Dennis R. Grayson

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

Reelin and glutamic acid decarboxylase (GAD)67 expressed by cortical γ-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 mmol/kg 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 prepulse/startle interval increased in mice receiving l-methionine. Valproic acid (2 mmol/kg 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.

DNA-methyltransferase 1 mRNA is selectively overexpressed in telencephalic GABAergic interneurons of schizophrenia brains

A down-regulation of reelin and glutamic acid decarboxylase (GAD) 67 mRNAs was detected in γ-aminobutyric acid (GABA)ergic cortical interneurons of schizophrenia (SZ) postmortem brains (10), suggesting that the availability of GABA and reelin may be decreased in SZ cortex. In situ hybridization of the mRNA encoding for DNA-methyltransferase 1, which catalyzes the methylation of promoter CpG islands, shows that the expression of this mRNA is increased in cortical GABAergic interneurons but not in pyramidal neurons of SZ brains. Counts of reelin mRNA-positive neurons in Brodmanns area 10 of either nonpsychiatric subjects or SZ patients show that the expression of reelin mRNA is decreased in layer-I, -II, and -IV GABAergic interneurons of SZ patients. These findings are consistent with the hypothesis that the increase of DNA-methyltransferase 1 expression in telencephalic GABAergic interneurons of SZ patients causes a promoter hypermethylation of reelin and GAD67 and perhaps of other genes expressed in these interneurons. It is difficult to decide whether this dysfunction of GABAergic neurons detected in SZ is responsible for this disease or is a consequence of this disorder. Although at present we cannot differentiate between these two alternatives, it is important to consider that so far a molecular pathology of cortical GABAergic neurons appears to be the most consistent finding associated with SZ morbidity.

GABAergic dysfunction in schizophrenia: new treatment strategies on the horizon

RationaleCortical γ-aminobutyric acid (GABA)ergic neurons contribute to the orchestration of pyramidal neuron population firing as follows: (1) by releasing GABA on GABAA and GABAB receptors, (2) by releasing reelin in the proximity of integrin receptors located on cortical pyramidal neuron dendritic spines, and (3) through reelin contributing to the regulation of dendritic spine plasticity by modulating dendritic resident mRNA translation. In schizophrenia (SZ) and bipolar (BP) postmortem brains, the downregulation of mRNAs encoding glutamic acid decarboxylase 67 (GAD67) and reelin decreases the cognate proteins coexpressed in prefrontal cortex (PFC) GABAergic neurons. This finding has been replicated in several laboratories. Such downregulation suggests that the neuropil hypoplasticity found in the PFC of SZ and BP disorder patients may depend on a downregulation of GABAergic function, which is associated with a decrease in reelin secretion from GABAergic neuron axon terminals on dendrites, somata, or axon initial segments of pyramidal neurons. Indirectly, this GABAergic neuron downregulation may play a key role in the expression of positive and negative symptoms of SZ and BP disorders.ObjectivesThe above described GABAergic dysfunction may be addressed by pharmacological interventions to treat SZ and BP disorders using specific benzodiazepines (BZs), which are devoid of intrinsic activity at GABAA receptors including α1 subunits but that act as full positive allosteric modulators of GABA action at GABAA receptors containing α2, α3, or α5 subunits. These drugs are expected to enhance GABAergic signal transduction without eliciting sedation, amnesia, and tolerance or dependence liabilities.Results and conclusionsBZs, such as diazepam, although they are efficient in equilibrating GABAA receptor signal transduction in a manner beneficial in the treatment of positive and negative symptoms of SZ, may not be ideal drugs, because by mediating a full positive allosteric modulation of GABAA receptors containing the α1 subunit, they contribute to sedation and to the development of tolerance after even a brief period of treatment. In contrast, other BZ-binding site ligands, such as 6-(2bromophenyl)-8-fluoro-4H-imidazo [1,5-a][1,4] benzodiazepine-3-carboxamide (imidazenil), which fail to allosterically and positively modulate the action of GABA at GABAA receptors with α1 subunits but that selectively allosterically modulate cortical GABAA receptors containing α5 subunits, contribute to the anxiolytic, antipanic, and anticonvulsant actions of these ligands without producing sedation, amnesia, or tolerance. Strong support for the use of imidazenil in psychosis emerges from experiments with reeler mice or with methionine-treated mice, which express a pronounced reelin and GAD67 downregulation that is also operative in SZ and BP disorders. In mice that model SZ symptoms, imidazenil increases signal transduction at GABAA receptors containing α5 subunits and contributes to the reduction of behavioral deficits without producing sedation or tolerance liability. Hence, we suggest that imidazenil may be considered a prototype for a new generation of positive allosteric modulators of GABAA receptors, which, either alone or in combination with neuroleptics, should be evaluated in GABAergic dysfunction operative in the treatment of SZ and BP disorders with psychosis.

