Schizophrenia

NRG1, DAOA/DAO

14-10-2010

Michele Colombo Lynn Kraak Svetlana Vuhman

Index
1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 2 2.1 2.2 2.3 2.4 Introduction ............................................................................................................................. 3 Incidence and Heritability ................................................................................................... 3 Candidate schizophrenia susceptibility genes ..................................................................... 4 Linkage studies as a starting point for GWAS .................................................................... 5 DAOA from linkage to GWAS ........................................................................................... 5 NRG1 from Linkage to GWAS ........................................................................................... 7 Genes related to NRG1........................................................................................................ 8 Comparison between Linkage studies and Association studies .......................................... 8 Obtaining information about NRG1 and DAOA genes........................................................... 9 Homology of the genes between the species ....................................................................... 9 Sequence and structure of the candidate genes ................................................................. 11 Microarray analysis for schizophrenia .............................................................................. 12 Animal models to define relationship between Schizophrenia, NRG1 and DAOA genes 12 Transgenic studies show behavioral effect of susceptibility genes such as NRG1 ... 13 The absence of animal models for DAOA (G72) ...................................................... 13

2.4.1 2.4.2 2.5

Biochemical pathway and its connection to schizophrenia ............................................... 14 NRG1erbB4 dysregulations in schizophrenia ......................................................... 15 Reduction of D-serine levels in schizophrenia .......................................................... 15

2.5.1 2.5.2 3 4

Research proposal.................................................................................................................. 16 References ............................................................................................................................. 18

1 Introduction
Schizophrenia is a common neuropsychiatric disorder, affecting 1% of the population that is characterized by positive symptoms such as delusions, paranoia and hallucinations, negative symptoms including apathy, and social withdrawal, and extensive cognitive impairments that may have the greatest impact on overall function. It is characterized by disturbances in sensorimotor gating and attentional processes which can be measured by prepulse inhibition (PPI) and latent inhibition (LI). The recent studies have implicated dysfunction of neurotransmission at the NMDA-type glutamate receptor in schizophrenia. A possible way to improve the treatment of schizophrenia is to identify genetic risk factors that might help to sub classify patients at a molecular level. The etiology of schizophrenia as is not well understood. While there are clear environmental contributors to disease, it is clear that genetic predisposition is the major determinant of who develops schizophrenia, with heritability estimates as high as 80%, placing schizophrenia amongst the most heritable of the common diseases.

1.1

Incidence and Heritability

Incidence refers to the number of new cases of disease that develop in a population during a

specific time period. High quality evidence suggests that the incidence of schizophrenia worldwide is between 11 and 15.2 per 100,000 with a 5.6 fold variance across regions (ongoing review of www.schizophreniaresearch.org). Schizophrenia has a complex and non-Mendelian inheritance pattern, involving the combined action of several genes 1. The risk of developing the disease increases exponentially with the degree of genetic relatedness to a positive case. Approximately risk of developing schizophrenia is well shown in the picture, although a bit old, still valid.

Figure 1: Risk of developing schizophrenia increases with degree of relatives. 2

Heritability is always relative to the genetic compared to environmental factors in the variance of a population, and cannot be interpreted as a direct contribution of genetic and environmental factors to a phenotype for a single individual.

Figure 2: Comparison of genetic effects and non-genetic effects of schizophrenia and bipolar disorder.

There has been debate on the overlapping of genetic factors between schizophrenia and bipolar disorder. The recent Swedish finding within 2 million families from the Swedish national register, allowed to compare around 35 000 schizophrenic patients and 40 000 BPD with their parents. The model underlying risk is the sum of several effects, including family-member type additive genetic, adult shared environmental, childhood shared environ-mental effects, along with a common non-shared environmental effect for both schizophrenia and bipolar disorder. 63% of familial co-aggregation between SZ and BP was due to additive genetic effects common to both disorders (Figure 2).

