Viviane Labrie

A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells

Protein lysine methyltransferases G9a and GLP modulate the transcriptional repression of a variety of genes via dimethylation of Lys9 on histone H3 (H3K9me2) as well as dimethylation of non-histone targets. Here we report the discovery of UNC0638, an inhibitor of G9a and GLP with excellent potency and selectivity over a wide range of epigenetic and non-epigenetic targets. UNC0638 treatment of a variety of cell lines resulted in lower global H3K9me2 levels, equivalent to levels observed for small hairpin RNA knockdown of G9a and GLP with the functional potency of UNC0638 being well separated from its toxicity. UNC0638 markedly reduced the clonogenicity of MCF7 cells, reduced the abundance of H3K9me2 marks at promoters of known G9a-regulated endogenous genes and disproportionately affected several genomic loci encoding microRNAs. In mouse embryonic stem cells, UNC0638 reactivated G9a-silenced genes and a retroviral reporter gene in a concentration-dependent manner without promoting differentiation.

5-hmC in the brain is abundant in synaptic genes and shows differences at the exon-intron boundary

The 5-methylcytosine (5-mC) derivative 5-hydroxymethylcytosine (5-hmC) is abundant in the brain for unknown reasons. Here we characterize the genomic distribution of 5-hmC and 5-mC in human and mouse tissues. We assayed 5-hmC by using glucosylation coupled with restriction-enzyme digestion and microarray analysis. We detected 5-hmC enrichment in genes with synapse-related functions in both human and mouse brain. We also identified substantial tissue-specific differential distributions of these DNA modifications at the exon-intron boundary in human and mouse. This boundary change was mainly due to 5-hmC in the brain but due to 5-mC in non-neural contexts. This pattern was replicated in multiple independent data sets and with single-molecule sequencing. Moreover, in human frontal cortex, constitutive exons contained higher levels of 5-hmC relative to alternatively spliced exons. Our study suggests a new role for 5-hmC in RNA splicing and synaptic function in the brain.

D-Serine Augments NMDA-NR2B Receptor-Dependent Hippocampal Long-Term Depression and Spatial Reversal Learning

The contributions of hippocampal long-term depression (LTD) to explicit learning and memory are poorly understood. Electrophysiological and behavioral studies examined the effects of modulating NMDA receptor-dependent LTD on spatial learning in the Morris water maze (MWM). The NMDA receptor co-agonist D-serine substantially enhanced NR2B-dependent LTD, but not long-term potentiation (LTP) or depotentiation, in hippocampal slices from adult wild type mice. Exogenous D-serine did not alter MWM acquisition, but substantially enhanced subsequent reversal learning of a novel target location and performance in a delayed-matching-to-place task. Conversely, an NR2B antagonist disrupted reversal learning and promoted perseveration. Endogenous synaptic D-serine likely saturates during LTP induction because exogenous D-serine rescued deficient LTP and MWM acquisition in Grin1D481N mutant mice having a lower D-serine affinity. Thus, D-serine may enhance a form of hippocampal NR2B-dependent LTD that contributes to spatial reversal learning. By enhancing this form of synaptic plasticity, D-serine could improve cognitive flexibility in psychiatric disorders characterized by perseveration of aberrant ideation or behaviors.

Serine racemase is associated with schizophrenia susceptibility in humans and in a mouse model

Abnormal N-methyl-d-aspartate receptor (NMDAR) function has been implicated in the pathophysiology of schizophrenia. d-serine is an important NMDAR modulator, and to elucidate the role of the d-serine synthesis enzyme serine racemase (Srr) in schizophrenia, we identified and characterized mice with an ENU-induced mutation that results in a complete loss of Srr activity and dramatically reduced d-serine levels. Mutant mice displayed behaviors relevant to schizophrenia, including impairments in prepulse inhibition, sociability and spatial discrimination. Behavioral deficits were exacerbated by an NMDAR antagonist and ameliorated by d-serine or the atypical antipsychotic clozapine. Expression profiling revealed that the Srr mutation influenced several genes that have been linked to schizophrenia and cognitive ability. Transcript levels altered by the Srr mutation were also normalized by d-serine or clozapine treatment. Furthermore, analysis of SRR genetic variants in humans identified a robust association with schizophrenia. This study demonstrates that aberrant Srr function and diminished d-serine may contribute to schizophrenia pathogenesis.