Valproate corrects the schizophrenia-like epigenetic behavioral modifications induced by methionine in mice

BACKGROUND Reelin and GAD(67) expression is downregulated in cortical interneurons of schizophrenia (SZ) patients. This downregulation is probably mediated by epigenetic hypermethylation of the respective promoters caused by the selective increase of DNA-methyltransferase 1 in GABAergic neurons. Mice receiving methionine (MET) provide an epigenetic model for neuropathologies related to SZ. We studied whether MET-induced epigenetic reelin promoter hypermethylation and the associated behavioral alterations can be reduced by valproate in doses that inhibit histone deacetylases (HDACs). METHODS Mice treated with either methionine (MET) (5.2 mmol/kg/SC/twice daily) or valproate (1.5 mmol/kg/SC/twice daily) or MET+ valproate combination were tested for prepulse inhibition of startle (PPI) and social interaction (SI). S-adenosylmethionine, acetylated histone 3, reelin promoter methylation, and reelin mRNA were assayed in the frontal cortex. RESULTS Valproate enhances acetylated histone 3 content, and prevents MET-induced reelin promoter hypermethylation, reelin mRNA downregulation, and PPI and SI deficits. Imidazenil, a positive allosteric modulator at GABA(A) receptors containing alpha(5) subunits but inactive at receptors including alpha(1) subunits, normalizes MET-induced behavioral changes. CONCLUSION This MET-induced epigenetic mouse models the neurochemical and behavioral aspects of SZ that can be corrected by positively modulating the action of GABA at alpha(5)-containing GABA(A) receptors with imidazenil or by inhibiting HDACs with valproate, thus opening exciting new avenues for treatment of epigenetically modified chromatin in SZ morbidity.

Characterization of NMDA receptor subunit-specific antibodies : distribution of NR2A and NR2B receptor subunits in rat brain and ontogenic profile in the cerebellum

Abstract: Selective antisera for NMDA receptor subunits NR2A and NR2B have been developed. Each antiserum identifies a single band on an immunoblot at ∼175 kDa that appears to be the appropriate subunit of the NMDA receptor. Using these antisera the relative densities of the subunits in eight areas of adult rat brain have been determined. The NR2A subunit was found to be at its highest level in hippocampus and cerebral cortex, to be at intermediate levels in striatum, olfactory tubercle, midbrain, olfactory bulb, and cerebellum, and to be at lowest levels in the pons‐medulla. The NR2B subunit was found to be expressed at its highest levels in the olfactory tubercle, hippocampus, olfactory bulb, and cerebral cortex. Intermediate levels were expressed in striatum and midbrain, and low levels were detected in the pons‐medulla. No signal for NR2B was found in the cerebellum. These regional distributions were compared with that for [3H]MK‐801 binding sites. It was found that although the distribution of the NR2A subunit corresponds well with radioligand binding, the distribution of the NR2B subunit does not. The ontogenic profiles of NR2A and NR2B subunits in the rat cerebellum were also determined. Just following birth [postnatal day (P) 2] NR2A subunits are undetectable, whereas NR2B subunits are expressed at amounts easily measurable. Beginning at about P12 the levels of NR2A rise rapidly to reach adult levels by P22. At the same time (P12), levels of NR2B protein begin to decline rapidly to reach undetectable levels by 22 days after birth. The results suggest that NMDA receptors are likely to be composed of different subunits in different parts of the brain and that even in the same tissue the receptors are likely to show different properties at various times during development due to alterations in the subunit composition of the receptor.

The Dynamics of DNA Methylation in Schizophrenia and Related Psychiatric Disorders

Major psychiatric disorders such as schizophrenia (SZ) and bipolar disorder (BP) with psychosis (BP+) express a complex symptomatology characterized by positive symptoms, negative symptoms, and cognitive impairment. Postmortem studies of human SZ and BP+ brains show considerable alterations in the transcriptome of a variety of cortical structures, including multiple mRNAs that are downregulated in both inhibitory GABAergic and excitatory pyramidal neurons compared with non-psychiatric subjects (NPS). Several reports show increased expression of DNA methyltransferases in telencephalic GABAergic neurons. Accumulating evidence suggests a critical role for altered DNA methylation processes in the pathogenesis of SZ and related psychiatric disorders. The establishment and maintenance of CpG site methylation is essential during central nervous system differentiation and this methylation has been implicated in synaptic plasticity, learning, and memory. Atypical hypermethylation of candidate gene promoters expressed in GABAergic neurons is associated with transcriptional downregulation of the corresponding mRNAs, including glutamic acid decarboxylase 67 (GAD67) and reelin (RELN). Recent reports indicate that the methylation status of promoter proximal CpG dinucleotides is in a dynamic balance between DNA methylation and DNA hydroxymethylation. Hydroxymethylation and subsequent DNA demethylation is more complex and involves additional proteins downstream of 5-hydroxymethylcytosine, including members of the base excision repair (BER) pathway. Recent advances in our understanding of altered CpG methylation, hydroxymethylation, and active DNA demethylation provide a framework for the identification of new targets, which may be exploited for the pharmacological intervention of the psychosis associated with SZ and possibly BP+.