1.2

Candidate schizophrenia susceptibility genes

Candidate genes are genes that are located in a chromosome region that is expected to be involved

in the expression of a disease such as schizophrenia. These genes are often identified by association studies and linkage studies. For schizophrenia, more than 2400 association studies are published in journals and these include more than 700 genes. Sun et al. propose two gene-based approaches for selecting and prioritizing candidate genes in 2009. The first one is combination-combination odds ratios (CCOR) in which they combine odds ratios of multiple markers in each study and then combine these ratios in multiple studies of a gene. The other method is called selection-combination odds ratios (SCOR), this method first selects the largest odds ratio of the marker in each association study and

then combines these odds ratios in multiple studies. Sun et al. also evaluated which method was best and concluded that the SCOR method generally surpasses the CCOR method. In table 1 a list of 75 top-ranking genes of schizophrenia, that were selected using this method, is shown. 3 Table 1: Candidate genes of schizophrenia, ranked by the SCOR method. 3

1.3

Linkage studies as a starting point for GWAS

Schizophrenia genetic research has traditionally focused on identifying linkage regions or on

candidate genes and polymorphisms. A first wave of linkage scans in 2002 used small family samples, identified chromosomal regions under the linkage peaks:13 q33 (a mood-disorder related region) and 13q34 by using linkage disequilibrium mapping ( LD). Specific genes in these regions have been related to schizophrenia by Genome Wide Association Studies: DAOA ( former called G72) and NRG1. The GWAS approach is based on linkage disequilibrium (LD), which aim to identify alleles associated with different loci with a non-random level of significance, thanks to a query based on known SNPs (single nucleotide polymorphisms) that compare common variation across the genome.

1.4

DAOA from linkage to GWAS

Using the known SNPs in the13q33 region, Chumakov et al (2002) made a systematic LD in two samples of Canadians and Russians, both with around 200 persons ( for each case of patients and controls) which evidenced the same locus on 13q associated with schizophrenia. Other studies followed the same protocol, providing stronger evidence for G72 as a gene whose variation acts as a risk factor for schizophrenia. The original report also found significant association for a cluster of SNPs in a region devoid of known genes (that has been somehow ignored in the following GWAS up to 2009).4 In 2008, the most comprehensive and updated meta-analysis of GWAS of the G72, resulting from 16 association articles containing 19 independent samples (around 4300 cases, 5400 controls and 1400 families)5, showed significant association for 2 SNPs in Asian population (rs778293, rs947267) and suggestive evidence for 1 SNP in an European population (rs1421292). From the 15 tested SNPs, only the Asian population showed a significant association of the alleles (an A in rs778293, a C in rs947267). This data remained significant at 0,001 level after the conservative Bonferroni correction for multiple testing. Vice versa complementary alleles, T in the first SNP, and G in the second , showed a small protective effect (OR<1). However the associated alleles or haplotypes vary across different studies, as also the markers used in the different studies meta-analyzed have strong heterogeneity. This may be due to study design (case/control vs family based), different ancestries ( Asian vs European), genotyping error rates, population-specific association ( all 3 SNPs are in different Haplotypes), big phonotypical variation. That is way the further studies of endo- or sub-phenotypes are required within the schizophrenia disease spectrum, gene-gene and gene-environment interaction (evidence is still lacking for DAOA). Three smaller and previous rank based genome scan meta-analysis failed to find evidence of any linkage on chromosome 13. Furthermore, among all the studies on DAOA and schizophrenia, 27 have found some positive associations and 12 did not (source: szgene on schizophreniaresearchforum.org ). Still, it has been the gene with the most widely reported evidence (review in 2006), and the recent discovery of its encoded protein, enabled biologists to obtain proofs of its involvement in the NMDA hypofunction. Some reviews look at this gene as the most promising current association for schizophrenia.6, 7

Figure 3: Meta-analysis of G vs. A polymorphism in DAOA gene which is associated with schizophrenia. (adapted from www.schizophreniaresearch.org)

Figure 4: Meta-analysis of T vs. C polymorphism in DAOA gene which is associated with schizophrenia. (adapted from www.schizophreniaresearch.org)

After the recently updated meta-analysis contained in the Szgene database, 2 up of 10 polymorphisms have showed statistical significance ( Figure 3, 4), even though other 66 SNPs have been published and were not included in the meta analysis because of strict methodological parameters.

