Acute imidazenil treatment after the onset of DFP-induced seizure is more effective and longer lasting than midazolam at preventing seizure activity and brain neuropathology.

Diazepam (DZ), the preferred anticonvulsant benzodiazepine (BZ) for the treatment of organophosphate (OP) nerve agent-induced seizures and neuronal damage, has been associated with unwanted effects such as sedation, amnesia, cardiorespiratory depression, anticonvulsant tolerance, and dependence liability. In a search for safer and more effective anticonvulsant BZs against OP-induced seizure and neuronal damage, we have previously shown that imidazenil (IMD), a low-intrinsic efficacy positive allosteric modulator of gamma-aminobutyric acid (GABA) action at α1-containing GABA(A) receptors, which has high intrinsic efficacy at α2-, α3-, and α5-containing GABA(A) receptors, is more potent and longer lasting than DZ pretreatment at protecting rats from diisopropyl fluorophosphate (DFP)-induced electrocorticographic (ECoG) seizures and neuronal damage. The effects of IMD were observed at doses that are devoid of sedative, amnestic, and anticonvulsant tolerance actions. In the present study, we compared the anticonvulsant and neuroprotective effects of a combination of atropine (2 mg/kg, ip) and pyridine-2-aldoxime methochloride (2-PAM, 20 mg/kg, ip) with IMD (0.5 mg/kg, ip) or midazolam (MDZ, 0.5-2 mg/kg, ip) administered after the onset of DFP (1.5 mg/kg, sc)-induced seizure activity. The severity of DFP-induced ECoG seizures was assessed by continuous radio telemetry recordings in unrestrained and freely moving rats. Furthermore, the extent of neuronal damage was evaluated using a neuron-specific nuclear protein immunolabeling and fluoro-jade B staining procedure. We report here that IMD is more efficacious and longer lasting than sedating doses of MDZ in protecting rats from DFP-induced ECoG seizures and neuronal damage.

L-methionine decreases dendritic spine density in mouse frontal cortex.

Schizophrenia postmortem brain is characterized by gamma aminobutyric acid downregulation and by decreased dendritic spine density in frontal cortex. Protracted L-methionine treatment exacerbates schizophrenia symptoms, and our earlier work (Tremolizzo et al. and Dong et al.) has shown that L-methionine decreases reelin and GAD67 transcription in mice which is prevented by co-administration of valproate. In this study, we observed a decrease in spine density following L-methionine treatment, which was prevented by co-administration of valproate. Together with our earlier findings conducted under the same experimental conditions, we suggest that downregulation of spine density in L-methionine-treated mice may be because of the decreased expression of reelin and that valproate may prevent spine downregulation by inhibiting the methylation induced decrease in reelin.

Anticonvulsant, anxiolytic, and non-sedating actions of imidazenil and other imidazo-benzodiazepine carboxamide derivatives.

Recent evidence suggests that alpha1-containing GABA(A) receptors mediate the sedative, amnestic, and to some extent the anticonvulsant actions of non-selective benzodiazepine (BZ) receptor ligands, such as diazepam (DZ). Anxiolytic and in part, anticonvulsant actions of BZ ligands are mediated by alpha2-, alpha3-, and alpha5-containing GABA(A) receptors. This has resulted in increasing interest in developing BZ ligands with selective actions at GABA(A) receptors, including alpha2-, alpha3-, and alpha5-subunits, but devoid of efficacy at alpha1-containing receptors. To refine their spectrum of pharmacological actions, efforts are being made to minimize unwanted effects such as sedation, amnesia, and tolerance liabilities. A prototype for such BZ ligands is imidazenil (IMD), an imidazo-benzodiazepine carboxylic acid derivative that elicits potent anticonvulsant and anxiolytic actions at doses virtually devoid of sedative, cardio-respiratory depressant and amnestic effects, and anticonvulsant tolerance liability. To define the pharmacological profile of IMD and its derivatives, we compared the anticonflict (anxiolytic), anti-proconflict (antipanic), anti-bicuculline (BIC), and maximal electroshock seizure (MES) effects, and the suppression of locomotor activity by imidazo-benzodiazepine carboxylic acid derivatives to those of DZ and bretazenil (BTZ). We report here that IMD and one of its derivatives (RO 25-2775) possess dose-dependent anticonflict, anti-proconflict, and anti-BIC actions but failed to suppress locomotor activity. Like DZ, the other IMD derivatives (enazenil, RO 25-2776, and RO 25-2847) not only elicit dose-dependent anticonflict, anti-proconflict, anti-BIC, anti-MES effects but also suppress locomotor activity. In contrast, none of the IMD derivatives studied shows any similarity to BTZ, which elicits anticonflict, anti-proconflict actions and suppresses locomotor activity but is virtually inactive against BIC-induced tonic-clonic convulsions.

