Nicotine improves cognitive performance and attention in both experimental animals and in human subjects, including patients affected by neuropsychiatric disorders. However, the specific molecular mechanisms underlying nicotine-induced behavioral changes remain unclear. We have recently shown in mice that repeated injections of nicotine, which achieve plasma concentrations comparable to those reported in high cigarette smokers, result in an epigenetically induced increase of glutamic acid decarboxylase 67 (GAD(67)) expression. Here we explored the impact of synthetic α(4)β(2) and α(7) nAChR agonists on GABAergic epigenetic parameters. Varenicline (VAR), a high-affinity partial agonist at α(4)β(2) and a lower affinity full agonist at α(7) neuronal nAChR, injected in doses of 1-5 mg/kg/s.c. twice daily for 5 days, elicited a 30-40% decrease of cortical DNA methyltransferase (DNMT)1 mRNA and an increased expression of GAD(67) mRNA and protein. This upregulation of GAD(67) was abolished by the nAChR antagonist mecamylamine. Furthermore, the level of MeCP(2) binding to GAD(67) promoters was significantly reduced following VAR administration. This effect was abolished when VAR was administered with mecamylamine. Similar effects on cortical DNMT1 and GAD(67) expression were obtained after administration of A-85380, an agonist that binds to α(4)β(2) but has negligible affinity for α(3)β(4) or α(7) subtypes containing nAChR. In contrast, PNU-282987, an agonist of the homomeric α(7) nAChR, failed to decrease cortical DNMT1 mRNA or to induce GAD(67) expression. The present study suggests that the α(4)β(2) nAChR agonists may be better suited to control the epigenetic alterations of GABAergic neurons in schizophrenia than the α(7) nAChR agonists.

Reelin is an extracellular matrix protein synthesized in cerebellar granule cells that plays an important role in Purkinje cell positioning during cerebellar development and in modulating adult synaptic function. In the cerebellum of schizophrenia (SZ) and bipolar (BP) disorder patients, there is a marked decrease ( approximately 50%) of reelin expression. In this study we measured Purkinje neuron density in the Purkinje cell layer of cerebella of 13 SZ and 17 BP disorder patients from the McLean 66 Cohort Collection, Harvard Brain Tissue Resource Center. The mean number of Purkinje neurons (linear density, neurons per millimeter) was 20% lower in SZ and BP disorder patients compared with nonpsychiatric subjects (NPS; n = 24). This decrease of Purkinje neuron linear density was unrelated to postmortem interval, pH, drugs of abuse, or to the presence, dose, or duration of antipsychotic medications. A comparative study in the cerebella of heterozygous reeler mice (HRM), in which reelin expression is down-regulated by approximately 50%, showed a significant loss in the number of Purkinje cells in HRM (10-15%) compared with age-matched (3-9 months) wild-type mice. This finding suggests that lack of reelin impairs GABAergic Purkinje neuron expression and/or positioning during cerebellar development.

Feeding mice, over 3 generations, an equicaloric diet in which alpha-linolenic acid, the dietary precursor of n-3 polyunsaturated fatty acids, was substituted by linoleic acid, the dietary precursor of n-6 polyunsaturated fatty acids, significantly increased body weight throughout life when compared with standard diet-fed mice. Adipogenesis observed in the low n-3 fatty acid mice was accompanied by a 6-fold upregulation of stearyl-coenzyme A desaturase 1 (Scd1), whose activity is correlated to plasma triglyceride levels. In total liver lipid and phospholipid extracts, the sum of n-3 fatty acids and the individual longer carbon chain acids, eicosapentaenoic acid (20:5n3), docosapentaenoic acid (22:5n3), and docosahexaenoic acid (22:6n3) were significantly decreased whereas arachidonic acid (20:4n6) was significantly increased. In addition, low n-3 fatty acid-fed mice had liver steatosis, heart, and kidney hypertrophy. Hence, reducing dietary alpha-linolenic acid, from 1.02 energy % to 0.16 energy % combined with raising linoleic acid intake resulted in obesity and had detrimental consequences on organ function.

The neuronal GABAergic mechanisms that mediate the symptomatic beneficial effects elicited by a combination of antipsychotics with valproate (a histone deacetylase inhibitor) in the treatment of psychosis (expressed by schizophrenia or bipolar disorder patients) are unknown. This prompted us to investigate whether the beneficial action of this combination results from a modification of histone tail covalent esterification or is secondary to specific chromatin remodeling. The results suggest that clozapine, or sulpiride associated with valproate, by increasing DNA demethylation with an unknown mechanism, causes a chromatin remodeling that brings about a beneficial change in the epigenetic GABAergic dysfunction typical of schizophrenia and bipolar disorder patients.

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 mumol/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 mumol/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.

In the cerebral prefrontal cortex (PFC), DNA-methyltransferase 1 (DNMT1), the enzyme that catalyzes the methylation of cytosine at carbon atoms in position 5 in CpG dinucleotides, is expressed selectively in GABAergic neurons and is upregulated in layers I and II of schizophrenia (SZ) and bipolar disorder patients with psychosis (BDP). To replicate these earlier findings and to verify whether overexpression of DNMT1 and the consequent epigenetic decrease of reelin and glutamic acid decarboxylase (GAD) 67 mRNA expression also occur in GABAergic medium spiny neurons of the caudate nucleus (CN) and putamen (PT) of SZ and BDP, we studied the entire McLean 66 Cohort (Harvard Brain Tissue Resource Center, McLean Hospital, Belmont, MA) including SZ and BDP, which were matched with nonpsychiatric subjects. The data demonstrate that in GABAergic medium spiny neurons of CN and PT, unlike in GABAergic neurons of layer I and II PFC, the increased expression of DNMT1 and the decrease of reelin and GAD67 occur in SZ but not in BDP. This suggests that different epigenetic mechanisms must exist in the pathogenesis underlying SZ and BDP and implies that these disorders might involve two separate entities that are characterized by a well-defined neuropathology.