1.5

NRG1 from Linkage to GWAS

The NRG1 gene has been isolated in an Icelandic population originally by Stefansson et al in 2002

as a gene at risk for schizophrenia8. In the NRG1 region have been identified different haplotypes and risk genotypes; however they vary between populations, mostly have low ODDS ratios ( <2). As usual in the schizophrenia genetic studies, no single causative allele has been isolated. The original Haplotype discovered is the HAPice, constituted by 5 SNPs and two microsatellites in the region that afterward identified as the 5' regulatory domain. He remarked that a SNP (snp8NRG243177) inside this haplotype is in disequilibrium with the other SNPs and may be considered as a functional polymorphism. Some findings, not all replicated, support this idea: its T/T allele is involved in impairment of frontal and temporal lobe activation, and a psychotic risk factor in individuals already susceptible for schizophrenia. However, other SNPs or microsatellite from the same haplotype is not involved. In normal individuals the same allele resulted in reduced spatial working memory and white matter in the internal capsule9. Different findings suggest that it affect transcription rate of NRG1, rather than coding the protein: no transcript is associated with its polymorphic site, but higher level of mRNA have been found in homozygotes (T/T).

Figure 5: Example of the meta-analysis of polymorphism in NRG1 gene associated with schizophrenia. (adapted from www.schizophreniaresearch.org)

After the recently systematic meta-analysis of the schizophrenia research forum, only one SNP over 13 included a proved to be significant, revealing it to be a protection factor (Figure 5). However, over 64 studies have been published about this gene, revealing other 440 polymorphism that were not excluded by strict criteria.

1.6

Genes related to NRG1

Of the whole family of Neuregulin, no other gene has been proved to be related with

schizophrenia, even though three SNPs in the locus of NRG3 have been associated in Jews with delusion, a typical phenotype of schizophrenia disease. Some evidence in a Japanese population show an increased risk for schizophrenia when considering the interaction of one SNP in NRG1 with another SNP of ErbB4, which is the NRG1 postsynaptic receptor. This study suggests that the signalling pathway of NRG1 can be disrupted at multiple sites while still leading to the same general phenotype. Several large GWAS have nonetheless failed to report association of neither NRG1 nor ErbB4; and some positive meta-analysis suggests that, even if significant, the association is weak. 10

1.7

Comparison between Linkage studies and Association studies

Linkage studies have an advantage over association studies because a chromosome region can be

pointed out from different families, even when those regional-genes involved are different. Moreover, different alleles of the same gene can sum up to a stronger signal. Indeed a linkage signal is composed both by common variation and some rare changes. That is why, despite the small number of subjects, linkage regions have popped out from these older studies, and in the meantime genetics browsed those genes under the peaks that showed biological compatibility with the disease. So, having these genes as a starting point, a biological analysis of other genes bonded in the same pathway led to discovery of other candidate genes: in this way neuregulin led to ErbB4; another

example is dysbindin. Dysbindin set researchers onto genes involved into vesicle movement and in the stability of dopamine receptor, and from these findings, using some SNPs known in the three dysbindin binding partners, allowed the discovery of the gene for MUTED. Once the genes coding for these proteins are validated, also the plausibility of dysbindin being truly involved an etiological factor for schizophrenia is increased, being closely related to the same cellular function In this way, biological research took advantage of the poor genetic evidence, improving its expansion and giving it a rational direction in which research could continue, in a virtuous loop fashion.11 Such studies have found many genes and variants; however these genes are not accepted as definitively associated with schizophrenia.11 It is now possible to represent the majority of common genetic variation by genotyping a selected set of tagging SNPs. Such hypothesis-free genome-wide association studies (GWAS) allow the discovery of new genes and pathways affecting complex traits such as schizophrenia with much greater power to detect small effects than linkage studies. These studies show that many genes are involved, each of which contributes a small risk, interacting with each other or with environmental risk factors to cause schizophrenia.11 Many of the susceptibility genes that have been identified for schizophrenia are known to regulate neuronal connectivity, synaptogenesis, and N-methyl-D-aspartate (NMDA) glutamate receptor functions. This includes genes for brain-derived neurotrophic factor (BDNF), dystrobrevin-binding protein 1, neuregulin, disrupted in schizophrenia-1 (DISC-1), D-amino acid oxidase activator (DAOA), and regulator of G-protein signaling (RGS4).

