Contribution of cystine-glutamate antiporters to the psychotomimetic effects of phencyclidine

Abstract

Altered glutamate signaling contributes to a myriad of neural disorders, including schizophrenia. While synaptic levels are intensely studied, nonvesicular release mechanisms, including cystine-glutamate exchange, maintain high steady-state glutamate levels in the extrasynaptic space. The existence of extrasynaptic receptors, including metabotropic group II glutamate receptors (mGluR), pose nonvesicular release mechanisms as unrecognized targets capable of contributing to pathological glutamate signaling. We tested the hypothesis that activation of cystine-glutamate antiporters using the cysteine prodrug N-acetylcysteine would blunt psychotomimetic effects in the rodent phencyclidine (PCP) model of schizophrenia. First, we demonstrate that PCP elevates extracellular glutamate in the prefrontal cortex, an effect that is blocked by N-acetylcysteine pretreatment. To determine the relevance of the above finding, we assessed social interaction and found that N-acetylcysteine reverses social withdrawal produced by repeated PCP. In a separate paradigm, acute PCP resulted in working memory deficits assessed using a discrete trial t-maze task, and this effect was also reversed by N-acetylcysteine pretreatment. The capacity of N-acetylcysteine to restore working memory was blocked by infusion of the cystine-glutamate antiporter inhibitor (S)-4-carboxyphenylglycine into the prefrontal cortex or systemic administration of the group II mGluR antagonist LY341495 indicating that the effects of N-acetylcysteine requires cystine-glutamate exchange and group II mGluR activation. Finally, protein levels from postmortem tissue obtained from schizophrenic patients revealed significant changes in the level of xCT, the active subunit for cystine-glutamate exchange, in the dorsolateral prefrontal cortex. These data advance cystine-glutamate antiporters as novel targets capable of reversing the psychotomimetic effects of PCP.

Figure 1

N-acetylcysteine (NAC) prevents phencyclidine-evoked glutamate release in the rodent prefrontal cortex. Extracellular glutamate in the prefrontal cortex is depicted as change from baseline across 20 min samples. (a) A low dose of phencyclidine (PCP; 1 mg/kg, SC, N=8) failed to alter extracellular glutamate levels in the prefrontal cortex. (b) Rats injected with saline followed by PCP (3 mg/kg, SC; n=13) exhibited an increase in extracellular glutamate that was not observed in rats pretreated with N-acetylcysteine (90 mg/kg, IP; n = 13). ANOVA yielded an interaction between time and NAC treatment (F_15,360=1.74, P=.04) such that there was a main effect of time in rats pretreated with saline (0 mg/kg NAC; F_15,180=1.97, P=.02) but not NAC (90 mg/kg; F_15,180=0.87, P=0.596). (c) Extracellular glutamate levels in the prefrontal cortex expressed as mean (± SEM) area under the curve (AUC) following the PCP injection; ANOVA yielded a main effect of treatment (F_1,24 = 4.86, P = 0.037); * indicates an increase in AUC in rats pretreated with NAC relative to saline pretreated rats, Tukey HSD, p<.05.

Figure 2

A representative coronal section illustrating the placement of microdialysis probes within the prefrontal cortex. The tract created by the insertion of a microdialysis probe is evident by the tissue damage on the medial aspect of each hemisphere. Note, the left hemisphere of each coronal section is marked with a tissue punch, which is evident as a cavity in the right side of the above, inverted section.

Figure 3

N-acetylcysteine (NAC) blocks PCP-evoked t-maze deficits. (a) Mean (± SEM) accuracy in a discrete-trial T-maze task following an injection of PCP (0–3 mg/kg, SC; n = 4–13/group as indicated in the bars of the graph). Pretreatment (60 min) with PCP (1–3 mg/kg, SC) dose-dependently produced working memory deficits evident as a decrease in the percent of correct choices during ten daily trials; ANOVA yielded a main effect of treatment (F_3,33 = 41.99, P < 0.001). * indicates a difference from controls, Tukey HSD, p<.05. (b) Mean (± SEM) accuracy in a discrete-trial T-maze task in rats pretreated (90 min) with NAC (90 mg/kg, IP; n = 7–29/group) prior to PCP (0–3 mg/kg, SC). NAC administration reverses PCP-induced working memory deficits; ANOVA yielded a main effect of treatment (F_5,92 = 25.5, P < 0.001). * indicates a difference relative to rats receiving saline alone (sal + 0 PCP), Tukey HSD, p<.05. # indicates a difference relative to rats receiving saline and respective dose of PCP, Tukey HSD, p<.05.