Lower number of cerebellar Purkinje neurons in psychosis is associated with reduced reelin expression.

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.

SSRIs act as selective brain steroidogenic stimulants (SBSSs) at low doses that are inactive on 5-HT reuptake.

Brain principal glutamatergic neurons synthesize 3alpha-hydroxy-5alpha-pregnan-20-one (Allo), a neurosteroid that potently, positively, and allosterically modulates GABA action at GABA(A) receptors. Cerebrospinal fluid (CSF) Allo levels are decreased in patients with posttraumatic stress disorder (PTSD) and major depression. This decrease is corrected by fluoxetine in doses that improve depressive symptoms. Emotional-like behavioral dysfunctions (aggression, fear, and anxiety) associated with a decrease of cortico-limbic Allo content can be induced in mice by social isolation. In socially isolated mice, fluoxetine and analogs stereospecifically normalize the decrease of Allo biosynthesis and improve behavioral dysfunctions by a mechanism independent from 5-HT reuptake inhibition. Thus, fluoxetine and related congeners facilitate GABA(A) receptor neurotransmission and effectively ameliorate emotional and anxiety disorders and depression by acting as selective brain steroidogenic stimulants (SBSSs).

Imidazenil, a non-sedating anticonvulsant benzodiazepine, is more potent than diazepam in protecting against DFP-induced seizures and neuronal damage.

Organophosphate (OP)-nerve agent poisoning may lead to prolonged epileptiform seizure activity, which can result in irreversible neuronal brain damage. A timely and effective control of seizures with pharmacological agents can minimize the secondary and long-term neuropathology that may result from this damage. Diazepam, the current anticonvulsant of choice in the management of OP poisoning, is associated with unwanted effects such as sedation, amnesia, cardio-respiratory depression, anticonvulsant tolerance, and dependence liabilities. In search for an efficacious and safer anticonvulsant benzodiazepine, we studied imidazenil, a potent anticonvulsant that is devoid of sedative action and has a low intrinsic efficacy at alpha1- but is a high efficacy positive allosteric modulator at alpha5-containing GABA(A) receptors. We compared the potency of a combination of 2 mg/kg, i.p. atropine with: (a) imidazenil 0.05-0.5 mg/kg i.p. or (b) equipotent anti-bicuculline doses of diazepam (0.5-5 mg/kg, i.p.), against diisopropyl fluorophosphate (DFP; 1.5 mg/kg, s.c.)-induced status epilepticus and its associated neuronal damage. The severity and frequency of seizure activities were determined by continuous radio telemetry recordings while the extent of neuronal damage and neuronal degeneration were assessed using the TUNEL-based cleaved DNA end-labeling technique or neuron-specific nuclear protein (NeuN)-immunolabeling and Fluoro-Jade B (FJB) staining, respectively. We report here that the combination of atropine and imidazenil is at least 10-fold more potent and longer lasting than the combination with diazepam at protecting rats from DFP-induced seizures and the associated neuronal damage or ongoing degeneration in the anterior cingulate cortex, CA1 hippocampus, and dentate gyrus. While 0.5 mg/kg imidazenil effectively attenuated DFP-induced neuronal damage and the ongoing neuronal degeneration in the anterior cingulate cortex, dentate gyrus, and CA1 hippocampus, 5 mg/kg or a higher dose of diazepam is required to produce similar protective effects. These finding suggests that imidazenil, a non-sedating anticonvulsant BZ ligand, is a more potent, effective, and safer drug than diazepam in protecting rats from DFP-induced seizures and the associated neuronal damage and/or ongoing neuronal degeneration.

GABAergic promoter hypermethylation as a model to study the neurochemistry of schizophrenia vulnerability.

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.

Characterization of the action of antipsychotic subtypes on valproate-induced chromatin remodeling.