2 Obtaining information about NRG1 and DAOA genes

2.1 Homology of the genes between the species
Homology of genes indicates that these genes are derived from a common ancestor. Homology of genes is mostly based on sequence similarity. If two genes have highly similar DNA sequences, they are probably homologous. This similarity in DNA sequences can be calculated and is, in the bioinformatics, usually indicated by the E-value. An E-value of 0 indicates nearly exact sequence similarity in an alignment, and the larger the E-value, the less similarity. The NRG1 has homologous genes in many different species, these homologs and their E-values were found on Homologene and are presented in table 2 (E-values were calculated by using the pair wise alignment). In figure 1 the proteins and their conserved domains are shown.

Table 2: Homologous genes of NRG1 and their E-values when aligned to NRG1 (Homo sapiens) adapted from Homologene.

Genes

Organism

Proteins

E-value

NRG1 NRG1 NRG1 Nrg1 Nrg1 NRG1 Nrg1

Homo sapiens Pan troglodytes Canis lupus familiaris Mus musculus Rattus norvegicus Gallus gallus Danio rerio

NP_039258.1 XP_001168800.1 XP_858187.1 NP_848706.2 NP_113776.1 NP_989448.1 NP_001038376.1

0.0 0.0 1x10-168 0.0 3x10-141 1x10-87

Figure 6: NRG1 homologous proteins and their conserved domains (obtained and adapted from Homologene).

In the Homologene database there are not yet homologous genes to be found for the DAOA gene. In front of this gene on chromosome 13 is a gene that does have a homologous gene in mice (Mus musculus), this is the SLC10A2 gene (solute carrier family 10, sodium/bile acid cotransporter family, member 2). This gene is in mice located on chromosome 8(location 8 A1.1; 8 2.0 cM). After the DAOA gene there is also a gene that does have a homologous gene in mice, that is the EFNB2 (ephrin 2) gene, this gene is also on chromosome 8 in mice(location 8 A1.1; 8 2.0 cM). These genes are thus similarly aligned on the chromosome 8 in mice as there are in chromosome 13 in humans, in a way that the SLC10A2 gene is in front/top and the EFNB2 is in the back/lower part of the chromosome. This order is shown in figure 2 which shows the order of some genes on chromosome 8 in mice.

Figure 7: Gene order of some genes on chromosome 8 in mice. In the table it indicated where the DAOA gene would be expected (in between SLC10A2 and EFBN2), adapted from NCBI.

2.2

Sequence and structure of the candidate genes

An elaborate variety of different isoforms of the NRG1 gene are produced by alternative splicing.

These isoforms are tissue specific and have a different structure. The gene structure can be seen in figure 8. Steinthorsdottir et al. showed in 2004 that there are several major isoforms. For example Isoform I include the heregulins (HRGs), the NEU differentiation factor (NDF) or the acetylcholine receptor inducing activity (ARIA), isoform II include the glial growth factors (GGFs) and isoform III include the sensory and motor neuron-derived factors (SMDFs). 12, 13

Figure 8: Gene structure of the NRG1 gene. Splice variants and haplotypes associated with risk for schizophrenia are displayed. The exon-intron structure of the human NNRGRG1 gene locus. Indicated are the locations of the at-risk haplotypes (HAPice, HAPchinese and HAPportugese), a SNP (SNP8NRG243177) and a missense mutation in the exon that encodes the transmembrane domain (TM, identified in Costa Rica). B shows the schematic view of different promoters and alternative splicing which underlie the NRG1 isoform diversity.13

Indicated are the locations of the at-risk haplotypes (HAPice, HAPchinese and HAPportugese), a SNP (SNP8NRG243177) and a missense mutation in the exon that encodes the transmembrane domain (TM, identified in Costa Rica). B shows the schematic view of different promoters and alternative splicing which underlie the NRG1 isoform diversity. 13

