Acute Glutathione Depletion Restricts Mitochondrial ATP Export in Cerebellar Granule Neurons

Abstract

Decreases in GSH pools detected during ischemia sensitize neurons to excitotoxic damage. Thermodynamic analysis predicts that partial GSH depletion will cause an oxidative shift in the thiol redox potential. To investigate the acute bioenergetic consequences, neurons were exposed to monochlorobimane (mBCl), which depletes GSH by forming a fluorescent conjugate. Neurons transfected with redox-sensitive green fluorescent protein showed a positive shift in thiol redox potential synchronous with the formation of the conjugate. Mitochondria within neurons treated with mBCl for 1 h failed to hyperpolarize upon addition of oligomycin to inhibit their ATP synthesis. A decreased ATP turnover was confirmed by monitoring neuronal oxygen consumption in parallel with mitochondrial membrane potential (⌬ m ) and GSH-mBCl formation. mBCl progressively decreased cell respiration, with no effect on mitochondrial proton leak or maximal respiratory capacity, suggesting adequate glycolysis and a functional electron transport chain. This approach to "state 4" could be mimicked by the adenine nucleotide translocator inhibitor bongkrekic acid, which did not further decrease respiration when administered after mBCl. The cellular ATP/ADP ratio was decreased by mBCl, and consistent with mitochondrial ATP export failure, respiration could not respond to an increased cytoplasmic ATP demand by plasma membrane Na ؉ cycling; instead, mitochondria depolarized. More prolonged mBCl exposure induced mitochondrial failure, with ⌬ m collapse followed by cytoplasmic Ca 2؉ deregulation. The initial bioenergetic consequence of neuronal GSH depletion in this model is thus an inhibition of ATP export, which precedes other forms of mitochondrial dysfunction.A balance between the formation of reactive oxygen species, as normal byproducts of mitochondrial respiration (1), and the actions of antioxidants prevents oxidative stress and is crucial to neuronal survival (2). Together with the overactivation of glutamate receptors (excitotoxicity), oxidative stress is a result of the bioenergetic crisis that characterizes ischemia and plays a central role in the pathophysiology of the consequent neuronal damage (3). Neurons are particularly sensitive to oxidative damage and can be strongly sensitized to other injurious stimuli by levels of oxidative stress that are nontoxic per se (4). Similarly, Ca 2ϩ homeostasis is lost more quickly in cerebellar granule neurons (CGNs), 2 which show higher superoxide levels prior to the application of toxic concentrations of glutamate (5).The tripeptide glutathione is a key antioxidant that maintains protein thiols in a reduced state and scavenges H 2 O 2 in a reaction catalyzed by glutathione peroxidase (6, 7). In vivo, mitochondrial glutathione is partially lost during ischemia (8), and its supplementation in the form of glutathione ethyl ester can reduce the infarct size (9). Although mitochondria within cells deprived of GSH can eventually release cytochrome c and undergo opening of the permeability transition pore (PTP) (10...

Glutathionylation of Adenine Nucleotide Translocase Induced by Carbon Monoxide Prevents Mitochondrial Membrane Permeabilization and Apoptosis

“…In contrast, higher concentrations of GSSG (100 M) inhibit ADP/ATP translocation by ANT (Fig. 7A), which is in agreement with the findings of Vesce et al (40), who have shown that acute GSH depletion decreases ATP transport through the inner membrane.…”

Spare Respiratory Capacity Rather Than Oxidative Stress Regulates Glutamate Excitotoxicity after Partial Respiratory Inhibition of Mitochondrial Complex I with Rotenone

“…Thus, studies with CGNs have shown that the bioenergetic failure associated with DCD causes a dramatic increase in O 2 ⅐Ϫ levels in the deregulated cells (Vesce et al, 2004). Additionally, oxidative stress induced by acute glutathione depletion restricts CGN ATP generation by inhibiting mitochondrial ATP export to the cytoplasm (Vesce et al, 2005). As confirmed in Figure 1, oxidative stress, or at least an increase in matrix O 2 ⅐Ϫ levels, occurs in the presence of even low nanomolar concentrations of rotenone.…”

“…It is established that energetic insufficiency can exacerbate excitotoxicity , and, in this study, the ability to monitor the respiration of coverslip-attached neurons has allowed us to test the contribution of oxidative stress and respiratory insufficiency to the rotenone-enhanced cell death associated with chronic NMDA receptor activation in primary cultures of rat cerebellar granule neurons (CGNs). It must be emphasized that the conclusions apply specifically in the context of glutamate excitotoxicity and that because oxidative stress can damage ATP generating capacity (Vesce et al, 2005), it should not be assumed that these are mutually opposing concepts.…”

Glutathionylation Acts as a Control Switch for Uncoupling Proteins UCP2 and UCP3

“…GSH is the major regulator of cellular redox status due to its high concentration, reductive potential, and involvement in quenching oxidative stress (50). Covalent modification of proteins with GSH is also emerging as a key post-translational modification required for protein regulation in response to changes in redox environment.…”