Recent advances in schizophrenia (SZ) research indicate that the telencephalic gamma-aminobutyric acid (GABA)ergic neurotransmission deficit associated with this psychiatric disorder probably is mediated by the hypermethylation of the glutamic acid decarboxylase 67 (GAD(67)), reelin and other GABAergic promoters. A pharmacological strategy to reduce the hypermethylation of GABAergic promoters is to induce a DNA-cytosine demethylation by altering the chromatin remodeling with valproate (VPA). When co-administered with VPA, the clinical efficacy of atypical antipsychotics is enhanced. This prompted us to investigate whether this increase in drug efficacy is related to a modification of GABAergic-promoter methylation via chromatin remodeling. Our previous and present results strongly indicate that VPA facilitates chromatin remodeling when it is associated with clozapine or sulpiride but not with haloperidol or olanzapine. This remodeling might contribute to reelin- and GAD(67)-promoter demethylation and might reverse the GABAergic-gene-expression downregulation associated with SZ morbidity.

Antipsychotic subtypes can be characterized by differences in their ability to modify GABAergic promoter methylation.

Recent advances in schizophrenia and bipolar disorder research suggest that a dysfunction of GABAergic neurotransmission that is operative in telencephalic structures may be an important dynamic mechanism associated with psychosis. We propose that this dysfunction is probably mediated by the hypermethylation of glutamic acid decarboxylase (GAD67), reelin and other gene promoters expressed in GABAergic neurons. A pharmacological strategy that reduces the hypermethylation of GABAergic promoters is to administer drugs (i.e., valproate [VPA]) that induce DNA demethylation by facilitating chromatin remodeling. The enhanced clinical efficacy of atypical antipsychotics when co-administered with VPA prompted us to investigate whether this increased drug efficacy is related to a modification of GABAergic promoter methylation via chromatin remodeling. Our previous and present results strongly suggest that when associated with VPA, clozapine or sulpiride, but not haloperidol or olanzapine, facilitate chromatin remodeling. This molecular remodeling may contribute to the induction of reelin (RELN) and GAD(67) (GAD1) promoter demethylation, and may reverse the downregulation of various GABAergic mRNAs and proteins detected in the telencephalon of patients with schizophrenia or bipolar disorders.

The combination of huperzine A and imidazenil is an effective strategy to prevent diisopropyl fluorophosphate toxicity in mice.

Diisopropyl fluorophosphate (DFP) causes neurotoxicity related to an irreversible inhibition of acetylcholinesterase (AChE). Management of this intoxication includes: (i) pretreatment with reversible blockers of AChE, (ii) blockade of muscarinic receptors with atropine, and (iii) facilitation of GABA(A) receptor signal transduction by benzodiazepines. The major disadvantage associated with this treatment combination is that it must to be repeated frequently and, in some cases, protractedly. Also, the use of diazepam (DZP) and congeners includes unwanted side effects, including sedation, amnesia, cardiorespiratory depression, and anticonvulsive tolerance. To avoid these treatment complications but safely protect against DFP-induced seizures and other CNS toxicity, we adopted the strategy of administering mice with (i) small doses of huperzine A (HUP), a reversible and long-lasting (half-life approximately 5 h) inhibitor of AChE, and (ii) imidazenil (IMI), a potent positive allosteric modulator of GABA action selective for alpha(5)-containing GABA(A) receptors. Coadministration of HUP (50 microg/kg s.c., 15 min before DFP) with IMI (2 mg/kg s.c., 30 min before DFP) prevents DFP-induced convulsions and the associated neuronal damage and mortality, allowing complete recovery within 18-24 h. In HUP-pretreated mice, the ED(50) of IMI to block DFP-induced mortality is approximately 10 times lower than that of DZP and is devoid of sedation. Our data show that a combination of HUP with IMI is a prophylactic, potent, and safe therapeutic strategy to overcome DFP toxicity.

Imidazenil: a low efficacy agonist at alpha1- but high efficacy at alpha5-GABAA receptors fail to show anticonvulsant cross tolerance to diazepam or zolpidem.