2.3

Microarray analysis for schizophrenia

A DNA-microarray is a little chip with a large amount of spots on it. In every spot there is a

specific DNA sequence attached to the bottom of the spot. This is called a probe; it's a DNA element that is used to hybridize a cDNA sample. This hybridization can be detected and quantified by detection of fluorophorelabeled targets, this way you can determine the relative abundance of nucleic acid sequence in the target. One array can contain tens of thousands of probes and microarray can be used to measure changes in expression level, detect single nucleotide polymorphisms (SNPs) or to genotype or resequence mutant genomes. There have been some studies in which genes involved in schizophrenia are identified using microarray analysis. Mirnics et al. did an elaborate microarray study in 2000. They did the gene expression profiling for 250 gene groups, but more than 98% of the gene groups didnt differ significantly between schizophrenic and control subjects. Only one group of genes, encoding proteins involved in the regulation of presynaptic function, were decreased in all subjects with schizophrenia in comparison to the control subjects. These data were verified by in situ hybridization. No microarray data are found that describe a difference in gene expression of NRG1 or DAOA in schizophrenic subjects compared to control subjects. 14

2.4

Animal models to define relationship between Schizophrenia, NRG1 and DAOA genes
Modeling a human psychiatric disorder like schizophrenia in animals has many difficulties.

Behavioral symptoms involving human communication and language are hardly possible to stimulate in animals. Moreover, heterogeneity in symptoms, course and etiology of schizophrenia, likely involving the complex interaction of genetic and environmental factors, presents challenge to identify such an isomorphic model of the disorder in animals. Accordingly, to the current animal model of schizophrenia are often designed to test specific hypothesis on the genetic or cellular level. Pharmacological models are used to understand the alteration in various neurotransmitter systems. For instance, the negative symptoms of schizophrenia can be mimicked by administration of phencyclidine (PCP) which is NMDA receptor antagonist. PCP and other NMDA receptor antagonists induce schizophrenia-like symptoms in healthy subjects and exacerbate several psychotic symptoms in schizophrenia patients 15. This has brought up the view that schizophrenia is related to an altered glutamatergic neurotransmission resulting in an altered intracellular Ca-homeostasis (title of the article: Genetic findings in schizophrenia patients related to alterations in the intracellular Cahomeostasis). Attempts to mimic these effects in rats revealed parallels between schizophrenia and

molecular, cellular, functional and behavioral abnormalities in these animal models. In an animal model based on chronic, low-dose treatment with the NMDA receptor antagonist MK-801 described by the expression of NMDA receptor subunits was altered in a pattern similar to schizophrenia 16. On a cellular level, the number of parvalbumin positive interneurons was selectively decreased, a finding which exactly parallels observations in post mortem brain from schizophrenic patients 17 and on a functional level, recurrent inhibition of pyramidal cells was altered, as postulated from the histological findings. Thus, this pharmacologic model of NMDA receptor hypofunction has a significant potential as an animal model of the pathobiology of schizophrenia as well as the assumption of disturbed intracellular Ca-homeostasis in schizophrenia as physiologically, parvalbumin acts as a calcium buffering protein (CABP).16, 17

2.4.1 Transgenic studies show behavioral effect of susceptibility genes such as NRG1
The current animal models of schizophrenia are often designed to test specific hypothesis on the genetic or cellular level. Transgenic approaches are important tool to evaluate the behavioral effect of susceptibility genes. Several transgenic lines have been developed for NRG1 gene and NRG1-Erb4 signalling. These include Mice mutant for NRG1 and/or ErbB4 receptor genes also exhibit behavioural alterations. Disruption of prepulse inhibition and latent inhibition, two models of information processing deficits in schizophrenia, has been reported in these mutants. NRG1 (+/-, EGF), transgenic line with a mutation in the EGF like domain (S.L. Erickson, et al., ErbB3 is required for normal cerebellar and cardiac development: a comparison with ErbB2- and heregulin-deficient mice). Other transgenic line was development which contains NRG1 (+/-, TM), with a deletion in trans-membrane domain. Another transgenic line manifesting dyregulated NRG1 is the BASE null, in which the betaSite APP-cleaving enzyme 1 (BACE1), responsible for proteolytic processing of NRG1, was targeted. These transgenic mouse models have been examined for behavioral phenotypes utilizing comprehensive testing batteries. Overall, the studies show that the mutations of various domains are associated with several endophenotypic behavioral characteristics of schizophrenia. Such characteristics include altered locomotor activity, which was suppressed by antipsychotic treatment, deficits in PPI, mismatch negativity, contextual fear conditioning, cognitive impairment, and social behaviors. Mutants for erbB4 showed hyperactivity similar to NRG1 mutants, yet exhibited no deficits in PPI. However, when erbB4 was perturbed in a conditional knockout paradigm specific for the CNS, the animals showed an overall decreased level of activity, the opposite of other mutants behavioral trait. These studies in transgenic animals support the notion that perturbations in NRG1erbB4 signaling contribute to the behavioral phenotypes of schizophrenia. It appears that alterations in NRG1erbB4 signaling can lead to different behavioral manifestations, depending on the affected domains, time, and biological context of the dysregulation.8, 18

