Methamphetamine induces Shati/Nat8L expression in the mouse nucleus accumbens via CREB- and dopamine D1 receptor-dependent mechanism

Shati/Nat8L significantly increased in the nucleus accumbens (NAc) of mice after repeated methamphetamine (METH) treatment. We reported that Shati/Nat8L overexpression in mouse NAc attenuated METH-induced hyperlocomotion, locomotor sensitization, and conditioned place preference. We recently found that Shati/Nat8L overexpression in NAc regulates the dopaminergic neuronal system via the activation of group II mGluRs by elevated N-acetylaspartylglutamate following N-acetylaspartate increase due to the overexpression. These findings suggest that Shati/Nat8L suppresses METH-induced responses. However, the mechanism by which METH increases the Shati/Nat8L mRNA expression in NAc is unclear. To investigate the regulatory mechanism of Shati/Nat8L mRNA expression, we performed a mouse Shati/Nat8L luciferase assay using PC12 cells. Next, we investigated the response of METH to Shati/Nat8L expression and CREB activity using mouse brain slices of NAc, METH administration to mice, and western blotting for CREB activity of specific dopamine receptor signals in vivo and ex vivo. We found that METH activates CREB binding to the Shati/Nat8L promoter to induce the Shati/Nat8L mRNA expression. Furthermore, the dopamine D1 receptor antagonist SCH23390, but not the dopamine D2 receptor antagonist sulpiride, inhibited the upregulation of Shati/Nat8L and CREB activities in the mouse NAc slices. Thus, the administration of the dopamine D1 receptor agonist SKF38393 increased the Shati/Nat8L mRNA expression in mouse NAc. These results showed that the Shati/Nat8L mRNA was increased by METH-induced CREB pathway via dopamine D1 receptor signaling in mouse NAc. These findings may contribute to development of a clinical tool for METH addiction.


Introduction
Addiction and abuse of drugs such as methamphetamine (METH) are social problems worldwide [1]. It is well-known that METH induces specific behavioral responses such as PLOS  Animals Male C57BL/6J inbred mice were acquired from Nihon SLC Inc. Japan (Shizuoka, Japan) were 8 weeks old. Mice and kept in the animal institute of University of Toyama were in a temperature-and humidity-controlled environment under a 12-h light/12-h dark cycle (lights on at 8:00) and had ad libitum access to food and water. The health and welfare of the animals was monitored by staff at least once a day. All mice were quickly decapitated by animal guillotine without feeling any suffering, since the fresh brain tissues were needed for the isolation of mRNA or brain slices. This procedure were done without anesthesia to avoid the effect of anesthetic drugs. All procedures followed the National Institute of Health Guideline for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised in 1996) and were approved by the committee for Animal Experiments of the University of Toyama (Permit Number A2015-PHA23). In the permission, It has been stated that even during the experiment period, if an animal experiences symptoms of torture (self-injury behavior, abnormal posture, crying etc.) and rapid weight loss (more than 20% in several days), take measures of euthanasia, with sodium pentobarbital (120mg/kg). However no mice were observed in such a situation in this study.

Transfection and dual luciferase assay
Dual luciferase assay was performed according to a method previously reported [18]. pGL3-Basic vector containing Shati/Nat8L promoter was transfected into PC12 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's recommendations. In brief, cells were incubated to confluency in 24-well plates for 24 h and exposed to a mixture of 2 μl/well of lipofectamine 2000 and 0.8 μg/well of plasmid DNA (Shati/Nat8L promoter-driven pGL3-Basic Vector 0.5 μg/well and CMV-Renilla luciferase 0.3 μg/well). Twenty-four hours after the transfections, the cells were incubated in a medium containing phosphate-buffered saline or METH (1 μM) for 2 h. The mediums were changed to the normal one, and then the cells were incubated for 22 h. A reporter assay was performed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) following the instructions in the manual. The activity of CREB and NF-κB were determined same protocol as Shati/Nat8L promoter's one using CRE-luc and κB-luc vectors instead of Shati/Nat8L vector.