Whereas advances in the molecular biology of GABA(A) receptor complex using knock-out and knock-in mice have been valuable in unveiling the structure, composition, receptor assembly, and several functions of different GABA(A) receptor subtypes, the mechanism(s) underlying benzodiazepine (BZ) tolerance and withdrawal remain poorly understood. Studies using specific GABA(A) receptor subunit knock-in mice suggest that tolerance to sedative action of diazepam requires long-term activation of alpha1 and alpha5 GABA(A) receptor subunits. We investigated the role of long-term activation of these GABA(A) receptor subunits during anticonvulsant tolerance using high affinity and high intrinsic efficacy ligands for GABA(A) receptors expressing the alpha5 subunit (imidazenil) or alpha1 subunit (zolpidem), and a non-selective BZ recognition site ligand (diazepam). We report here that long-term activation of GABA(A) receptors by zolpidem and diazepam but not by imidazenil elicits anticonvulsant tolerance. Although anticonvulsant cross-tolerance occurs between diazepam and zolpidem, there is no cross-tolerance between imidazenil and diazepam or zolpidem. Furthermore, diazepam or zolpidem long-term treatment decreased the expression of mRNA encoding the alpha1 GABA(A) receptor subunit in prefrontal cortex by 43% and 20% respectively. In addition, diazepam but not zolpidem long-term treatment produced a 30% increase in the expression of the alpha5 GABA(A) receptor subunit mRNA in prefrontal cortex. In contrast, imidazenil which is devoid of anticonvulsant tolerance does not elicit significant changes in the expression of alpha1 or alpha5 GABA(A) receptor subunit. These findings suggest that long-term activation of GABA(A) receptors containing the alpha1 or other subunits but not the alpha5 receptor subunit is essential for the induction of anticonvulsant tolerance.

Neurosteroid biosynthesis regulates sexually dimorphic fear and aggressive behavior in mice.

The neurosteroid allopregnanolone is a potent positive allosteric modulator of GABA action at GABA(A) receptors. Allopregnanolone is synthesized in the brain from progesterone by the sequential action of 5alpha-reductase type I (5alpha-RI) and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD). 5alpha-RI and 3alpha-HSD are co-expressed in cortical, hippocampal, and olfactory bulb glutamatergic neurons and in output neurons of the amygdala, thalamus, cerebellum, and striatum. Neither 5alpha-RI nor 3alpha-HSD mRNAs is expressed in glial cells or in cortical or hippocampal GABAergic interneurons. It is likely that allopregnanolone synthesized in principal output neurons locally modulates GABA(A) receptor function by reaching GABA(A) receptor intracellular sites through lateral membrane diffusion. This review will focus on the behavioral effects of allopregnanolone on mouse models that are related to a sexually dimorphic regulation of brain allopregnanolone biosynthesis. Animal models of psychiatric disorders, including socially isolated male mice or mice that receive a long-term treatment with anabolic androgenic steroids (AAS), show abnormal behaviors such as altered fear responses and aggression. In these animal models, the cortico-limbic mRNA expression of 5alpha-RI is regulated in a sexually dimorphic manner. Hence, in selected glutamatergic pyramidal neurons of the cortex, CA3, and basolateral amygdala and in granular cells of the dentate gyrus, mRNA expression of 5alpha-RI is decreased, which results in a downregulation of allopregnanolone content. In contrast, 5alpha-RI mRNA expression fails to change in the striatum medium spiny neurons and in the reticular thalamic nucleus neurons, which are GABAergic.By manipulating allopregnanolone levels in glutamatergic cortico-limbic neurons in opposite directions to improve [using the potent selective brain steroidogenic stimulant (SBSS) S-norfluoxetine] or induce (using the potent 5alpha-RI inhibitor SKF 105,111) behavioral deficits, respectively, we have established the fundamental role of cortico-limbic allopregnanolone levels in the sexually dimorphic regulation of aggression and fear. By selectively targeting allopregnanolone downregulation in glutamatergic cortico-limbic neurons, i.e., by improving the response of GABA(A) receptors to GABA, new therapeutics would offer appropriate and safe management of psychiatric conditions, including impulsive aggression, irritability, irrational fear, anxiety, posttraumatic stress disorders, and depression.

Decreased corticolimbic allopregnanolone expression during social isolation enhances contextual fear: A model relevant for posttraumatic stress disorder.

