Behavioral and molecular neuroepigenetic alterations in prenatally stressed mice: relevance for the study of chromatin remodeling properties of antipsychotic drugs.

We have recently reported that mice born from dams stressed during pregnancy (PRS mice), in adulthood, have behavioral deficits reminiscent of behaviors observed in schizophrenia (SZ) and bipolar (BP) disorder patients. Furthermore, we have shown that the frontal cortex (FC) and hippocampus of adult PRS mice, like that of postmortem chronic SZ patients, are characterized by increases in DNA-methyltransferase 1 (DNMT1), ten-eleven methylcytosine dioxygenase 1 (TET1) and exhibit an enrichment of 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC) at neocortical GABAergic and glutamatergic gene promoters. Here, we show that the behavioral deficits and the increased 5MC and 5HMC at glutamic acid decarboxylase 67 (Gad1), reelin (Reln) and brain-derived neurotrophic factor (Bdnf) promoters and the reduced expression of the messenger RNAs (mRNAs) and proteins corresponding to these genes in FC of adult PRS mice is reversed by treatment with clozapine (5 mg kg(-1) twice a day for 5 days) but not by haloperidol (1 mg kg(-1) twice a day for 5 days). Interestingly, clozapine had no effect on either the behavior, promoter methylation or the expression of these mRNAs and proteins when administered to offspring of nonstressed pregnant mice. Clozapine, but not haloperidol, reduced the elevated levels of DNMT1 and TET1, as well as the elevated levels of DNMT1 binding to Gad1, Reln and Bdnf promoters in PRS mice suggesting that clozapine, unlike haloperidol, may limit DNA methylation by interfering with DNA methylation dynamics. We conclude that the PRS mouse model may be useful preclinically in screening for the potential efficacy of antipsychotic drugs acting on altered epigenetic mechanisms. Furthermore, PRS mice may be invaluable for understanding the etiopathogenesis of SZ and BP disorder and for predicting treatment responses at early stages of the illness allowing for early detection and remedial intervention.

Toward the identification of peripheral epigenetic biomarkers of schizophrenia.

Schizophrenia (SZ) is a heritable, nonmendelian, neurodevelopmental disorder in which epigenetic dysregulation of the brain genome plays a fundamental role in mediating the clinical manifestations and course of the disease. The authors recently reported that two enzymes that belong to the dynamic DNA methylation/demethylation network-DNMT (DNA methyltransferase) and TET (ten-eleven translocase; 5-hydroxycytosine translocator)-are abnormally increased in corticolimbic structures of SZ postmortem brain, suggesting a causal relationship between clinical manifestations of SZ and changes in DNA methylation and in the expression of SZ candidate genes (e.g., brain-derived neurotrophic factor [BDNF], glucocorticoid receptor [GCR], glutamic acid decarboxylase 67 [GAD67], reelin). Because the clinical manifestations of SZ typically begin with a prodrome followed by a first episode in adolescence with subsequent deterioration, it is obvious that the natural history of this disease cannot be studied only in postmortem brain. Hence, the focus is currently shifting towards the feasibility of studying epigenetic molecular signatures of SZ in blood cells. Initial studies show a significant enrichment of epigenetic changes in lymphocytes in gene networks directly relevant to psychiatric disorders. Furthermore, the expression of DNA-methylating/demethylating enzymes and SZ candidate genes such as BDNF and GCR are altered in the same direction in both brain and blood lymphocytes. The coincidence of these changes in lymphocytes and brain supports the hypothesis that common environmental or genetic risk factors are operative in altering the epigenetic components involved in orchestrating transcription of specific genes in brain and peripheral tissues. The identification of DNA methylation signatures for SZ in peripheral blood cells of subjects with genetic and clinical high risk would clearly have potential for the diagnosis of SZ early in its course and would be invaluable for initiating early intervention and individualized treatment plans.

DNA-methylation gene network dysregulation in peripheral blood lymphocytes of schizophrenia patients.

