Joachim D. Uys

S-Glutathionylation: From Molecular Mechanisms to Health Outcomes

Redox homeostasis governs a number of critical cellular processes. In turn, imbalances in pathways that control oxidative and reductive conditions have been linked to a number of human disease pathologies, particularly those associated with aging. Reduced glutathione is the most prevalent biological thiol and plays a crucial role in maintaining a reduced intracellular environment. Exposure to reactive oxygen or nitrogen species is causatively linked to the disease pathologies associated with redox imbalance. In particular, reactive oxygen species can differentially oxidize certain cysteine residues in target proteins and the reversible process of S-glutathionylation may mitigate or mediate the damage. This post-translational modification adds a tripeptide and a net negative charge that can lead to distinct structural and functional changes in the target protein. Because it is reversible, S-glutathionylation has the potential to act as a biological switch and to be integral in a number of critical oxidative signaling events. The present review provides a comprehensive account of how the S-glutathionylation cycle influences protein structure/function and cellular regulatory events, and how these may impact on human diseases. By understanding the components of this cycle, there should be opportunities to intervene in stress- and aging-related pathologies, perhaps through prevention and diagnostic and therapeutic platforms.

The role of glutathione S-transferase P in signaling pathways and S-glutathionylation in cancer

Glutathione S-transferase P is abundantly expressed in some mammalian tissues, particularly those associated with malignancies. While the enzyme can catalyze thioether bond formation between some electrophilic chemicals and GSH, novel nondetoxification functions are now ascribed to it. This review summarizes recent material that implicates GSTP in mediating S-glutathionylation of specific clusters of target proteins and in reactions that define a negative regulatory role in some kinase pathways through ligand or protein:protein interactions. It is becoming apparent that GSTP participates in the maintenance of cellular redox homeostasis through a number of convergent and divergent mechanisms. Moreover, drug platforms that have GSTP as a target have produced some interesting preclinical and clinical candidates.

Early maternal separation alters the response to traumatization: resulting in increased levels of hippocampal neurotrophic factors.

Early life adversity predisposes individuals to the development of psychopathology in later life, especially depression and anxiety disorders. Prior history of stressors may also be a vulnerability factor for developing posttraumatic stress disorder (PTSD) in response to trauma. We examined the mechanisms underlying this phenomenon by employing two animal stress models, early maternal separation followed by later time-dependent sensitization (TDS). In animals exposed to adult TDS, those with prior early adversity did not differ from controls on tests of anxiety (elevated plus maze, open field), or HPA function (ACTH and corticosterone levels). However, those with prior early adversity had increased levels of neurotrophic factors (BDNF, NGF and NT-3) in both the dorsal and ventral hippocampus. Although early adversity is known to be associated with negative effects on neuronal function, it may also be associated with an increased ability to respond to subsequent stressors with compensatory mechanisms such as increased neurotrophic factor release.

Cocaine-Induced Adaptations in Cellular Redox Balance Contributes to Enduring Behavioral Plasticity

Impaired glutamate homeostasis in the nucleus accumbens has been linked to cocaine relapse in animal models, and results in part from cocaine-induced downregulation of the cystine–glutamate exchanger. In addition to regulating extracellular glutamate, the uptake of cystine by the exchanger is a rate-limiting step in the synthesis of glutathione (GSH). GSH is critical for balancing cellular redox in response to oxidative stress. Cocaine administration induces oxidative stress, and we first determined if downregulated cystine–glutamate exchange alters redox homeostasis in rats withdrawn from daily cocaine injections and then challenged with acute cocaine. Among the daily cocaine-induced changes in redox homeostasis were an increase in protein S-glutathionylation and a decrease in expression of GSH-S-transferase pi (GSTpi). To mimic reduced GSTpi, a genetic mouse model of GSTpi deletion or pharmacological inhibition of GSTpi by administering ketoprofen during daily cocaine administration was used. The capacity of cocaine to induce conditioned place preference or locomotor sensitization was augmented, indicating that reducing GSTpi may contribute to cocaine-induced behavioral neuroplasticity. Conversely, an acute cocaine challenge after withdrawal from daily cocaine elicited a marked increase in accumbens GSTpi, and the expression of behavioral sensitization to a cocaine challenge injection was inhibited by ketoprofen pretreatment; supporting a protective effect by the acute cocaine-induced rise in GSTpi. Together, these data indicate that cocaine-induced oxidative stress induces changes in GSTpi that contribute to cocaine-induced behavioral plasticity.

AKAP Signaling in Reinstated Cocaine Seeking Revealed by iTRAQ Proteomic Analysis

To identify candidate proteins in the nucleus accumbens (NAc) as potential pharmacotherapeutic targets for treating cocaine addition, an 8-plex iTRAQ (isobaric tag for relative and absolute quantitation) proteomic screen was performed using NAc tissue obtained from rats trained to self-administer cocaine followed by extinction training. Compared with yoked-saline controls, 42 proteins in a postsynaptic density (PSD)-enriched subfraction of the NAc from cocaine-trained animals were identified as significantly changed. Among proteins of interest whose levels were identified as increased was AKAP79/150, the rat ortholog of human AKAP5, a PSD scaffolding protein that localizes signaling molecules to the synapse. Functional downregulation of AKAP79/150 by microinjecting a cell-permeable synthetic AKAP (A-kinase anchor protein) peptide into the NAc to disrupt AKAP-dependent signaling revealed that inhibition of AKAP signaling impaired the reinstatement of cocaine seeking. Reinstatement of cocaine seeking is thought to require upregulated surface expression of AMPA glutamate receptors, and the inhibitory AKAP peptide reduced the PSD content of protein kinase A (PKA) as well as surface expression of GluR1 in NAc. However, reduced surface expression was not associated with changes in PKA phosphorylation of GluR1. This series of experiments demonstrates that proteomic analysis provides a useful tool for identifying proteins that can regulate cocaine relapse and that AKAP proteins may contribute to relapse vulnerability by promoting increased surface expression of AMPA receptors in the NAc.