Mice subjected to social isolation (3-4 weeks) exhibit enhanced contextual fear responses and impaired fear extinction. These responses are time-related to a decrease of 5alpha-reductase type I (5alpha-RI) mRNA expression and allopregnanolone (Allo) levels in selected neurons of the medial prefrontal cortex, hippocampus, and basolateral amygdala. Of note, the cued fear response was not different between group housed and socially isolated mice. In socially isolated mice, S-norfluoxetine, a selective brain steroidogenic stimulant (SBSS), in doses (0.45-1.8 mumol/kg) that increase brain Allo levels but fail to inhibit serotonin reuptake, greatly attenuates enhanced contextual fear response. SKF 105,111 (a potent 5alpha-RI inhibitor) decreases corticolimbic Allo levels and enhances the contextual fear response in group housed mice, which suggests that social isolation alters emotional responses by reducing the positive allosteric modulation of Allo at GABA(A) receptors in corticolimbic circuits. Thus, these procedures model emotional hyperreactivity, including enhanced contextual fear and impaired contextual fear extinction, which also is observed in posttraumatic stress disorder (PTSD) patients. A recent clinical study reported that cerebrospinal fluid Allo levels also are down-regulated in PTSD patients and correlate negatively with PTSD symptoms and negative mood. Thus, protracted social isolation of mice combined with tests of fear conditioning may be a suitable model to study emotional behavioral components associated with neurochemical alterations relating to PTSD. Importantly, drugs like SBSSs, which rapidly increase corticolimbic Allo levels, normalize the exaggerated contextual fear responses resulting from social isolation, suggesting that selective activation of neurosteroidogenesis may be useful in PTSD therapy.

Down-regulation of neurosteroid biosynthesis in corticolimbic circuits mediates social isolation-induced behavior in mice.

Allopregnanolone (ALLO), synthesized by pyramidal neurons, is a potent positive allosteric modulator of the action of GABA at GABA(A) receptors expressing specific neurosteroid binding sites. In the brain, ALLO is synthesized from progesterone by the sequential action of two enzymes: 5alpha-reductase type I (5alpha-RI) and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD). In the cortex, hippocampus, and amygdala, these enzymes are colocalized in principal glutamatergic output neurons [Agís-Balboa RC, Pinna G, Zhubi A, Maloku E, Veldic M, Costa E, Guidotti A (2006) Proc Natl Acad Sci USA 103:14602-14607], but they are not detectable in GABAergic interneurons. Using RT-PCR and in situ hybridization, this study compares 5alpha-RI and 3alpha-HSD mRNA brain expression levels in group housed and in socially isolated male mice for 4 weeks. In these socially isolated mice, the mRNA expression of 5alpha-RI was dramatically decreased in hippocampal CA3 glutamatergic pyramidal neurons, dentate gyrus granule cells, glutamatergic neurons of the basolateral amygdala, and glutamatergic pyramidal neurons of layer V/VI frontal (prelimbic, infralimbic) cortex (FC). In contrast, 5alpha-RI mRNA expression failed to change in CA1 pyramidal neurons, central amygdala neurons, pyramidal neurons of layer II/III FC, ventromedial thalamic nucleus neurons, and striatal medium spiny and reticular thalamic nucleus neurons. Importantly, 3alpha-HSD mRNA expression was unchanged by protracted social isolation (Si). These data suggest that, in male mice, after 4 weeks of Si, the expression of 5alpha-RI mRNA, which is the rate-limiting-step enzyme of ALLO biosynthesis, is specifically down-regulated in glutamatergic pyramidal neurons that converge on the amygdala from cortical and hippocampal regions. In socially isolated mice, this down-regulation may account for the appearance of behavioral disorders such as anxiety, aggression, and cognitive dysfunction.

Reviewing the role of DNA (cytosine-5) methyltransferase overexpression in the cortical GABAergic dysfunction associated with psychosis vulnerability.

In this review, we discuss changes in the regulation of gene expression in the central nervous system (CNS) associated with DNA (cytosine-5) methylation, chromatin remodeling and post-translational covalent modifications of histones. During brain development, abnormal intrinsic or extrinsic cues may compromise epigenetic processes regulating neural stem cell proliferation and differentiation and thus directly or indirectly could contribute to altered epiphenotypes leading to psychiatric disorders. These mechanisms, that include chromatin remodeling and reversible changes in promoter methylation patterns, are largely expressed by terminally differentiated cortical GABAergic neurons. These neurons are unique among various brain cell subtypes because they express high levels of DNA-methyltransferase-1 (DNMT1). Moreover, DNMT1 expression is further increased in schizophrenia (SZ) and bipolar (BP) disorder brains. To unravel how this pathological DNMT1 overexpression induces GABAergic neuronal dysfunction in SZ and in other psychoses, we report on how alterations in methylation modify the expression of susceptible vulnerability genes such as reelin or GAD67 in these neurons. The results encourage the view that promoter hypermethylation in GABAergic neurons that occurs in SZ represents a testable target for novel therapeutic strategies to treat this disorder.