Selective epigenetic alteration of layer I GABAergic neurons isolated from prefrontal cortex of schizophrenia patients using laser-assisted microdissection

Among the most consistent results of studies of post-mortem brain tissue from schizophrenia patients (SZP) is the finding that in this disease, several genes expressed by GABAergic neurons are downregulated. This downregulation may be caused by hypermethylation of the relevant promoters in affected neurons. Indeed, increased numbers of GABAergic interneurons expressing DNA methyltransferase 1 (DNMT1) mRNA have been demonstrated in the prefrontal cortex (PFC) of SZP using in situ hybridization. The present study expands upon these findings using nested competitive reverse transcription-polymerase chain reaction combined with laser-assisted microdissection to quantitate the extent of DNMT1 mRNA overexpression in distinct populations of GABAergic neurons obtained from either layer I or layer V of the PFC of SZP. In a cohort of eight SZP and eight non-psychiatric subject (NPS) post-mortem BA9 tissue samples, DNMT1 mRNA was found to be selectively expressed in GABAergic interneurons and virtually absent in pyramidal neurons. DNMT1 mRNA expression was approximately threefold higher in GABAergic interneurons microdissected from layer I of SZP relative to the same neurons microdissected from NPS. GABAergic interneurons obtained from layer V of the same samples displayed no difference in DNMT1 mRNA expression between groups. In the same samples, the GABAergic neuron-specific glutamic acid-decarboxylase67 (GAD67) and reelin mRNAs were underexpressed twofold in GABAergic interneurons isolated from layer I of SZP relative to GABAergic interneurons microdissected from layer I of NPS, and unaltered in GABAergic interneurons of layer V. These findings implicate an epigenetically mediated layer I GABAergic dysfunction in the pathogenesis of schizophrenia, and suggest novel strategies for treatment of the disease.

Is There a Future for Histone Deacetylase Inhibitors in the Pharmacotherapy of Psychiatric Disorders

In recent years, it has become widely recognized that a comprehensive understanding of chromatin biology is necessary to better appreciate its role in a wide range of diseases. The histone code has developed as a new layer of our appreciation of transcription factor-based mechanisms of gene expression. Although epigenetic regulation refers to a host of chromatin modifications that occur at the level of DNA, histones, and histone-associated proteins, how this regulation is orchestrated is still incompletely understood. Of those processes that comprise the epigenetic regulatory machinery, DNA methylation and histone acetylation/deacetylation have been the most thoroughly studied. Compounds that act as inhibitors of DNA methyltransferases or histone deacetylases (HDACs) activate a variety of intracellular signaling pathways that ultimately affect the coordinated expression of multiple genes. The altered patterns of mRNA and protein expression collectively converge on pathways linked to apoptosis and cell cycle arrest, among others. This has prompted a widespread search for epigenetic inhibitors that could be used as chemotherapeutic agents, and several are undergoing clinical evaluation. More recently, there has been interest in the use of HDAC inhibitors to activate the expression of mRNAs that are down-regulated in various neurological and psychiatric conditions. Considerably less is known regarding the effect these drugs have on postmitotic cells such as neurons. Before we consider the clinical use of additional HDAC inhibitors to treat schizophrenia or unipolar depression, there are a number of key issues that need to be resolved.

Histone deactylase 1 expression is increased in the prefrontal cortex of schizophrenia subjects: analysis of the National Brain Databank microarray collection.

Histone deactylase enzymes are responsible for the deacetylation of histone tails, and consequently influence gene regulation through their ability to modify chromatin structure surrounding promoter regions. We analyzed the microarray collection of the National Brain Databank to investigate differential expression of these enzymes in the prefrontal cortices of control, schizophrenia and bipolar subjects. HDAC1 expression levels were significantly higher in schizophrenia versus normal subjects. The mRNA expression level of an epigenetically regulated schizophrenia candidate gene GAD67 was strongly and negatively correlated with the mRNA expression levels of HDAC1, HDAC3 and HDAC4 levels. These findings provide additional support for the proposal that epigenetic factors are operative in the brain pathology of patients with schizophrenia.

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.