Glutathione and redox signaling in substance abuse

Throughout the last couple decades, the cause and consequences of substance abuse has expanded to identify the underlying neurobiological signaling mechanisms associated with addictive behavior. Chronic use of drugs, such as cocaine, methamphetamine and alcohol leads to the formation of oxidative or nitrosative stress (ROS/RNS) and changes in glutathione and redox homeostasis. Of importance, redox-sensitive post-translational modifications on cysteine residues, such as S-glutathionylation and S-nitrosylation could impact on the structure and function of addiction related signaling proteins. In this commentary, we evaluate the role of glutathione and redox signaling in cocaine-, methamphetamine- and alcohol addiction and conclude by discussing the possibility of targeting redox pathways for the therapeutic intervention of these substance abuse disorders.

Nitrosative Stress-Induced S-Glutathionylation of Protein Disulfide Isomerase

Oxidative and nitrosative stress result in the accumulation of reactive oxygen and nitrogen species (ROS/RNS) which trigger redox-mediated signaling cascades through posttranslational modifications on cysteine residues, including S-nitrosylation (P-SNO) and S-glutathionylation (P-SSG). Protein disulfide isomerase (PDI) is the most abundant chaperone in the endoplasmic reticulum and facilitates protein folding via oxidoreductase activity. Prolonged or acute nitrosative stress blunts the activity of PDI through the formation of PDI-SNO and PDI-SSG. The functional implication is that reduced activity for the period of time leads to an accumulation of misfolded or unfolded proteins and activation of the unfolded protein response. Redox regulation of PDI and downstream signaling events provides an integration point for the functional determination of cell survival pathways. Herein, we describe the methodologies to globally identify S-glutathionylated targets of ROS/RNS; validate and identify the specific cysteine targets and characterize the structural and functional consequences.

Glutamatergic neuroplasticity in cocaine addiction.

Neuroadaptations among glutamatergic projections within the mesocorticolimbic circuits engaged by drugs of abuse have been described since the 1990s. There is now substantial evidence that drugs of abuse lead to long-term changes in glutamatergic signaling and encompass multiple levels of analysis. For example, cocaine induces changes in extracellular glutamate concentrations and in synaptic glutamatergic transmission. In addition, glutamate receptors are required for the expression of cocaine-related behaviors, and long-term changes have been reported in the expression of proteins at glutamatergic synapses, in glutamate-related redox regulation of neurons, and in glutamatergic synaptic and structural plasticity following chronic exposure to cocaine. In this chapter, we will describe the neurocircuitry involved, and will summarize evidence for adaptations in glutamatergic neuroplasticity as a mechanism for cocaine addiction. Finally, we will discuss progress in the development of glutamate-mediated pharmacotherapies for the treatment of cocaine dependence.

The development of behavioral and endocrine abnormalities in rats after repeated exposure to direct and indirect stress.

The present study compared the effects of direct and indirect stress on the behavior and hypothalamic-pituitary-adrenal axis of rats. Animals were placed in a two compartment box. In one compartment the direct stressed rat was subjected to electric foot shocks randomly applied for 10 minutes (0.5 mA of 1 s duration). In the adjacent compartment, the indirect stressed rats witnessed the application of these electric foot shocks. Our data showed substantial behavioral changes in the open field test, but limited effects in the elevated plus maze. The findings suggested that single and repeated stress exposure may have different consequences, that the effects of stress exposure may develop over time and persist for an extended period, and that both direct and indirect stressed rats displayed a hyposensitive HPA axis following acute restraint stress. Overall our observations moderately indicate direct exposure to elicit behavioral changes, and both direct and indirect exposure to stress to result in aberrations within the neuroendocrine system. With additional development our stress models may be considered for studying the complex interrelationship between an external stressor, and the experience of the organism.

Chronic intermittent ethanol exposure and withdrawal leads to adaptations in nucleus accumbens core postsynaptic density proteome and dendritic spines.

Alcohol use disorder is a chronic relapsing brain disease characterized by the loss of ability to control alcohol (ethanol) intake despite knowledge of detrimental health or personal consequences. Clinical and pre‐clinical models provide strong evidence for chronic ethanol‐associated alterations in glutamatergic signaling and impaired synaptic plasticity in the nucleus accumbens (NAc). However, the neural mechanisms that contribute to aberrant glutamatergic signaling in ethanol‐dependent individuals in this critical brain structure remain unknown. Using an unbiased proteomic approach, we investigated the effects of chronic intermittent ethanol (CIE) exposure on neuroadaptations in postsynaptic density (PSD)‐enriched proteins in the NAc of ethanol‐dependent mice. Compared with controls, CIE exposure significantly changed expression levels of 50 proteins in the PSD‐enriched fraction. Systems biology and functional annotation analyses demonstrated that the dysregulated proteins are expressed at tetrapartite synapses and critically regulate cellular morphology. To confirm this latter finding, the density and morphology of dendritic spines were examined in the NAc core of ethanol‐dependent mice. We found that CIE exposure and withdrawal differentially altered dendrite diameter and dendritic spine density and morphology. Through the use of quantitative proteomics and functional annotation, these series of experiments demonstrate that ethanol dependence produces neuroadaptations in proteins that modify dendritic spine morphology. In addition, these studies identified novel PSD‐related proteins that contribute to the neurobiological mechanisms of ethanol dependence that drive maladaptive structural plasticity of NAc neurons.