Allopregnanolone (ALLO) and tetrahydrodeoxycorticosterone (THDOC) are potent positive allosteric modulators of GABA action at GABAA receptors. ALLO and THDOC are synthesized in the brain from progesterone or deoxycorticosterone, respectively, by the sequential action of two enzymes: 5alpha-reductase (5alpha-R) type I and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD). This study evaluates 5alpha-R type I and 3alpha-HSD mRNA expression level in mouse brain by using in situ hybridization combined with glutamic acid decarboxylase 67/65, vesicular glutamate transporter 2, glial fibrillary acidic protein, and S100beta immunohistochemistry. We demonstrate that 5alpha-R type I and 3alpha-HSD colocalize in cortical, hippocampal, and olfactory bulb glutamatergic principal neurons and in some output neurons of the amygdala and thalamus. Neither 5alpha-R type I nor 3alpha-HSD mRNAs are expressed in S100beta- or glial fibrillary acidic protein-positive glial cells. Using glutamic acid decarboxylase 67/65 antibodies to mark GABAergic neurons, we failed to detect 5alpha-R type I and 3alpha-HSD in cortical and hippocampal GABAergic interneurons. However, 5alpha-R type I and 3alpha-HSD are significantly expressed in principal GABAergic output neurons, such as striatal medium spiny, reticular thalamic nucleus, and cerebellar Purkinje neurons. A similar distribution and cellular location of neurosteroidogenic enzymes was observed in rat brain. Taken together, these data suggest that ALLO and THDOC, which can be synthesized in principal output neurons, modulate GABA action at GABAA receptors, either with an autocrine or a paracrine mechanism or by reaching GABAA receptor intracellular sites through lateral membrane diffusion.

The association of the histone deacetylase (HDAC) inhibitor valproate (VPA) with atypical antipsychotics has become a frequent treatment strategy for schizophrenia and bipolar disorder. Because the VPA doses administered are elevated, one cannot assume that the benefits of the VPA plus antipsychotic treatment are exclusively related to the covalent modifications of nucleosomal histone tails. We compared the actions of N-(2-aminophenyl)-4-[N-(pyridin-3-yl-methoxycarbonyl)aminomethyl]benzamide derivative (MS-275), which is a potent HDAC inhibitor in vitro, with the actions of VPA for their ability to (i) increase the acetylated status of brain nucleosomal histone tail domains and (ii) to regulate brain histone-RELN and histone-GAD67 promoter interactions. MS-275 increases the content of acetylhistone 3 (Ac-H3) in the frontal cortex. Whereas this response peaks after a s.c. injection of 15 micromol/kg, the increase in Ac-H3 content in the hippocampus becomes significant only after an injection of 60 micromol/kg, suggesting that MS-275 is 30- to 100-fold more potent than VPA in increasing Ac-H3 in these brain regions. In contrast to VPA, MS-275, in doses up to 120 micromol/kg, fails to increase Ac-H3 content in the striatum. Chromatin immunoprecipitation shows that MS-275 increases Ac-H3-RELN and Ac-H3-GAD67 promoter interaction in the frontal cortex. These results suggest that MS-275 is a potent brain region-selective HDAC inhibitor. It is likely that, in addition to MS-275, other benzamide derivatives, such as sulpiride, are brain-region selective inhibitors of HDACs. Hence, some benzamide derivatives may express a greater efficacy than VPA as an adjunctive to antipsychotics in the treatment of epigentically induced psychiatric disorders.

Cortical DNA-methyltransferase 1 (DNMT1) is preferentially expressed in interneurons secreting GABA where it very likely contributes to promoter CpG island hypermethylation, thus causing a down-regulation of promoter functions. To consolidate and expand on previous findings that, in the cortex of schizophrenia (SZ) brains, glutamic acid decarboxylase 67 (GAD67) expression is down-regulated whereas that of DNMT1 is up-regulated, we studied both parameters in Brodmann's area (BA) 9 from the McLean 66 Cohort Collection (Harvard Brain Tissue Resource Center, Belmont, MA). In BA9 of SZ and bipolar disorder patients with psychosis, DNMT1 mRNA and protein expression preferentially increases in layer I, II, and IV interneurons, and this increase is paralleled by a decreased number of GAD67 mRNA-positive neurons. The increase in DNMT1 and the decrease in GAD67-expressing neurons were unrelated to postmortem interval, pH, RNA quality, or to the presence, dose, or duration of antipsychotic (APS) medication, with the exception of a subgroup of SZ patients treated with a combination of valproate and APS in which the expression of DNMT1 failed to change. The DNMT1 increase and the GAD67 decrease in BA9 interneurons are significant features of SZ and bipolar disorder with psychosis. Interestingly, the DNMT1 increase failed to occur when patients with psychosis received a combination of valproate and APS treatment but not APS monotherapy.