The epigenetic dysregulation of the brain genome associated with the clinical manifestations of schizophrenia (SZ) includes altered DNA promoter methylation of several candidate genes. We and others have reported that two enzymes that belong to the DNA-methylation/demethylation network pathways-DNMT1 (DNA-methyltransferase) and ten-eleven translocator-1(TET1) methylcytosine deoxygenase are abnormally increased in corticolimbic structures of SZ postmortem brain. The objective of this study was to investigate whether the expression of these components of the DNA-methylation-demethylation pathways known to be altered in the brain of SZ patients are also altered in peripheral blood lymphocytes (PBL). The data show that increases in DNMT1 and TET1 and in glucocorticoid receptor (GCortR) and brain derived neurotrophic factor (BDNF) mRNAs in PBL of SZ patients are comparable to those reported in the brain of SZ patients. The finding that the expressions of DNMT1 and TET1 are increased and SZ candidate genes such as BDNF and GCortR are altered in the same direction in both the brain and PBL together with recent studies showing highly correlated patterns of DNA methylation across the brain and blood, support the hypothesis that a common epigenetic dysregulation may be operative in the brain and peripheral tissues of SZ patients.

Epigenetic GABAergic targets in schizophrenia and bipolar disorder.

It is becoming increasingly clear that a dysfunction of the GABAergic/glutamatergic network in telencephalic brain structures may be the pathogenetic mechanism underlying psychotic symptoms in schizophrenia (SZ) and bipolar (BP) disorder patients. Data obtained in Costa's laboratory (1996-2009) suggest that this dysfunction may be mediated primarily by a downregulation in the expression of GABAergic genes (e.g., glutamic acid decarboxylase67[GAD67] and reelin) associated with DNA methyltransferase (DNMT)-dependent hypermethylation of their promoters. A pharmacological strategy to reduce the hypermethylation of GABAergic promoters is to administer drugs, such as the histone deacetylase (HDAC) inhibitor valproate (VPA), that induce DNA-demethylation when administered at doses that facilitate chromatin remodeling. The benefits elicited by combining VPA with antipsychotics in the treatment of BP disorder suggest that an investigation of the epigenetic interaction of these drugs is warranted. Our studies in mice suggest that when associated with VPA, clinically relevant doses of clozapine elicit a synergistic potentiation of VPA-induced GABAergic promoter demethylation. Olanzapine and quetiapine (two clozapine congeners) also facilitate chromatin remodeling but at doses higher than used clinically, whereas haloperidol and risperidone are inactive. Hence, the synergistic potentiation of VPA's action on chromatin remodeling by clozapine appears to be a unique property of the dibenzepines and is independent of their action on catecholamine or serotonin receptors. By activating DNA-demethylation, the association of clozapine or its derivatives with VPA or other more potent and selective HDAC inhibitors may be considered a promising treatment strategy for normalizing GABAergic promoter hypermethylation and the GABAergic gene expression downregulation detected in the postmortem brain of SZ and BP disorder patients. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Enhanced dizocilpine efficacy in heterozygous reeler mice relates to GABA turnover downregulation.

Reelin synthesized by cortical GABAergic interneurons throughout the telencephalon is secreted into the extracellular matrix (ECM) and binds with nM affinity to integrin receptors located at dendritic spine postsynaptic densities and positively modulates Arc and other dendritic resident mRNAs translation, thereby facilitating the onset of synaptic plasticity and LTP consolidation. Accordingly, the reelin haploinsufficient heterozygous reeler mice (HRM) express a marked decrease of cortical thickness, of cortical and hippocampal dendritic spine density, and of cortical GAD67 expression. Behaviorally, HRM manifest a sensorimotor deficit, an exaggerated response to fear, and a deficit in olfactory discrimination learning. HRM and wild-type mice (WTM) were trained to retrieve to criterion palatable chocolate-flavored food pellets in an eight-arm radial maze. In 9-14 days of training HRM and WTM learned the task equally well committing only a few errors. However, HRM, when compared with WTM, show a greater cognitive impairment following the administration of dizocilpine. Also, HRM are more susceptible to the increased locomotion and stereotypic behavior elicited by dizolcipine. The enhanced dizocilpine susceptibility of HRM is not due to differences in pharmacokinetics because the levels of dizocilpine in cortices of HRM and WTM were virtually equal. We also failed to detect differences between HRM and WTM in glutamate brain content and in the rate of 13C-glucose incorporation into the glutamate brain pools. In contrast we found that the conversion index of glutamate into GABA (an indirect measurement of GABA turnover rate) is decreased in cortex, hippocampus and striatum of HRM when compared to WTM. Thus, HRM recapitulate several neurochemical and behavioral endophenotypes reminiscent of schizophrenia and these mice can be proposed as a relevant animal model for the study of pharmacological treatments aimed at alleviating the sensory-motor and cognitive dysregulation associated with schizophrenia.