2.4.2

The absence of animal models for DAOA (G72)

The absence of a known rodent homologue of G72 has hindered work on the biology of this gene. G72 gene is only present in higher primates for instance genomic sequence analysis identifies some regions of homology to human G72 in chimpanzee, gorilla genomic sequences. The study of mutant mice lacking DAO showed that homozygous DAO -/- mice had high levels of D-serine and significantly reduced stereotypy and rotational activity after administration of NMDA receptor antagonists than did wild-type and DAO -/- mice (Hashimoto et al 2003). Furthermore, DAO -/- mice have indiscernible levels of D-serine in the cerebellum but that DAO -/- mice display high levels of the co-agonist. 19, 20

2.5

Biochemical pathway and its connection to schizophrenia

The glutamergic model of schizophrenia, which hypothesizes that NMDA hypofunction is

involved in the pathophysiology of schizophrenia symptoms. This results decrease of intracellular calcium and enhanced oxidative stress and excitotoxicity and may ultimately lead to adverse long-term adaptive changes or neurodegeneration21. Glutamergic model was published after it was found that PCP an katamine can induce symptoms that resemble negative symptoms by blocking the binding of the glutamate to the NMDA receptors (NMDAR). The NMDAR is one of the glutamate receptor in the central nervous system and it contains glutamate, glycine and/ or D-serine binding domains. NMDAR is ligand-gated and voltage-dependent, activation of NMDAR lead to calcium-ion flow into the cell. 21, 22

Figure 9: The primary cellular and subcellular location(s) of the proteins encoded by schizophrenia susceptibility genes (NRG1 and DAO/DAOA). (Genetic findings in schizophrenia
patients related to alterations in theintracellular Ca-homeostasis)

NRG1 and DAOA genes are associated with the NMDA hypofunction and they can possibly help to understand better the biochemical pathway on the genetic level.

The neuregulins (NRG1) are a family of growth and differentiation factors whose effects are mediated via four neuregulin (NRG1-4) genes that bind to the ErbB family of tyrosine kinase transmembrane receptors (ErbB1-4). NRG1 expression in the central nervous system (CNS) has been detected in many regions including the prefrontal cortex (PFC), hippocampus, cerebellum and substantia nigra, in both humans 23 and rodents. Numerous roles for NRG1 in CNS development and function have been identified, including synapse formation, neuronal migration, synaptic plasticity and the regulation of neurotransmitter expression and function. 24

2.5.1 NRG1erbB4 dysregulations in schizophrenia

Norton et al. (2005) reported an interaction between variants of genes encoding NRG1and its ErbB4 receptor for neuregulin-1 increases risk for schizophrenia. This finding is consistent with the suggestion that defects in NRG1ErbB signalling may contribute to the pathogenesis of the disease.8 ErbB4 and glutamatergic receptors are highly concentrated in the postsynaptic density (PSD) and are physically associated, albeit indirectly (Figure 9). ErbB4 can impact the establishment and activity of glutamatergic receptors and alter glutamatergic receptor function. NMDAR in particular, can also impact on erbB4. The relationship between erbB4 and NMDAR is of particular important, considering the increasing evidence supporting NMDAR hypofunction as a pathophysiologic mechanism for schizophrenia.

