Genetics and Schizophrenia

7/27/2019 Genetics and Schizophrenia

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Schizophrenia

NRG1, DAOA/DAO

14-10-2010

Michele Colombo

Lynn Kraak

Svetlana Vuhman


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Index

1 Introduction ............................................................................................................................. 3

1.1 Incidence and Heritability ................................................................................................... 3

1.2 Candidate schizophrenia susceptibility genes ..................................................................... 4

1.3 Linkage studies as a starting point for GWAS .................................................................... 5

1.4 DAOA from linkage to GWAS ........................................................................................... 5

1.5 NRG1 from Linkage to GWAS ........................................................................................... 7

1.6 Genes related to NRG1 ........................................................................................................ 8

1.7 Comparison between Linkage studies and Association studies .......................................... 8

2 Obtaining information about NRG1 and DAOA genes ........................................................... 9

2.1 Homology of the genes between the species ....................................................................... 9

2.2 Sequence and structure of the candidate genes ................................................................. 11

2.3 Microarray analysis for schizophrenia .............................................................................. 12

2.4 Animal models to define relationship between Schizophrenia, NRG1 and DAOA genes 12

2.4.1 Transgenic studies show behavioral effect of susceptibility genes such as NRG1 ... 13

2.4.2 The absence of animal models for DAOA (G72) ...................................................... 13

2.5 Biochemical pathway and its connection to schizophrenia ............................................... 14

2.5.1 NRG1erbB4 dysregulations in schizophrenia ......................................................... 15

2.5.2 Reduction of D-serine levels in schizophrenia .......................................................... 15

3 Research proposal .................................................................................................................. 16

4 References ............................................................................................................................. 18


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1 IntroductionSchizophrenia 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 HeritabilityIncidence 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 genes1. 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.

Figure1: Risk of developing schizophrenia increases with degree of relatives.2


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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.

Figure2: 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 genesCandidate 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 combinethese 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


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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 GWASSchizophrenia 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) and13q34 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


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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


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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 GWASThe 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 (


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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 NRG1Of 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 studiesLinkage 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 geneticsbrowsed 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


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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 genes2.1 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.


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Genes

Organism Proteins E-value

NRG1 Homo sapiens NP_039258.1 -

NRG1 Pan troglodytes XP_001168800.1 0.0

NRG1 Canis lupus familiaris XP_858187.1 0.0

Nrg1 Mus musculus NP_848706.2 1x10-

Nrg1 Rattus norvegicus NP_113776.1 0.0

NRG1 Gallus gallus NP_989448.1 3x10-141

Nrg1 Danio rerio NP_001038376.1 1x10-87

Figure6: 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.


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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 genesAn 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

Figure8: 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, HAPchineseand 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


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Indicated are the locations of the at-risk haplotypes (HAPice, HAPchineseand 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 schizophreniaA 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 DAOAgenes

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 Ca-

homeostasis).Attempts to mimic these effects in rats revealed parallels between schizophrenia and


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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 schizophrenia16

. 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 17and 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 NRG1The 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 beta-

Site 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)


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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 schizophreniaThe 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-serinebinding 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 byschizophrenia 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.


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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 23and 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 schizophreniaNorton 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 (Figure9). 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 schizophreniaD-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 co-

immunoprecipation 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, D-

serine is not the only substrate that might be affected by an increase in DAO activity For example, D-

alanine 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


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3 Research proposalConverging 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).

Figure10: 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


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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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4 References

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