Adaptations in glutathione-based redox protein signaling pathways and alcohol drinking across species

Abstract

Alcohol use disorder (AUD) is the most prevalent substance use disorder but there is incomplete knowledge of the underlying molecular etiology. Here, we examined the cytosolic proteome from the nucleus accumbens core (NAcC) of ethanol drinking rhesus macaques to identify ethanol-sensitive signaling proteins. The targets were subsequently investigated using bioinformatics, genetic, and pharmacological manipulations in mouse models of ethanol drinking. Of the 1000+ cytosolic proteins identified in our screen, 50 proteins differed significantly between control and ethanol drinking macaques. Gene Ontology analysis of the differentially expressed proteins identified enrichment in pathways regulating metabolic processes and proteasome activity. Because the family of Glutathione S-transferases (GSTs) was enriched in these pathways, validation studies targeted GSTs using bioinformatics and genetically diverse mouse models. Gstp1 and Gstm2 were identified in Quantitative Trait Loci and published gene sets for ethanol-related phenotypes (e.g., ethanol preference, conditioned taste aversion, differential expression), and recombinant inbred strains that inherited the C57BL/6J allele at the Gstp2 interval consumed higher amounts of ethanol than those that inherited the DBA/2J allele. Genetic deletion of Gstp1/2 led to increased ethanol consumption without altering ethanol metabolism or sucrose preference. Administration of the pharmacologic activator of Gstp1/2, carnosic acid, decreased voluntary ethanol drinking. Proteomic analysis of the NAcC cytosolic of heavy drinking macaques that were validated in mouse models indicate a role for glutathione-mediated redox regulation in ethanol-related neurobiology and the potential of pharmacological interventions targeting this system to modify excessive ethanol drinking.

Keywords: Alcohol; Carnosic acid; GSTP; Nucleus accumbens; Posttranslational modification; S-glutathionylation.

Fig. 1.. Ethanol drinking in rhesus macaques across 12 months of open access.

(A) Schedule of polydipsia model in female ethanol drinking (n = 4) and control (n = 3) rhesus macaques. (B) Daily and (C) averaged ethanol consumption across 12-month open access drinking sessions. (D) Average BECs acquired at ~7 hours into drinking sessions. Data shown are mean ± SEM. <include color in figure>

Fig 2.. Proteomic analysis of ethanol drinking induced changes in cytosolic proteins from the rhesus macaque nucleus accumbens core.

(A) Volcano plot showing differentially expression proteins in the nucleus accumbens core from control and ethanol drinking macaques. (B) Gene Ontology enrichment analysis of down-regulated DEPs. (C) Enrichment analysis of up-regulated DEPs. <include color in figure>

Fig 3.. Enrichment of differentially expressed proteins in gene families and inhibitory neurons.

(A) Gene family enrichment analysis of the DEPs. (B) Dot plot showing enrichment of DEPs in inhibitory interneuron subclasses in the nucleus accumbens core of alcohol drinking monkeys. (C) Venn diagram of DEPs across 4 subclasses of interneurons. <include color in figure>

Fig 4.. BXD recombinant inbred strains of mice that inherit the B allele at the Gstp2 interval consume more ethanol than BXD mice that inherit the D allele.

(A) Daily ethanol (10%, v/v) consumption in male and female BXD mice in a home cage drinking model. Female strains are identified with ♀. (B) Ethanol drinking in BXD mice that inherit the B or D allele at the Gstp2 interval. ***p < 0.001. <include color in figure>

Fig 5.. Increased ethanol drinking in GstP1/P2^(−/−) mice.

(A) Ethanol intake in wildtype and GstP1/P2^(−/−) mice. (B) Correlational between ethanol drinking between wildtype and GstP1/P2^(−/−) mice. (C) Blood ethanol concentrations in wildtype and GstP1/P2^(−/−) mice measured after 2 h of ethanol access. (D) Blood ethanol concentrations following injection of ethanol (3 g/kg, i.p.). (E) Sucrose intake in wildtype and GstP1/P2^(−/−) mice. *p < 0.05. <include color in figure>

Fig 6.. Unmodified thiols in the NAcC of C57BL/6J mice drinking ethanol.

*p < 0.05.

Fig 7.. The GSTP activator carnosic acid reduced drinking and increased GSTP activity in the NAcC.

(A) Carnosic acid reduced drinking in C57BL/6J mice (B) without reducing body weight and (C) increased GSTP activity in the NAcC. (D) Ambulatory activity on days 1 and 7 of carnosic acid treatment. (E) Total distance traveled in locomotor activity chambers. *p < 0.05, **p < 0.01. <include color in figure>