2.5.2 Reduction of D-serine levels in schizophrenia

D-Amino Acid Oxidase Activator (DAOA or also called G72) in interaction with D-amino acid oxidase (DAO) first was found by Chumakov in 2002 on the basis of yeast two-hybrid and coimmunoprecipation assays. DAOA interact directly with DAO leading to it activation. DAO is known as an upstream effecter on NMDA receptors. DAO oxidizes D-serine, an endogenous co-agonist of the NMDA receptor (). It was reported a reduction in serum levels of D-serine in schizophrenia supporting the hypofunction hypothesis of NMDAR20, 25. Altered of D-serine in schizophrenia may be explained, in part, by involvement of DAO, which activity is augmented in postmortem cortex of schizophrenia26. These findings indicate that decreased levels of D-serine in the nervous system of schizophrenia patients may be induced by increased D-amino acid degradation by DAO. However, Dserine is not the only substrate that might be affected by an increase in DAO activity For example, Dalanine is present in the cerebellum, is an NMDAR modulator, and may be therapeutically beneficial in schizophrenia. Overall, whilst a primary effect on D-serine, and thence NMDARs, is an attractive interpretation of the DAO increase in schizophrenia, further studies are needed to confirm the biochemical consequences. 4

3 Research proposal
Converging pharmacological, genetic, neuropathological and other data have led to the widely supported NMDAR hypofunction model of schizophrenia. A more specific variant of this hypothesis envisages that a deficiency of D-serine signaling contributes to NMDAR hypofunction. The research proposal is to asses more about mechanism of deficiency of D-serine signaling by modifying its upstream pathway. Previous studies show that D-amino acid oxidase (DAO) metabolises the NMDA receptor (NMDAR) modulator D-serine (Mothet JP, Parent AT, Wolosker H, Brady RO, Linden DJ, Ferris CD, et al. Proc
Natl Acad Sci USA. 2000; 97:492631). Changes in DAO activity thus affect D-serine and NMDAR

functioning. Enhanced DAO activity might be a potential cause of reduced D-serine and thence impaired NMDAR functioning which has been seen in schizophrenic patients (Tsai GC, Coyle JT. Annu Rev
Pharmacol Toxicol. 2002; 42:16579).

Figure 10: Process overview and detailed schematic process paths

DAO mRNA is detectable in forebrain regions, both in rodents and human as assed by a group of Japanese researchers in various articles from 2004 to 2007. However, the absence of a known rodent homologue of G72 has hindered work on the biology of this gene. The main goal of this research proposal is to investigate whether DAOA gene has the same functional effect on DAO in mice as in humans. If DAOA has the same effect in mice as in human, that will provide more possibilities to investigate the role of DAO and to test the NMDA hypofunction hypothesis in schizophrenia. The way this will be done is by first inducing the DAOA gene in mice by using a vector. In the experiment four different phenotypes will be tested: wildtype mice as control , transgenic mice with the DAOA gene, mice with DAO over-expression and a negative control mice with PCP. The mice with the over-expression will serve as a control to see if the transgenic mice will show the same results

as these mice. This is expected because DAOA in humans activates DAO and so will show a similar result as to when DAO itself is over expressed. The negative control mice will show NMDA hypofunction because PCP is an antagonist of NMDA receptor and can be used to decrease activity of NMDAR. To test the hypothesis of our research proposal we would like to look at the differences in intracellular calcium levels, which we expect will be lower in the negative mice control, mice with the DAOA gene and over expression of DAO in comparing to wildtype mice. This will be visualized using two-photon excitation microscopy. This is a fluorescence imaging technique, which allows us to look at living tissue up to very high depths. This technique provides depth and field resolution comparable to that produced by a confocal laser scanning microscopes and it also reduces photobleaching and phototoxicity. 27 Besides doing the two photon imaging an immunohistochemistry for the DAO protein would be advised to look at the difference in distribution and localization of the protein. The photo imaging can be also used in combination with patch clamp. This provides to visualize and measure calcium influx at the same time. If these data will show that the results of the control with PCP are similar to transgenic mice and to the mice with overexpression of DAO and different to control mice without PCP then this suggest that these transgenic mice will be a good model for further research on the role of DAOA in schizophrenia.

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