ABSTRACT
Loss-of-function parkin mutations cause oxidative stress and degeneration of dopaminergic neurons in the substantia nigra. Several consequences of parkin mutations have been described; to what degree they contribute to selective neurodegeneration remains unclear. Specific factors initiating excessive reactive oxygen species production, inefficient antioxidant capacity, or a combination are elusive. Identifying key oxidative stress contributors could inform targeted therapy. The absence of Drosophila parkin causes selective degeneration of a dopaminergic neuron cluster that is functionally homologous to the substantia nigra. By comparing observations in these to similar non-degenerating neurons, we may begin to understand mechanisms by which parkin loss of function causes selective degeneration. Using mitochondrially targeted redox-sensitive GFP2 fused with redox enzymes, we observed a sustained increased mitochondrial hydrogen peroxide levels in vulnerable dopaminergic neurons of parkin-null flies. Only transient increases in hydrogen peroxide were observed in similar but non-degenerating neurons. Glutathione redox equilibrium is preferentially dysregulated in vulnerable neuron mitochondria. To shed light on whether dysregulated glutathione redox equilibrium primarily contributes to oxidative stress, we supplemented food with folic acid, which can increase cysteine and glutathione levels. Folic acid improved survival, climbing, and transiently decreased hydrogen peroxide and glutathione redox equilibrium but did not mitigate whole-brain oxidative stress.
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.