The involvement of the NMDA receptor d-serine/glycine site in the pathophysiology and treatment of schizophrenia

Hypofunction of the N-methyl-D-aspartate receptor (NMDAR) has been implicated in the pathophysiology of schizophrenia. The NMDAR contains a D-serine/glycine site on the NR1 subunit that may be a promising therapeutic target for psychiatric illness. This review outlines the complex regulation of endogenous NMDAR D-serine/glycine site agonists and explores their contribution to schizophrenia pathogenesis and their potential clinical utility. Genetic studies have associated genes influencing NMDAR D-serine/glycine site activation with an increased susceptibility to schizophrenia. Postmortem studies have identified abnormalities in several transcripts affecting D-serine/glycine site activity, consistent with in vivo reports of alterations in levels of endogenous D-serine/glycine site agonists and antagonists. Genetically modified mice with aberrant NMDAR D-serine/glycine site function model certain features of the negative and cognitive symptoms of schizophrenia, and similar behavioral abnormalities have been observed in other candidate genes models. Compounds that directly activate the NMDAR D-serine/glycine site or inhibit glycine transport have demonstrated beneficial effects in preclinical models and clinical trials. Future pharmacological approaches for schizophrenia treatment may involve targeting enzymes that affect D-serine synthesis and metabolism.

Contributions of the d-serine pathway to schizophrenia

The glutamate neurotransmitter system is one of the major candidate pathways for the pathophysiology of schizophrenia, and increased understanding of the pharmacology, molecular biology and biochemistry of this system may lead to novel treatments. Glutamatergic hypofunction, particularly at the NMDA receptor, has been hypothesized to underlie many of the symptoms of schizophrenia, including psychosis, negative symptoms and cognitive impairment. This review will focus on D-serine, a co-agonist at the NMDA receptor that in combination with glutamate, is required for full activation of this ion channel receptor. Evidence implicating D-serine, NMDA receptors and related molecules, such as D-amino acid oxidase (DAO), G72 and serine racemase (SRR), in the etiology or pathophysiology of schizophrenia is discussed, including knowledge gained from mouse models with altered D-serine pathway genes and from preliminary clinical trials with D-serine itself or compounds modulating the D-serine pathway. Abnormalities in D-serine availability may underlie glutamatergic dysfunction in schizophrenia, and the development of new treatments acting through the D-serine pathway may significantly improve outcomes for many schizophrenia patients.

Epigenetics of major psychosis: progress, problems and perspectives

Understanding the origins of normal and pathological behavior is one of the most exciting opportunities in contemporary biomedical research. There is increasing evidence that, in addition to DNA sequence and the environment, epigenetic modifications of DNA and histone proteins may contribute to complex phenotypes. Inherited and/or acquired epigenetic factors are partially stable and have regulatory roles in numerous genomic activities, thus making epigenetics a promising research path in etiological studies of psychiatric disease. In this article, we review recent epigenetic studies examining the brain and other tissues, including those from individuals with schizophrenia (SCZ) and bipolar disorder (BPD). We also highlight heuristic aspects of the epigenetic theory of psychiatric disease and discuss the future directions of psychiatric epigenetics.