Western blotting
Mice were decapitated 2 h after the last METH-treatment (2 mg/kg, 6 days), and a NAc fraction was taken by brain slice. In the brain slice experiments, we also took the slices immediately after drug stimulations [artificial cerebrospinal fluid (aCSF) or METH]. NAc tissues were added to RIPA buffer (50 mM Tris-HCl pH 7.5, 152 mM NaCl, 5 mM EDTA, 1% Tri-tonX-100, 0.5% sodium deoxy cholate, 1 mM PMSF, 2% protease inhibitor cocktail, 1% phosphatase inhibitor cocktail), sonicated on ice, and then centrifuged at 20,000g for 15 min at 4˚C. A volume of sample buffer (312.5 mM Tris-HCl, 25% 2-mercaptoethanol, 10% SDS, 25% sucrose, 0.025% bromophenol blue) five times the supernatant volume was added. The mixture was then altered by thermal denaturation at 95˚C for 5 min. The target proteins were isolated using a SDS-polyacrylamide gel electrophoresis (PAGE) method and removed from the polyacrylamide gel to a polyvinylidene fluoride membrane (Immobilon-P Trans Membrane, Merck Millipore, Darmstadt, Germany) using a semi-dry transfer method. The blots were blocked for 1 h at room temperature using 5% skim milk in Trisbuffered saline solution containing 0.1% Tween-20 (TBS-T). The membranes were incubated with polyclonal antibodies (CREB 48H2 Rabbit mAb, phospho-CREB Ser133, or 87G3 Rabbit mAb; Cell Signaling Technology) and diluted 1:1,000 in TBS-T containing 2% skim milk at 4˚C for 16 h. The blots were then washed and incubated with the secondary antibody, horseradish peroxidase (HRP)-linked goat-rabbit IgG (Cell Signaling Technology). The HRP was detected using an Amersham ECL Plus Western Blotting Reagent Pack (GE Healthcare).

Experiments using mouse brains
Brain slice experiments were performed following the protocol provided with the ChIP assay kit as described previously [19]. C57BL/6J mice were quickly decapitated, and their brains were removed and placed into ice-cold aCSF saturated with oxygen (95% O2-5% CO2 mixture, pH 7.4). Coronal mice brains were cut (400 μm) in iced aCSF and transferred to a recovery chamber containing aCSF for 50 min at room temperature. Brain slices were stimulated with METH (100 μM or 1 mM) or aCSF for 1 h after the stimulation of aCSF, PKA inhibitor KT5720 (3μM), dopamine D1 receptor antagonist SCH23390 (10 μM) or dopamine D2 receptor antagonist sulpiride (10 μM) in aCSF for 30 min, and then NAc tissues were taken for measurement of Shati/Nat8L mRNA. The accurate location of NAc structure was based on visual inspection of each section using a stereomicroscope and compared with the stereotaxic atlas of mouse brain [20]. NAc structures were placed on dry ice and stored at -80˚C until use.

Statistical analysis
All data were expressed as the mean ±SEM. Statistical differences between the two groups were determined using Student's t-test. Statistically significant differences among values for individual groups were determined by analysis of variance, followed by the Student-Newmann-Keuls post hoc test when F ratios were significant (p < 0.05).

Results
Shati/Nat8L' mRNA was increased by METH treatment in NAc and PC12 cells  Fig 1C). These results were consistent with those of our previous report [8,21], which indicated that PC12 cells could upregulate Shati/Nat8L treated with METH. Therefore, PC12 cells were used for the luciferase assay in this study to clarify the regulatory system of Shati/Nat8L production.
Increased cAMP by forskolin potentiated Shati/Nat8L expression in PC12 cells and brain slices The ChIP assay results showed that CREB was necessary for potentiation of Shati/Nat8L expression. To investigate the upstream of CREB in the Shati/Nat8L site, we focused on cAMP, which activates PKA following CREB activity. Stimulation of forskolin (10 μM), an inducer for cAMP signaling, increased Shati/Nat8L mRNA in PC12 cells (Fig 4A). Moreover, the luciferase assay results supported the possibility that CREB induced Shati/Nat8l expression in PC12 cells (Fig 4B). Furthermore, stimulation of KT5720 (3 μM) were significantly inhibited the expression of Shati/Nat8L mRNA expression induced by METH in mice brain slices ( Fig  4C: aCSF (Fig 5C  and 5D). However, dopamine D2 agonist quinpirole hydrochloride had no effects on Shati/ Nat8L mRNA expression in NAc (Fig 5E and 5F). Alternatively in PC12 cells SCH23390, but not sulpiride, inhibited the increase in Shati/Nat8L mRNA ( These results supported a putative mechanism in which Shati/Nat8L expression is regulated by activation of dopamine D1 receptor signaling.

