruptions in mitochondrial redox activity are implicated in maladies ranging from those in which cells degenerate to those in which cell division is unregulated. This is not surprising given the pivotal role of mitochondria as ATP producers, reactive oxygen species (ROS) generators, and gatekeepers of apoptosis. While increased ROS are implicated in such a wide variety of disorders, pinpointing the cause of their hyperproduction is challenging. Elevated levels of ROS can result from increases in their production and/or decreases in their turnover. Disruptions in and/or hyperactivity of NADH-ubiquinone oxidoreductase or ubiquinone-cytochrome c oxidoreductase can cause excessive ROS generation. Alternatively, if respiration is functioning in a homeostatic manner, decreases in levels or activity of antioxidants like glutathione, CuZn- and Mn-superoxide dismutase, and catalase could result in excessive ROS. Because of the diversity of disorders in which oxidative damage occurs, the most effective therapeutic strategies may be those that address the putatively diverse causes of increased ROS. Strategies for determining antioxidant activity typically involve semiquantitative measurement of relative protein levels using immunochemistry and mass spectrometry. These methods can be applied to a variety of samples, but they do not lend themselves to detection of cell-specific analyses within tissue like brain.

Because we are interested in elucidating the cause of oxidative stress in selectively vulnerable brain neurons, we have taken advantage of the easily manipulatable genetics and high fecundity of the fly. Using a cell type-targeting approach, we have driven redox sensitive green fluorescent proteins (roGFP2 ) into the mitochondria of tyrosine hydroxylase-producing (dopaminergic) neurons. In oxidizing conditions, the fluorophore’s maximal excitation wavelength reversibly shifts. Therefore, the relative amount of mitochondrial protein oxidation can be determined by taking the ratio of fluorescence excited with two different lasers. In addition, these GFPs have been independently fused to human glutaredoxin-1 (mito-roGFP2-Grx1) and yeast oxidant receptor peroxidase (mito-roGFP2-Orp1), facilitating measurements of relative mitochondrial glutathione redox potential and H₂O₂ levels, respectively. In order to obtain a more comprehensive observation of redox states, we capture 3D images of roGFP2 excited by two different lasers. Mito- and cytoplasmic-roGFP2 -Grx1 and -Orp1 expression can be driven by hundreds of genetic drivers in Drosophila , facilitating fixed or living whole organism or tissue- and cell-specific redox measurements.