Nicotine decreases DNA methyltransferase 1 expression and glutamic acid decarboxylase 67 promoter methylation in GABAergic interneurons

Abstract

Tobacco smoking is frequently abused by schizophrenia patients (SZP). The major synaptically active component inhaled from cigarettes is nicotine, hence the smoking habit of SZP may represent an attempt to use nicotine self-medication to correct (i) a central nervous system nicotinic acetylcholine receptor (nAChR) dysfunction, (ii) DNA-methyltransferase 1 (DMT1) overexpression in GABAergic neurons, and (iii) the down-regulation of reelin and GAD(67) expression caused by the increase of DNMT1-mediated hypermethylation of promoters in GABAergic interneurons of the telencephalon. Nicotine (4.5-22 micromol/kg s.c., 4 injections during the 12-h light cycle for 4 days) decreases DNMT1 mRNA and protein and increases GAD(67) expression in the mouse frontal cortex (FC). This nicotine-induced decrease of DNMT1 mRNA expression is greater (80%) in laser microdissected FC layer I GABAergic neurons than in the whole FC (40%), suggesting selectivity differences for the specific nicotinic receptor populations expressed in GABAergic neurons of different cortical layers. The down-regulation of DNMT1 expression induced by nicotine in the FC is also observed in the hippocampus but not in striatal GABAergic neurons. Furthermore, these data show that in the FC, the same doses of nicotine that decrease DNMT1 expression also (i) diminished the level of cytosine-5-methylation in the GAD(67) promoter and (ii) prevented the methionine-induced hypermethylation of the same promoter. Pretreatment with mecamylamine (6 micromol/kg s.c.), an nAChR blocker that penetrates the blood-brain barrier, prevents the nicotine-induced decrease of FC DNMT1 expression. Taken together, these results suggest that nicotine, by activating nAChRs located on cortical or hippocampal GABAergic interneurons, can up-regulate GAD(67) expression via an epigenetic mechanism. Nicotine is not effective in striatal medium spiny GABAergic neurons that primarily express muscarinic receptors.

Fig. 1.

Nicotine reduces DNMT1 mRNA expression in mouse FC and hippocampus but not in striatum. Mice were injected with nicotine s.c. four times a day during 12-h light cycle for 4 days. DNMT1 mRNA was measured 2 h after the last nicotine injection. Each value is the mean ± SE of five mice. Overall one-way ANOVA for DNMT1 mRNA levels in vehicle and nicotine treatment yielded a P < 0.003 for the FC and hippocampus. *, P < 0.01; **, P < 0.003 for Student-Newman-Keuls comparison between vehicle and nicotine. #, Data are expressed as fmol DNMT1mRNA/0.1 pmol NSE mRNA.

Fig. 2.

DNMT1 and GAD₆₇ colocalize in layers I and II of mouse frontal cortex. Double fluorescence microscopy images show DNMT1 immunoreactivity (green) and GAD₆₇ immunoreactivity (red). (Scale bars: 10 μm.)

Fig. 3.

Nicotine-induced reduction of DNMT1 mRNA is expressed at a higher level in laser-microdissected layer I FC than in the whole FC samples. Mice were treated with nicotine (22 μmol/kg s.c. four times a day for 4 days) and were killed 2 h after the last injection. DNMT1 mRNA values in vehicle treated mice are as follows: whole FC, 3.2 fmol DNMT1 mRNA/0.1 pmol NSE mRNA; layer I, 18 fmol DNMT1 mRNA/0.1 pmol NSE mRNA. Each value is the mean ± SE of five mice. *, P < 0.01; **, P < 0.001, Student's t test between vehicle and nicotine treatment.

Fig. 4.

DNMT1 in situ hybridization in the FC layer I/II (Upper) and layer V (Lower) in vehicle (Left) and nicotine-treated (Right) mice. Nicotine was given 22 mmol/kg s.c. four times per 12 h for 4 days. Brains were fixed 2 h after the last injection. (Scale bars: 10 μm).

Fig. 5.

Nicotine increases GAD₆₇ expression in FC but not in striatum. Mice were treated with nicotine four times a day during the 12 h of light cycle for 4 days. GAD₆₇ was measured 2 h after the last nicotine injection. Each value is the mean ± SE of 5–8 mice. **, P < 0.02; *, P < 0.05 Student's t test between vehicle (VEH) and nicotine treatment. (Inset) Typical Western immunoblot of GAD₆₇ and β-actin after 4–12% SDS/PAGE. Comparison between FC vehicle (lanes 1–3, serial dilutions of the same sample) and nicotine-treated (lanes 4–6, serial dilutions of the same sample) mice.

Fig. 6.

Nicotine reduces GAD₆₇ promoter methylation in the FC. V: received saline for four times a day for 5 days. N: received nicotine (4.5, 9, or 22 μmol/kg s.c. four times a day for 4 days) and saline on the fifth day. V+M: received saline for 2 days followed by methionine (5 mmol/kg, twice a day) for 3 days. N+M: received nicotine (22 μmol/kg) for 4 days. On the third and fourth days of nicotine treatment, mice also received methionine. On the fifth day, nicotine was suspended and mice received only methionine. Each value is the mean ± SE of three mice. Overall one-way ANOVA for GAD₆₇ promoter methylation yielded a P < 0.001. **, P < 0.01 for Student-Newman-Keuls multiple comparison between V versus V+M and V+M versus N+M; *, P < 0.05 for V versus N and V versus N+M.