Research ReportNrf2 gene deletion fails to alter psychostimulant-induced behavior or neurotoxicity
Introduction
Repeated cocaine treatment produces enduring behavioral and neurochemical changes that arise, in part, from cocaine-induced plasticity in glutamate neurotransmission in the nucleus accumbens, a brain region critically involved in cocaine's addictive properties (Kalivas et al., 2005, Wolf, 1998). Among the long-term changes shown to be associated with cocaine-seeking in self-administration and reinstatement paradigms is an increase in synaptic glutamate release and a reduction in basal nonsynaptic extracellular levels of glutamate (Baker et al., 2003, McFarland et al., 2003). In the accumbens of rats, the basal extracellular level of glutamate is maintained by the cystine/glutamate exchanger (system xc-) (Baker et al., 2002), which promotes the substrate-dependent exchange of one extracellular cystine for one intracellular glutamate and is the rate-limiting step in synthesis of the intracellular antioxidant glutathione (McBean, 2002). Repeated cocaine treatment in rats reduces the affinity of the cystine/glutamate exchanger leading to lowered basal extracellular levels of glutamate in the accumbens and disinhibition of synaptic glutamate release (Baker et al., 2003, Moran et al., 2005). The restoration of system xc- by cysteine pro-drugs prevents the reduction in basal glutamate levels in the accumbens, as well as cocaine-seeking in the reinstatement model of relapse (Baker et al., 2003).
System xc- is a heterodimeric protein complex consisting of a catalytic chain (xCT), unique to the exchanger, and a heavy chain (4F2hc) common to many amino acid transporters and probably important for membrane localization (Bassi et al., 2001, Sato et al., 1999, Shih and Murphy, 2001). The expression of the gene encoding xCT is controlled, in part, by a transcription factor called NF-E2 related factor 2 (Nrf2) (Sasaki et al., 2002). Nrf2 is the major transcriptional regulator of phase 2 detoxifying enzymes by binding the antioxidant responsive element (ARE) (Itoh et al., 1997, Sasaki et al., 2002). Oxidative stress regulates Nrf2 transcriptional activity by controlling its intracellular distribution. Under basal conditions, Nrf2 is bound to Keap1 (Kelch-like ECH associating protein 1) and sequestered in the cytoplasm where it is targeted for ubiquitin-mediated degradation (Itoh et al., 1999, Katoh et al., 2005). Nrf2 and Keap1 exist as a complex that can move between the nucleus and the cytoplasm. In response to oxidative stress the Nrf2–Keap1 complex accumulates in the nucleus, where it dissociates leaving Nrf2 free to dimerize with Maf proteins to form a transcriptional activation complex capable of binding the ARE promotor region and increasing the expression of xCT and other phase 2 enzymes (Dinkova-Kostova et al., 2005, Itoh et al., 1997, Katsuoka et al., 2005).
Nrf2 gene deletion prevents the induction of cystine uptake produced by agents inducing oxidative stress (e.g. diethyl maleate, paraquat, CdCl2) in embryonic fibroblast (Sasaki et al., 2002) and macrophage cultures (Ishii et al., 2000). Also, in primary neuronal or glial cultures the loss of Nrf2 reduces the constitutive and inducible expression of cytoprotective genes (Lee et al., 2003a, Lee et al., 2003b, Shih et al., 2005), and Nrf2 over-expression in glia cells protects neurons from oxidative stress (Shih et al., 2003). Finally, constitutive deletion of the Nrf2 gene in vivo reduces the induction of glutathione-dependent enzymes in the liver, and augments cortical and striatal lesions induced by the mitochondrial complex II inhibitor 3-nitropropionic acid (Calkins et al., 2005, Chanas et al., 2002, Shih et al., 2005). Therefore, it has been concluded from in vitro and in vivo studies that Nrf2 controls the expression of xCT as part of a cellular response that protects cells against oxidative stress.
Based upon the predominantly in vitro data showing a role for Nrf2 in regulating xCT expression and the in vivo role of system xc- in the enduring behavioral and neurochemical effects of repeated cocaine we tested three hypotheses. 1) Homozygous deletion of the Nrf2 gene in mice (Nrf2 KO) will decrease system xc- basal activity by reducing xCT expression. 2) Nrf2 KO will show alterations in cocaine-induced locomotion, locomotor sensitization and conditioned place preference compared to wild-type littermates (WT). 3) Nrf2 KO will be more susceptible to methamphetamine(METH)-induced neurotoxicity. This latter hypothesis was based on evidence that METH-induced toxicity to dopamine (DA) terminals results, at least in part, from the induction of reactive oxygen species (ROS), such as superoxides and free hydroxyradicals (Pubill et al., 2005), that ROS promote Nrf2 induction of detoxifying enzymes (Kita et al., 2003), and that treatment with antioxidants attenuates METH toxicity (Hashimoto et al., 2004).
Section snippets
Nrf2 KO and WT mice have equivalent basal system xc- activity and levels of xCT
Fig. 1a shows that in ventral striatal slices (containing nucleus accumbens), homozygous Nrf2 deletion did not alter system xc- basal activity as measured by Na+-independent l-[3H]-glutamate uptake in pmoles/mg protein/minute. A two-way ANOVA using genotype and l-glutamate concentration as main factors, showed a significant effect of l-glutamate concentration (F(2,16) = 133.56, p < 0.0001) but no genotypic differences between Nrf2 KO and WT. Consistent with the apparent lack of effect by Nrf2
Discussion
The data show that constitutive homozygous deletion of the Nrf2 gene did not affect baseline activity of system xc- or protein levels of xCT in the ventral striatum. Correspondingly, Nrf2 deletion did not modify cocaine-induced behavioral effects either after acute or repeated administration, which have been shown to be associated with reduced system xc- activity in rats (Baker et al., 2003). Furthermore, Nrf2 deletion did not alter vulnerability to METH-induced DA depletion.
The lack of
Experimental subjects
Nrf2 KO adult mice (8–10 weeks age; 129sv × C57BL/6 background) originated from the laboratory of Dr. Yuet Wai Kan (Chan et al., 1996). Mice bred from heterozygous mating pairs resulted in WT, KO and heterozygous mice in the litter. At the age of 4 weeks, each mouse was genotyped. DNA was isolated from their tails and amplified by PCR using the following primers; primer 1 5′ GGAATGGAAAATAGCTCCTGCC 3′, primer 2 5′ GCCTGAGAGCTGTAGGCCC 3′, primer 3 5′ GGGTTTTCCCAGTCACGAC 3′. PCR conditions were as
Acknowledgments
This work was supported in part by USPHS grants DA-05369 and DA-03906. A.Y.S was supported by studentships from the Canadian Institute of Health Research (CIHR) and the Michael Smith Foundation for Health Research (MSFHR). T.H.M. is a CIHR investigator and MSFHR senior scholar. T.H.M. is also supported by grants from the Heart and Stroke Foundation of British Columbia and Yukon and the Canadian Stroke Network.
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