the involvement of the nmda receptor d-serine/glycine site in the

Review

The involvement of the NMDA receptor D-serine/glycine site in thepathophysiology and treatment of schizophrenia

Viviane Labrie a,b,*, John C. Roder a,b

a Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canadab Institute of Medical Science, University of Toronto, Toronto, ON M5S 1A8, Canada

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352

2. The NMDAR: structure and regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352

3. Endogenous modulators of the NMDAR D-serine/glycine site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352

3.1. Glycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353

3.2. D-Serine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353

4. The predominant physiological co-agonist of the NMDAR D-serine/glycine site. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354

5. Dysfunction of the NMDAR in schizophrenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355

6. Genetic studies: G72, DAO, and SRR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356

7. Evidence for abnormal modulation of the NMDAR D-serine/glycine site in patients with schizophrenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356

8. Pharmacological treatments targeting the NMDAR D-serine/glycine site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

9. Animal models based on diminished NMDAR function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360

10. Animal models relevant to the NMDAR D-serine/glycine site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361

11. Comparison of animal models examining genes related to the NMDAR D-serine/glycine site and other genetic models based on

schizophrenia susceptibility genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363

12. The future of NMDAR D-serine/glycine site modulators in the treatment of schizophrenia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365

Neuroscience and Biobehavioral Reviews 34 (2010) 351–372

A R T I C L E I N F O

Article history:

Received 28 May 2009

Received in revised form 10 August 2009

Accepted 11 August 2009

Keywords:

Schizophrenia

NMDA receptor

D-Serine/glycine site agonists

Susceptibility genes

Animal models

D-Serine

Behavioral phenotype

A B S T R A C T

Hypofunction of the N-methyl-D-aspartate receptor (NMDAR) has been implicated in the pathophysiol-

ogy 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.

� 2009 Elsevier Ltd. All rights reserved.

Contents lists available at ScienceDirect

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journa l homepage: www.e lsev ier .com/ locate /neubiorev

* Corresponding author at: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Ave., rm 860, Toronto, ON M5G 1X5, Canada.

E-mail address: [email protected] (V. Labrie).

0149-7634/$ – see front matter � 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.neubiorev.2009.08.002

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http://dx.doi.org/10.1016/j.neubiorev.2009.08.002

V. Labrie, J.C. Roder / Neuroscience and Biobehavioral Reviews 34 (2010) 351–372352

1. Introduction

Schizophrenia is a chronic and severely debilitating psychiatricdisorder affecting nearly 1% of the population worldwide. It ischaracterized by positive symptoms that include hallucinations,delusions, and thought disorder, by negative symptoms comprisedof affective flattening and social isolation, and by profoundcognitive deficits in attention, learning, memory, and behavioralflexibility (Ross et al., 2006; Lewis and Gonzalez-Burgos, 2006).Symptoms of schizophrenia typically emerge during adolescenceor early adulthood and while positive symptoms often fluctuate,negative and cognitive symptoms are more enduring, causing greatdisability and deterioration in the quality of life of patients(Yamauchi et al., 2008; Milev et al., 2005). Current antipsychotictreatments for schizophrenia show success in reducing the severityof positive symptoms, but have limited efficacy in amelioratingnegative and cognitive deficits (Ross et al., 2006). Furthermore,antipsychotic regimes are often poorly tolerated, leading to poorcompliance and symptomatic relapse (Lewis and Gonzalez-Burgos,2006). In order to develop effective therapies, much effort has beenmade to further understand the molecular alterations involved inthe pathophysiology of schizophrenia.

Abnormalities in several neurotransmitter systems have beenimplicated in the pathophysiological processes underlying schizo-phrenia. The predominant theory has been the dopaminehypothesis, which postulates that schizophrenic symptoms arisefrom excessive dopaminergic transmission, particularly in thestriatum, and the presence of dopaminergic deficits in prefrontalbrain regions (Davis et al., 1991). This theory was based on theobservation that blockade of D2 receptors is a mechanism of actionfor antipsychotics (Seeman et al., 1975; Creese et al., 1976) and theability of dopamine-releasing stimulants, such as amphetamine, toinduce psychosis (Janowsky and Risch, 1979). Amphetamine elicitsonly the positive symptoms of schizophrenia, consistent with thegreater efficacy of antipsychotics in relieving the positivesymptoms rather than the negative symptoms, cognitive impair-ments, and cortical atrophy seen in schizophrenia patients. Inaddition to the dopaminergic abnormalities, NMDA receptor(NMDAR) hypofunction has been proposed to be involved inschizophrenia. This theory originated from studies demonstratingthat non-competitive NMDAR antagonists like phencyclidine (PCP)reliably and immediately induce a syndrome similar to schizo-phrenia in healthy individuals and exacerbate symptoms inschizophrenia patients (Javitt and Zukin, 1991; Krystal et al.,1994). Serum concentrations of PCP that are able to producepsychiatric symptoms correspond to the level that blocks NMDARs(Javitt and Zukin, 1991). Moreover, NMDAR inhibitors generate thenegative and cognitive disturbances as well as the psychoticsymptoms characteristic of the disorder (Javitt and Zukin, 1991;Krystal et al., 1994). Negative symptoms induced by NMDARantagonists are not worsened by amphetamine administration inhealthy humans and differences in the psychotic states producedby psychostimulants and NMDAR inhibitors have been observed(Krystal et al., 2005).