An epigenetic mouse model for molecular and behavioral neuropathologies related to schizophrenia vulnerability.

Reelin and glutamic acid decarboxylase (GAD)67 expressed by cortical gamma-aminobutyric acid-ergic interneurons are down-regulated in schizophrenia. Because epidemiological studies of schizophrenia fail to support candidate gene haploinsufficiency of Mendelian origin, we hypothesize that epigenetic mechanisms (i.e., cytosine hypermethylation of CpG islands present in the promoter of these genes) may be responsible for this down-regulation. Protracted l-methionine (6.6 mmolkg for 15 days, twice a day) treatment in mice elicited in brain an increase of S-adenosyl-homocysteine, the processing product of the methyl donor S-adenosyl-methionine, and a marked decrease of reelin and GAD67 mRNAs in both WT and heterozygous reeler mice. This effect of l-methionine was associated with an increase in the number of methylated cytosines in the CpG island of the reelin promoter region. This effect was not observed for GAD65 or neuronal-specific enolase and was not replicated by glycine doses 2-fold greater than those of l-methionine. Prepulse inhibition of startle declined at a faster rate as the prepulsestartle interval increased in mice receiving l-methionine. Valproic acid (2 mmolkg for 15 days, twice a day) reverted l-methionine-induced down-regulation of reelin and GAD67 in both WT and heterozygous reeler mice, suggesting an epigenetic action through the inhibition of histone deacetylases. The same dose of valproate increased acetylation of histone H3 in mouse brain nearly 4-fold. This epigenetic mouse model may be useful in evaluating drug efficacy on schizophrenia vulnerability. Hence the inhibition of histone deacetylases could represent a pharmacological intervention mitigating epigenetically induced vulnerability to schizophrenia in individuals at risk.

The phenotypic characteristics of heterozygous reeler mouse.

Histological and behavioral traits are associated with reelin (Reln) haplo-insufficiency in heterozygous reeler mouse (rl+/-). These phenotypic traits are an approximately 50% decrease of brain Reln mRNA and Reln protein, an accumulation of nicotinamide-adenine dinucleotide phosphate-diaphorase (NADPH-d)-positive neurons in subcortical white matter, an age-dependent decrease in prepulse inhibition of startle (PPI), and neophobic behavior on the elevated plus-maze. Possible analogies between these rl+/- phenotypic traits and signs of psychosis vulnerability are discussed.

A decrease of reelin expression as a putative vulnerability factor in schizophrenia.

Postmortem prefrontal cortices (PFC) (Brodmann's areas 10 and 46), temporal cortices (Brodmann's area 22), hippocampi, caudate nuclei, and cerebella of schizophrenia patients and their matched nonpsychiatric subjects were compared for reelin (RELN) mRNA and reelin (RELN) protein content. In all of the brain areas studied, RELN and its mRNA were significantly reduced (approximately 50%) in patients with schizophrenia; this decrease was similar in patients affected by undifferentiated or paranoid schizophrenia. To exclude possible artifacts caused by postmortem mRNA degradation, we measured the mRNAs in the same PFC extracts from gamma-aminobutyric acid (GABA)A receptors alpha1 and alpha5 and nicotinic acetylcholine receptor alpha7 subunits. Whereas the expression of the alpha7 nicotinic acetylcholine receptor subunit was normal, that of the alpha1 and alpha5 receptor subunits of GABAA was increased when schizophrenia was present. RELN mRNA was preferentially expressed in GABAergic interneurons of PFC, temporal cortex, hippocampus, and glutamatergic granule cells of cerebellum. A protein putatively functioning as an intracellular target for the signal-transduction cascade triggered by RELN protein released into the extracellular matrix is termed mouse disabled-1 (DAB1) and is expressed at comparable levels in the neuroplasm of the PFC and hippocampal pyramidal neurons, cerebellar Purkinje neurons of schizophrenia patients, and nonpsychiatric subjects; these three types of neurons do not express RELN protein. In the same samples of temporal cortex, we found a decrease in RELN protein of approximately 50% but no changes in DAB1 protein expression. We also observed a large (up to 70%) decrease of GAD67 but only a small decrease of GAD65 protein content. These findings are interpreted within a neurodevelopmental/vulnerability "two-hit" model for the etiology of schizophrenia.