Figure 4

The capacity of N-acetylcysteine (NAC) to reverse PCP-evoked t-maze deficits is blocked by inhibitors of cystine-glutamate exchange and group II mGluRs. (a) Mean (± SEM) accuracy in a discrete-trial T-maze task assessed during a drug-free session (sham) and a test day in which the cystine-glutamate antiporter inhibitor (S)-4-carboxyphenylglycine (CPG; 1 μM) was infused into the dorsolateral prefrontal cortex for 180 min before testing. Rats also received a systemic injection of N-acetylcysteine (0 or 90 mg/kg, IP; 160 min prior to testing) and phencyclidine (PCP; 0 or 2 mg/kg, SC; 60 min prior to testing) on the test day. Rats treated with PCP alone exhibited a deficit in working memory evident as a reduction in t-maze performance on the test day relative to the sham day (PCP, 2 mg/kg, SC; n = 4); this deficit was reversed by co-administration of N-acetylcysteine (90 mg/kg, IP; n = 5). Rats receiving CPG + N-acetylcysteine + PCP (n = 6) exhibited deficits in performance comparable to rats receiving only PCP or PCP + CPG (n = 6). ANOVA yielded a day x treatment interaction (F_5,25 = 5.235, P = .002). * indicates a difference relative to performance on respective sham day (T-test, P<.05). (b) Mean (± SEM) accuracy in a discrete-trial T-maze task in rats pretreated with NAC (90 mg/kg, IP; 90 min) and the group II mGluR antagonist LY341495 (1 mg/kg, IP; 70 min) prior to PCP (2 mg/kg, SC; n = 7–16/group). PCP produced a reduction in t-maze performance evident as a decrease in the percent of correct choices during ten daily trials, and this deficit was reversed by NAC pretreatment. Rats receiving NAC + LY341495 prior to PCP exhibited deficits in performance comparable to rats receiving PCP alone; ANOVA yielded a main effect of treatment (F_4,47 = 14.42, P < 0.001). * indicates a difference relative to vehicle controls, Tukey HSD, p<.05; # indicates a difference from rats treated with PCP + NAC, Tukey HSD, p<.05.

Figure 5

N-acetylcysteine (NAC) blocks PCP-evoked social withdrawal in a group II mGluR-dependent manner. Social interaction was assessed as the mean (± SEM) time unfamiliar rat pairs spent within 20 cm. PCP (3 mg/kg, SC; n = 6–14) produced a decrease in interaction time and this effect that was reversed by NAC (90 mg/kg, IP) pretreatment (90 min). LY341495 (1 mg/kg, IP) reversed the protective effects of NAC indicating a role for mGluR II receptors in the effects of NAC; ANOVA yielded a main effect of treatment (F_3,34 = 5.217, P = 0.005). * indicates a difference relative to vehicle controls, Tukey HSD, p<.05; # indicates a difference relative to rats treated with NAC + PCP, Tukey HSD, p<.05.

Figure 6

Protein expression of xCT is significantly higher in the dorsal lateral prefrontal cortex (DLPFC) of schizophrenic patients. (a) Levels of the catalytic subunit for cystine-glutamate antiporters, xCT protein, are expressed as a change (mean ± SEM) from controls (n = 27) in the ratio of xCT/β-tubulin measured from human DLPFC (n = 23; ANCOVA: F_1,45 = 5.6, P = 0.022), anterior cingulate cortex (ACC; n = 23; ANCOVA: F_1,45 = 0.03, P = 0.870), and hippocampus (n = 23; ANCOVA: F_1,45 = 0.69, P = 0.41). * indicates a difference from control tissue, p<.05. (b) Representative immunoblots illustrate bands with an apparent molecular mass of 55 kDa. (c) The mean (± SEM) levels of xCT in patients that received antipsychotic therapy within the last six weeks (n = 12) relative to those that had not (n = 10).

Figure 7

Knockdown or overexpression of xCT altered xCT RNA or protein levels as determined using semi-quantitative multi-plex RT-PCR (a), western blotting (b), and immunofluorescence (c). (a) The expression of hxCT RNA (724 bp) was equivalent between mock and control siRNA transfected cells. Cells transfected with hxCT siRNA (empty vector) exhibited a >90% reduction of hxCT transcript relative to mock controls. Cells co-transfected with mouse xCT (mxCT) cDNA plus hxCT siRNA expressed an increase in mxCT (182 bp) and a reduction in hxCT. The expression of the internal control, 18S rRNA, was equivalent or higher across all cells relative to mock controls. (b) The expression of xCT protein (55kD) was equivalent between mock and control siRNA transfected cells. Cells transfected with siRNA (+ empty vector) exhibited an 80% reduction of hxCT relative to mock controls. Transfection of cDNA mxCT restored xCT protein. β-actin levels were unchanged across all transfection conditions. (c) xCT labeling was evident in the mock and control siRNA transfected cells. Depletion of xCT labeling was observed in hxCT siRNA (+ empty vector) transfected cells. Transfection with mxCT cDNA along with hxCT siRNA restored xCT labeling. Collectively, these results demonstrated antibody specificity against the xCT protein for both human and mouse species.