Cellular metabolism is associated
with oxido-reductive (redox) reactions, in which electrons flow among various chemical species—according to a gradient in their redox potential (Eh
)—to reach the final acceptor, oxygen. Under physiological conditions, the cell maintains redox homeostasis by controlling the ratio of oxidized to reduced chemical species. While fluctuations in this balance occur under normal conditions, an excessive shift toward more oxidized states—oxidative stress—can be lethal for the cell (28
). The redox homeostasis of the cell is tightly controlled and the responsible regulatory mechanisms principally rely on thiol groups of cysteine (cys) residues. Because of their unique chemical properties, this functional group is extremely reactive toward reactive oxygen species (ROS), rendering cys primary ROS sensors (62
). Thiol groups buffer the oxidation in the cellular environment by undergoing an oxidative condensation of two thiols to form a disulfide. Therefore, in a given redox state, cys-containing species coexist in a dynamic equilibrium between the reduced and oxidized forms, in a ratio that reflects the intracellular redox state (59
). Extensive attention has been dedicated to the role of the small tri-peptide glutathione (GSH) in redox homeostasis. However, it is now very clear that protein thiols (pr-SH)—which are present in specialized proteins such as thioredoxins and in other proteins that possess primary functions other than controlling the redox state—also significantly contribute to redox homeostasis (1
). In several cases, the reversible oxidation of pr-SH also performs a fundamental regulatory function and acts as a molecular switch, whereby protein activity is modulated through oxidation or reduction of thiols in critical positions. In this respect, thiol oxidation constitutes the major mechanism of integration between ROS and signaling (17
). In summary, understanding how ROS modulate the intracellular thiol redox state in health and disease is critical to unraveling the responses activated by the cell to preserve normal function and prevent pathogenesis under conditions of redox imbalance.
Dopamine (DA) neurons in the substantia nigra pars compacta
(SNpc) are part of the basal ganglia circuitry and selectively degenerate in Parkinson's disease (PD) (10
). The study of redox homeostasis in SNpc DA neurons is of extreme interest to understand the biology of these cells, as their particular physiology is intrinsically associated with elevated ROS production. DA neurons are spontaneous pacemaking cells, and generate rhythmic action potentials in the absence of synaptic inputs. While most neuronal types use Na+
to generate their action potentials, SNpc DA neurons rely on Ca2+
, which enters the cytoplasm through L-type channels (12
). Over time, the spontaneous activity of these neurons could lead to elevated and harmful concentrations of cytosolic Ca2+
. This potential harm can be prevented by the buffering activity of certain organelles, such as mitochondria. However, this activity is invariably associated with ROS production (14
). In addition, excess ROS could derive directly from DA metabolism, which generates harmful by-products such as semiquinones and hydrogen peroxide (33
). The overall result is an increase in the basal levels of ROS in SNpc DA neurons that impairs their ability to tolerate further oxidative insults. In fact, systemic administration of pro-oxidants—which in principle target every cell in the brain—results in the selective damage of the DA system, and chronic administration of ROS producers, such as rotenone and paraquat, successfully mimic PD pathogenesis (9
). Taken together, the above evidence supports the concept that SNpc DA neurons must manage ROS in a particular manner. We hypothesize that the effects of mild, physiological amounts of ROS on the cellular redox state will be different in SNpc DA neurons as compared with other types of neurons that do not share the same physiology.
This hypothesis is testable if the redox state can be determined in specific single neurons of interest, with a method sensitive enough to detect mild variations in the intracellular redox state, within the physiological range. For this purpose, we developed a novel histochemical strategy for fluorescence imaging, in which oxidized and reduced thiol residues are labeled differentially. The technique provides a sensitive and ratiometric read-out of the redox state, which is expressed as the ratio between the signals of oxidized and reduced thiols. We applied this method to study the physiological redox response in DA neurons, which was elicited by nonlethal doses of the pro-oxidant rotenone. Our results indicate that the response in DA neurons is distinctive, and presents features that were not observed in cortical neurons. Moreover, sublethal doses of rotenone induce a reaction in SNpc DA neurons, but not in those in the ventral tegmental area (VTA) or cortical ones. Finally, we studied simultaneously, in the same cells, the intracellular redox state and the activation of the mitogen activated protein kinase (MAPK) pathway. We found that signaling activation is multiphasic and synchronized with the redox variations, with oxidation anticipating MAPK phosphorylation. Importantly, these features are hallmarks of DA neurons, as they were not observed in cortical neurons.
In summary, we have developed a new imaging approach to study redox homeostasis, and provided direct evidence in primary culture, zebrafish larvae, and rat that pro-oxidants elicit a distinctive response in DA neurons. While our new methodology is of general interest, and is broadly applicable to any study involving redox biology, our findings provide new important elements to understand the biology of DA neurons in health and disease, and to test future treatments for PD.