Auditory evoked potentials, clinical vs. research applications.

Evidence of abnormal auditory evoked potentials (EPs) in patients suffering from schizophrenia has been accumulating. In spite of the magnitude of the EPs in schizophrenia literature, EPs have not been found to be clinically useful thus far. In this study we attempted to replicate the findings in a large sample of schizophrenia patients, and describe how auditory EPs may be used as supplemental tests in the differential diagnostic process. Five subject groups were formed; paranoid (PAR) and disorganized/undifferentiated (disorg/undiff) schizophrenics, schizoaffective (SA), bipolar, and a normal control group. All patients were stable on medications. Subjects underwent one EP recording session. Classification and regression trees (CART) based on EP amplitudes were used to classify subjects into subgroups. The optimal Bayes classification rule that minimizes the expected misclassification cost was then constructed for various misclassification cost functions. In a standard 'Odd Ball' paradigm the N100 amplitudes were significantly decreased in the disorg/undiff group than in the bipolar or normal subjects. The P200 amplitude was smaller in the PAR, disorg/undiff and the SA groups than in the normal controls. Both the disorg/undiff and the PAR groups had significantly lower P300 amplitudes than the normal controls. Classification rules used to classify subjects into normal or ill were sensitive to the relative cost of misclassifying a subject, as well as the prior clinical probability that this subject was ill. Our data largely agree with the existing literature showing abnormally decreased N100, P200, and P300 amplitudes in schizophrenic patients, particularly the disorg/undiff patients. We conclude that whether EP measures are clinically useful depends on the clinical situation. In particular, the prior probability of the diagnosis in question being present and the cost of misclassifying the patient are critical.

The P50 evoked potential component and mismatch detection in normal volunteers: implications for the study of sensory gating.

Sensory gating is a complex, multistage, multifaceted physiological function believed to be protecting higher cortical centers from being flooded with incoming irrelevant sensory stimuli. Failure of such mechanisms is hypothesized as one of the mechanisms underlying the development of psychotic states. Attenuation of the amplitude of the P50 evoked potential component with stimulus repetition is widely used to study sensory gating. In the current study, we investigated the responsiveness of the P50 component to changes in the physical characteristics of ongoing trains of auditory stimuli. Forty normal volunteers were studied in a modified oddball paradigm. At all cerebral locations studied, P50 amplitudes were higher in response to infrequent stimuli. We postulate that the increase in P50 amplitude reflects the system's recognition of novel stimuli or "gating in" of sensory input. The ratio of the amplitude of the responses to the infrequent stimuli to those of the frequent stimuli was significantly higher for the posterior temporal regions. This finding provides further evidence that the temporal lobes may be significantly involved in sensory gating processes. Although this study only included normal subjects, the data generated contribute to the understanding of sensory gating mechanisms that may be relevant to psychotic states.

Investigational drugs and research.

This chapter will define investigational use for clinicians and outline FDA policies in this area in a simple and pragmatic way. Instances in which investigational use would require application to the FDA for an investigational New Drug Exemption (IND) and instances in which their use would require approval by an Institutional Review Board (IRB) will be described and examples given.

Psychophysiological predictors of drug treatment response.

Few of the psychophysiologic findings reviewed above are diagnostically specific. Not all persons given the same psychiatric diagnosis are likely to share the biological characteristic while others with different diagnoses do. The first and most simple explanation commonly offered is to refer to inadequacies in clinical diagnostic procedures or in the particular diagnostic system that was used in the study. It is true that the reliability of psychiatric ratings are typically lower than the reliability of psychophysiologic measures, but the situation appears to be more complex. In considering the issue of the relationship between psychiatric diagnosis and psychophysiological data, it is important to realize that psychiatric diagnosis may have several purposes only one of which is predicting drug treatment response. In addition, the degree to which a biological characteristic is present before the illness and between episodes, or in relatives or normal subjects having traits suggestive of vulnerability to the disorder, are important aspects of psychophysiological investigations. The state vs trait nature of the measure is of frequent concern, and changes in a psychophysiologic measure as a function of drug treatment are considered a part of this broader issue. Furthermore, psychophysiologic measures seem to be tapping underlying dimensions which cross-cut current diagnostic boundaries to a greater or lesser degree depending upon the measure and the subject samples. Candidates for such underlying dimensions include illness severity, anxiety, arousal, attention, cognitive impairment, neuronal loss, intelligence and mood, to mention a few. Scores on these dimensions may predict drug response to a higher degree than diagnosis. The use of drug treatment response itself to validate subgroups of individuals identified by diagnosis is a common assumption in psychophysiology. Thus, patients who are responsive to a drug are assumed to have a different underlying illness than patients who are resistant. In clinical nosology, this assumption is not usually taken for granted. However, Brown and Hertz have recently argued for the need to pay more attention to identification and classification of patients along a neuroleptic response dimension. Diagnostic systems based on psychophysiological measures have been developed. One such system is "neurometrics" which involves comparing individuals on electrophysiological measurements, such as EEG power spectra, against a data base of normative values previously obtained from normal subjects. John et al. have shown that classifications based on neurometrics corroborate clinical diagnostic categories to a high degree, and can be used to independently validate current psychiatric nosology.(ABSTRACT TRUNCATED AT 400 WORDS)

