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 Nacetylaspartylglutamate 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

cAMP Response Element-Binding protein (CREB) binding to <i>Shati/Nat8L</i> promoter was activated by administration of Methamphetamine (METH).

<p>(<b>A</b> and <b>B</b>) ChIP (chromatin immunoprecipitation) assay was performed with antibodies for CREB and NF-κB on the nucleus accumbens (NAc) of mice repeatedly administered METH (2 mg/kg/day for 6 days. <i>s</i>.<i>c</i>.) For each group. ***<i>p</i> < 0.0001 vs control IgG (-380~-270), ^$$$<i>p</i> < 0.0001 vs saline, ###<i>p</i> < 0.0001 vs control IgG (-680~-270) (Newman–Keuls post hoc test). <i>n</i> = 9 (<b>C</b>) Repeated METH potentiated the immunoreactivity of p-CREB/CREB in NAc. *<i>p</i> < 0.05 vs saline group (Student’s <i>t</i>-test). <i>n</i> = 6. Error bars represent the SEM.</p

The effects of cAMP on <i>Shati/Nat8L</i> mRNA expression in PC12 cells and the nucleus accumbens slice.

<p>(<b>A</b>) PC12 cells treated with forskolin (10 μM) or DMSO as control for 2 h to perform qPCR. ***<i>p</i> < 0.0001 vs DMSO (Student’s <i>t</i>-test). (n = 4)(<b>B</b>) PC12 cells were transfected with PGl3-Basic Vector, including five kinds of promoter for <i>Shati/Nat8L</i> using luciferase assay. Detection of luciferase performed 22 h after 2-h forskolin stimulation (10 μM) using a Dual Luciferase Assay kit. ^##<i>p</i> < 0.001 vs –980/+120, −680/+120, and −380/+120 vector (Newman–Keuls post hoc test). (n = 4) (<b>C</b>) Brain slices treated with a PKA inhibitor, KT5720 (3 μM), for 30 min before methamphetamine (METH) treatment (1 mM) to perform qPCR. *<i>p</i> < 0.05 vs aCSF, ^#<i>p</i> < 0.05 vs METH (Newman–Keuls post hoc test). (n = 4–8) Error bars represent the SEM.</p

Dopamine D1 receptor potentiated expression of <i>Shati/Nat8L</i> mRNA by Methamphetamine (METH) in the Nucleus Accumbens (NAc).

<p>(<b>A</b>) The effects of pharmacological inhibition by dopamine D1 (SCH23390, 0.5 mg/kg) or D2 (sulpiride, 20 mg/kg) receptor antagonist in mice NAc were analyzed by qPCR. Dopamine D1 receptor antagonist inhibits METH-induced increases in <i>Shati/Nat8L</i> mRNA in NAc. *<i>p</i> < 0.05 vs saline, ^##<i>p</i> < 0.01 vs METH effects (Newman–Keuls post hoc test). n = 4–5. (<b>B</b>) Phosphorylation of cAMP response element-binding protein (CREB) in mice NAc were assayed by Western blotting. **<i>p</i> < 0.01 vs saline, ^#<i>p</i> < 0.005 vs METH effects (Newman–Keuls post hoc test) n = 5. The effects of Shati/Nat8L mRNA in mice NAc by (<b>C</b>) single and (<b>D</b>) repeated administrations of dopamine D1 receptor agonist, SKF38393 (0.5 mg/kg s.c.) were analyzed by real time RT-PCR. *<i>p</i> < 0.05 and **<i>p</i> < 0.01 vs saline in each condition (Student’s -<i>t</i>-test). n = 4. The influences of (<b>E</b>) single (n = 4) and (<b>F</b>) repeated administration of dopamine D2 agonist quinpirole hydrochloride (Quin) (0.05 and 0.5 mg/kg s.c.) assayed by qPCR. n = 5. (G) Dopamine D1 or D2 receptor antagonist (D1: SCH23390 10μM, D2: sulpiride 10μM) were pretreated before METH (1mM) stimulation in PC12 cells, followed by qPCR for Shati/Nat8L mRNA expression was determined. **<i>p</i> < 0.01 vs PBS, ^##<i>p</i> < 0.05 vs METH effects (Newman–Keuls post hoc test). n = 4. Error bars represent the SEM.</p

Luciferase assay using various fragments of <i>Shati/Nat8L</i> promoter regions in PC12 cells.

<p>(A) PC12 cells transfected with PGl3-Basic Vector containing five kinds of promoter region for the luciferase assay. Detection of luciferase 22 h after 2 h-methamphetamine (METH) stimulation using a Dual Luciferase Assay kit. ^##<i>p</i> < 0.05 and ^###<i>p</i> < 0.001 vs luciferase activities on PC12 cells comparing with −980 bp and −680 bp vector (Newman–Keuls post hoc test). <i>n</i> = 4. (<b>B</b>) METH induces activities of transcriptional factors in PC12 cells treated with METH.*<i>p</i> < 0.05 and **<i>p</i> < 0.01 vs PBS (Student’s <i>t</i>-test). <i>n</i> = 4. Error bars represent the SEM.</p

Effects of Methamphetamine (METH) on <i>Shati/Nat8L</i> mRNA in the Nucleus Accumbens (NAc) of mice and cultured cells.

<p><b>(A)</b><i>Shati/Nat8L</i> mRNA levels in NAc of mice repeatedly administered saline or METH (2 mg/kg/day) for 6 days. NAc samples taken 2 h after the last treatment. <i>n</i> = 4. *<i>p</i> < 0.05 vs saline group (Student’s <i>t</i>-test). Increasing levels of <i>Shati/Nat8L</i> mRNA induced by METH (1 μM) in (B) PC12 <b>(B)</b> and (C) Neuro2a cells. These cells were exposed to METH for 2 h. After the procedure, samples were taken for measurement of <i>Shati/Nat8L</i> mRNA **<i>p</i> < 0.001 vs PBS group (Student’s <i>t</i>-test). <i>n</i> = 4. Error bars represent the SEM.</p