Epigenetic mechanisms expressed in basal ganglia GABAergic neurons differentiate schizophrenia from bipolar disorder.

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.

S-adenosyl methionine and DNA methyltransferase-1 mRNA overexpression in psychosis.

Prefrontal cortex (Brodmann's area 9) levels of the methyl donor S-adenosyl methionine were increased by about two-fold in schizophrenia and bipolar disorder patients, but not in unipolar depressed patients compared with nonpsychiatric subjects from the Stanley Foundation Neuropathology Consortium (Bethesda, Maryland, USA). Neither age, brain weight and pH, hemisphere, post-mortem interval, disease onset/duration, nor cumulative dose of fluphenazine affected S-adenosyl methionine content. In schizophrenia and bipolar disorder patients, the increase of S-adenosyl methionine is associated with an overexpression of DNA methyltransferase-1 mRNA in Brodmann's area 9 GABAergic neurons. Hence, the increased expression of S-adenosyl methionine and DNA methyltransferase-1 may contribute to promoter cytosine 5-methylation and to downregulation of the expression of mRNAs encoding for reelin and GAD67 in cortical GABAergic neurons of schizhophrenia and bipolar disorder patients.

DNA methyltransferase inhibitors coordinately induce expression of the human reelin and glutamic acid decarboxylase 67 genes.

Reelin and glutamic acid decarboxylase 67 (GAD67) mRNAs and protein levels are substantially reduced in postmortem brains of patients with schizophrenia. Increasing evidence suggests that the observed down-regulation of reelin and GAD67 gene expression may be caused by dysfunction of the epigenetic regulatory mechanisms operative in cortical GABAergic interneurons. To explore whether human reelin and GAD67 mRNAs are coordinately regulated through DNA methylation-dependent mechanisms, we studied the effects of DNA methyltransferase inhibitors on reelin and GAD67 expression in NT-2 neuronal precursor cells. Competitive reverse transcription-polymerase chain reaction with internal standards was used to quantitate mRNA levels. The data showed that reelin and GAD67 mRNAs are induced in the same dose- and time-dependent manners. We further demonstrated that the activation of these two genes correlated with a reduction in DNA methyl-transferase activity and DNA methyltransferase 1 (DNMT1) protein levels. Time course Western blot analysis showed that DNMT1 protein down-regulation occurs temporally before the reelin and GAD67 mRNA increase. In addition, chromatin immunoprecipitation assays demonstrated that the activation of the reelin gene correlates with the dissociation of DNMT1 and methyl-CpG binding protein 2 (MeCP2) from the promoter, and an increased acetylation of histones H3 in the region. Together, our data strongly imply that human reelin and GAD67 genes are coordinately regulated through epigenetic mechanisms that include the action of DNMT1. Our study also suggests that negative regulation of the reelin gene involves methylation-dependent recruitment of DNMT1, MeCP2, and certain histone deacetylases, which most likely reduce the activity of the promoter by shifting the surrounding chromatin into a more compact state.

Valproic acid and chromatin remodeling in schizophrenia and bipolar disorder: preliminary results from a clinical population.

Levels of acetylated Histone 3 and 4 proteins are strongly predictive of a chromatin structure that is conducive to gene expression. In cell and animal studies, valproic acid is a potent inhibitor of histone deactylating enzymes, and consequently results in increased levels of acetylated Histone 3 (acH3) and acetylated Histone 4 proteins (acH4). To examine this effect in a clinical setting, 14 schizophrenic and bipolar patients were treated with valproic acid (Depakote ER), either as monotherapy or in combination with antipsychotics, over a period of 4 weeks. AcH3 and acH4 levels from lymphocyte nuclear protein extracts were measured by Western Blot. Treatment with Depakote ER resulted in a significant increase of acH3 and a trend-level increase of acH4. Levels of valproic acid were positively and significantly correlated with percent increase in acH3 but not acH4. Schizophrenia patients were significantly less likely to increase their acH3 and acH4 levels after 4 weeks on Depakote ER. The authors consider these results in the context of future application of HDAC inhibitors to the treatment of psychiatric disorders.