Genetic inactivation of D-amino acid oxidase enhances extinction and reversal learning in mice

Activation of the N-methyl-D-aspartate receptor (NMDAR) glycine site has been shown to accelerate adaptive forms of learning that may benefit psychopathologies involving cognitive and perseverative disturbances. In this study, the effects of increasing the brain levels of the endogenous NMDAR glycine site agonist D-serine, through the genetic inactivation of its catabolic enzyme D-amino acid oxidase (DAO), were examined in behavioral tests of learning and memory. In the Morris water maze task (MWM), mice carrying the hypofunctional Dao1(G181R) mutation demonstrated normal acquisition of a single platform location but had substantially improved memory for a new target location in the subsequent reversal phase. Furthermore, Dao1(G181R) mutant animals exhibited an increased rate of extinction in the MWM that was similarly observed following pharmacological administration of D-serine (600 mg/kg) in wild-type C57BL/6J mice. In contextual and cued fear conditioning, no alterations were found in initial associative memory recall; however, extinction of the contextual fear memory was facilitated in mutant animals. Thus, an augmented level of D-serine resulting from reduced DAO activity promotes adaptive learning in response to changing conditions. The NMDAR glycine site and DAO may be promising therapeutic targets to improve cognitive flexibility and inhibitory learning in psychiatric disorders such as schizophrenia and anxiety syndromes.

Mutant mice with reduced NMDA-NR1 glycine affinity or lack of d-amino acid oxidase function exhibit altered anxiety-like behaviors

Several compounds that promote activation of the N-methyl-d-aspartate receptor (NMDAR) glycine site have been proposed as treatments for schizophrenia, but the impact of these putative antipsychotics on anxiety remains unclear. In this study, we employed genetic and pharmacological mouse models of altered NMDAR glycine site function to examine the effects of these proposed treatments in unconditioned tests of anxiety. In the elevated plus-maze, open field, and novel object test, homozygous Grin1(D481N) mutant mice that have a five-fold reduction in NMDAR glycine affinity demonstrated an anxiolytic-like phenotype. In contrast, d-serine, a direct activator of the NMDAR glycine site, and ALX-5407, a glycine transporter-1 (GlyT-1) inhibitor, enhanced anxiety-like behaviors in wild-type and Grin1(D481N) mutant animals. Homozygous Dao1(G181R) mutant mice that lack function of the d-serine catabolic enzyme, d-amino acid oxidase (DAO), displayed an elevation in anxiety. Deficient DAO activity also reversed the anxiolytic effects of diminished NMDAR function in mice carrying both the homozygous Grin1(D481N) and Dao1(G181R) mutation. Thus, a direct agonist of the NMDAR glycine site, a GlyT-1 inhibitor, and suppression of DAO function induced anxiogenic-like behaviors. Consequently, application of these treatments for amelioration of schizophrenic symptoms necessitates caution as an enhancement of comorbid anxiety disorders may result.

Genetic loss of D-amino acid oxidase activity reverses schizophrenia-like phenotypes in mice

Reduced function of the N‐methyl‐d‐aspartate receptor (NMDAR) has been implicated in the pathophysiology of schizophrenia. The NMDAR contains a glycine binding site in its NR1 subunit that may be a useful target for the treatment of schizophrenia. In this study, we assessed the therapeutic potential of long‐term increases in the brain levels of the endogenous NMDAR glycine site agonist D‐serine, through the genetic inactivation of its catabolic enzyme D‐amino acid oxidase (DAO) in mice. The effects of eliminating DAO function were investigated in mice that display schizophrenia‐related behavioral deficits due to a mutation (Grin 1D481N) in the NR1 subunit that results in a reduction in NMDAR glycine affinity. Grin 1D481N mice show deficits in sociability, prolonged latent inhibition, enhanced startle reactivity and impaired spatial memory. The hypofunctional Dao 1G181R mutation elevated brain levels of D‐serine, but alone it did not affect performance in the behavioral measures. Compared to animals with only the Grin 1D481N mutation, mice with both the Dao1G181R and Grin 1D481N mutations displayed an improvement in social approach and spatial memory retention, as well as a reversal of abnormally persistent latent inhibition and a partial normalization of startle responses. Thus, an increased level of D‐serine resulting from decreased catalysis corrected the performance of mice with deficient NMDAR glycine site activation in behavioral tasks relevant to the negative and cognitive symptoms of schizophrenia. Diminished DAO activity and elevations in D‐serine may serve as an effective therapeutic intervention for the treatment of psychiatric symptoms.