Discussion
Shati/Nat8L has been shown to be increased in NAc of mice after repeated treatment of METH [8]. Important neuronal roles of Shati/Nat8L, in addition to the function of lipid turnover in brown adipocytes, were subsequently demonstrated [11,12,14,22,23]. Recently, it was reported that NAA produced from Shati/Nat8L was associated with Canavan disease [24]. Although these reports have shown that Shati/Nat8L has important physiological functions in the central nervous system and peripheral tissues, the regulatory mechanism of Shati/Nat8L expression has remained unknown. In the present study, we attempted to elucidate the mechanism of Shati/Nat8L production at the molecular level. First, we reconfirmed the activation of Shati/Nat8L mRNA expression in NAc of mice repeatedly treated with METH ( Fig 1A). This finding of inductive activity agreed with our previously reported findings [8]. We also demonstrated that the increase in Shati/Nat8L mRNA by METH was regulated by CREB activity via dopamine D1 receptor signaling in NAc of mice (Figs 3-5). Because PC12 cells have DAT, Tyrosine hydroxylase, and dopamine receptors, the cells mimicked NAc of mice [7]. Moreover, forskolin, an inducer of cAMP in cells, also mimicked METH-induced luciferase activity and Shati/Nat8L mRNA induction (Fig 4). These results indicated that Shati/Nat8L expression depended on the CREB and cAMP pathway. Shati/Nat8L mRNA was not increased by treatment with METH at Neuro2a (Fig 1B) because Neuro2a has not been considered to have tyrosine hydroxylase and D1R [25,26]. Furthermore, overexpression of the drd1a gene, but not the drd2 gene, in Neuro2a induced Shati/Nat8L mRNA (S1 Fig). These results also support our conclusion that Shati/Nat8L production is dependent on the dopamine D1 receptor pathway.
We have previously reported that Shati/Nat8L overexpression in NAc controlled the dopaminergic system via activation of mGluR3 by elevated NAAG following NAA production [11]. We had thought that overexpression of Shati/Nat8L in Nac interneurons indirectly affected sensitization to METH and dopamine release to NAc. Nevertheless, a previous report indicated that METH-induced Shati/Nat8L potentiation occurred at D1-MSN, which directly released GABA to the GABA interneurons of VTA [7]. Thus, D1R-signal-potentiated NAAG appeared to suppress GABA release from D1-MSN to GABA interneurons in VTA via activation of mGluR3 at D1-MSN.
Previously, we reported that Shati/Nat8l knockout caused D1R over localization on cell surfaces in NAc of mice and that the mice exhibited stronger responses to SKF38393 and METH than did WT mice [14]. Consistent with the present and previous results, overexpression of dominant negative CREB in D1R-expressed cells has been shown to increase the response to cocaine [27]. These results support our finding that Shati/Nat8L expression had a suppressive role in drug addiction downstream of dopamine D1 receptor signaling. Furthermore, these reports also indicated that Shati/Nat8L expression by CREB on D1R signaling was regulated via a cycle involving D1R signaling to CREB activity, CREB to Shati/Nat8L expression, and Shati/Nat8L to D1R localization.
In the present study, we also showed that Shati/Nat8L expression was regulated by NF-κB. Exogenous TNF-alpha in NAc has been shown to attenuate METH-induced addiction [28]. Previously, we also showed that Shati/Nat8L overexpression in PC12 cells increased TNFalpha mRNA and also that Shati/Nat8L KO mice had a lower level of LITAF upstream of TNFalpha in the brain [10]. The association of NF-κB with Shati/Nat8L promotor using luciferase assay could be a secondary effect (Fig 2A and 2B). The finding indicates that the promoter activity of NF-κB were regulated by TNF-alpha due to Shati/Nat8l upregulation induced by METH stimulation. These results indicated that the Shati/Nat8L gene may be associated with the cytokine family.
In conclusion, we demonstrated the importance of the Shati/Nat8L promoter region and of CREB to Shati/Nat8L expression, and that expression of Shati/Nat8L mRNA was regulated especially by activation of CREB. These results suggest that inactivation of this mechanism may be a risk factor of addiction.