ERPs and psychopathology. II. Biological issues.

We have briefly covered important issues and considerations in four topic areas likely to be of use to the ERP researcher who is also interested in biological psychiatry. We cannot ignore diagnosis because of its basic role in the field of psychiatry. Although there are reliability problems, especially in the field of psychiatric diagnosis, ERPs may help in the painstaking task of validating diagnosis based on behavioral observation. In addition, the ERP researcher can use diagnosis to obtain potentially more homogeneous samples of patients so as to reduce between-subject error variance in studies. We have considered research areas like PET scan and CT scan which provide data of more anatomical precision than ERPs for deep neurological sites. While the potential may be there, it is essentially unrealized as yet. Finally, neurochemistry and psychopharmacology seem to be the areas most likely to bear fruit in the immediate future. If this potential is to be realized, it is necessary to formulate new questions and a new methodology to handle these questions. An important question, for example, is whether a biological profile is a more useful criterion than a behavioral profile for predicting response to pharmacological intervention. In biological psychiatry, this question is a human suffering issue of considerable importance.

The effect of lithium on platelet monoamine oxidase activity in bipolar and schizoaffective disorders.

Platelet monoamine oxidase (MAO) activity was studied in 33 in-patients with bipolar and schizoaffective disorder who were treated with lithium. Platelet MAO activity was found to increase following 10-41 dys of lithium treatment compared to a prior drug free period, and the increase was positively correlated with the duration of lithium treatment. The increase in platelet MAO activity was not correlated wtih clinical improvement as measured by the Brief Psychiatric Rating Scale (BPRS) and the Global Assessment Scale (GAS). The number of platelets per unit of blood was also significantly correlated with the number of days of lithium treatment. However, the increase in the number of platelets in lithium-treated patients was not correlated with the increase in MAO activity and thus appears not to account for it. These results indicate that studies relating platelet MAO activity to psychiatric diagnosis should be interpreted cautiously if patients are receiving lithium carbonate.

On the independence of the CNV and the P300 components of the human averaged evoked potential.

We report an experiment designed to assess the interactions between the CNV and the P300 components of human event-related potential. Eight subjects were each presented with series of experimental trials on all of which either a 1200 c/sec or an 800 c/sec tone was presented. There were three independent variables: (a) The presence or absence of a warning flash 1000 msec prior to the tone. (b) The task assigned to the subject--that is subjects were either to make a discriminative response to the tone or, on half the series, to predict prior to the tiral which of the two tones would be presented. (c) The predictability of the tone frequency. On half the series high and low tones alternated from trial to trial. On the other series, tones were chosen randomly on each trial. The data show that the amplitude of the P300 component is not affected by the presence or absence of a warning stimulus. Furthermore, the distributions of P300 and the CNV over the scalp are quite different. These conclusions are supported by a principal component and a discriminant analysis of the data. We conclude that the CNV and the P300 reflect the activity of functionally distinct cortical mechanisms.

Information delivery and the sensory evoked potential.

The waveform of evoked responses recorded from human scalp is not determined solely by the physical eliciting stimulus, but also varies as a function of the effective information provided by the stimulus. There is a positive component whose latency is determined by the point in time at which ambiguity is reduced, and whose shape and amplitude are influenced by whether it is the presence or absence of an external event which delivers the information.