Since these initial observations, convergent evidence hassupported a role for aberrant NMDAR-mediated neurotransmis-sion in schizophrenia pathogenesis (Coyle, 2006; Millan, 2005) andthe glutamatergic system in schizophrenia is considered to be partof a larger complex framework involving the interaction ofmultiple neurotransmitters and risk genes. More recently, studieshave indicated that the NMDAR D-serine/glycine binding site andits modulatory enzymes may be crucially involved in theglutamatergic dysfunction thought to occur (Millan, 2005; Coyle,2006). Here, we examine the significance of the NMDAR D-serine/glycine site and its related modulators to the pathophysiology ofschizophrenia. Genetic, neurochemical, and postmortem studies in

humans have begun to distinguish pathogenetic events that mayalter glutamatergic transmission and plausibly lead to abnormalNMDAR D-serine/glycine site activity and the formation ofschizophrenic symptoms. Evidence from pharmacological andgenetic animal models have provided further support for aberrantNMDAR D-serine/glycine site function in endophenotypes relevantto schizophrenia. Finally, D-serine/glycine site agonists assessed inclinical studies have shown promising ameliorative effects. Over-all, these findings present novel molecular alterations andtherapeutic interventions for schizophrenia.

2. The NMDAR: structure and regulation

NMDARs in the central nervous system (CNS) are heteromericprotein complexes composed of at least one NR1 subunit togetherwith different combinations of NR2 and/or NR3 subunits (Cull-Candy et al., 2001). Alternative splicing of the Grin1 gene produceseight NR1 isoforms and by associating with different constellationsof NR2 (NR2A-D) and NR3 subunits (NR3A and NR3B) form amultitude of different NMDAR receptors with distinct biophysicalproperties (Monyer et al., 1994) and specific patterns of expressionduring development and in the mature mammalian CNS (Dumas,2005). NMDAR complexity is further enhanced through post-translational modifications, such as phosphorylation, glycosyla-tion, and ubiquitination, affecting cellular localization and functionof the receptor (Dingledine et al., 1999).

At resting membrane potential, the pore of the NMDAR channel isblocked by magnesium, and this block can be removed via a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated membrane depolarization (Dingledine et al., 1999). TheNMDAR also contains a glutamate recognition site on the NR2subunit and a glycine or D-serine modulatory site on the NR1 subunit(Fig. 1) (Johnson and Ascher, 1987; Clements and Westbrook, 1991).The D-serine/glycine site on the NMDAR must be occupied forglutamate to activate the receptor (Clements and Westbrook, 1991).The unique property of being both voltage-dependent and ligand-gated gives the NMDAR the ability to act as a coincidence detector forpresynaptic activity (glutamate release) and post-synaptic activity(adequate depolarization of the post-synaptic membrane). Onceactivated, the NMDAR channel permits the influx of calcium, whichstimulates intracellular signaling cascades that can subsequentlyaffect synaptic plasticity and gene transcription (Bading et al., 1993).Induction of NMDAR-dependent forms of synaptic plasticity, such aslong-term potentiation (LTP), is thought to underlie many types oflearning and memory formation (Nicoll, 2003).

NMDARs are present throughout the brain and are principallyneuronal, though they can also be expressed on astrocytes. Beyondthe glutamate and D-serine/glycine binding site, they containseveral regulatory sites sensitive to polyamines, Zn2+, protons, andglutathione (Cull-Candy et al., 2001; Dingledine et al., 1999). Thenumerous influences that converge on the NMDAR highlight theimportance of these receptors in diverse brain functions.Additionally, the NMDAR can also be modulated by severalartificially derived inhibitory compounds, including PCP, MK-801,and ketamine, which are high affinity open channel blockers(Cull-Candy et al., 2001; Dingledine et al., 1999).

3. Endogenous modulators of the NMDAR D-serine/glycine site

In NMDAR complexes containing NR1 and NR2 subunits,D-serine and glycine both have excitatory effects, with D-serinebeing up to three times more potent than glycine (Matsui et al.,1995). NMDARs with NR2/NR1 subunits have been implicated innumerous physiological processes, including synaptic plasticityand development, as well as in several pathological conditions,such as neurodegenerative and psychiatric diseases (Cull-Candy

Fig. 1. The NMDA receptor. Activation of the receptor requires membrane depolarization in order to remove the magnesium block, along with concurrent binding of glycine or

D-serine to the NR1 subunit and glutamate to the NR2 subunit.

V. Labrie, J.C. Roder / Neuroscience and Biobehavioral Reviews 34 (2010) 351–372 353

et al., 2001). In receptors composed of NR1 and NR3 subunits,glycine continues to act as an activator, while D-serine exerts weakpartial agonistic effects (Chatterton et al., 2002; Smothers andWoodward, 2007). To date, the role of NR3/NR1 heteromers in theadult brain is unclear and they display a peculiar resistance topsychotomimetic NMDAR antagonists (Smothers and Woodward,2007; Chatterton et al., 2002), thus bringing their relevance to theinduction of psychotic syndromes into question.

In addition to enabling NMDAR activation, endogenous D-serine/glycine site agonists have a role in neuromodulation.Binding to the D-serine/glycine site allosterically influences theNMDAR to enhance the affinity and efficacy of glutamate (Faddaet al., 1988), delays receptor desensitization to increase theduration and frequency of the open channel state (Vyklicky et al.,1990), and promotes NMDAR turnover through priming of thereceptor for internalization (Nong et al., 2003).

Examination of the crystal structure of the NR1 binding coreprovided further insight on the modes of interaction and selectivityof D-serine/glycine site agonists. D-Serine binds more tightly to thereceptor than glycine, due to its ability to make 3 additionalhydrogen bonds and displace a water molecule within the bindingpocket (Furukawa and Gouaux, 2003). The binding of the D-enantiomer to NR1 is selective, as L-serine contains a hydroxylgroup that interacts unfavorably with a phenyl ring in the bindingsite (Furukawa and Gouaux, 2003).

Whether the D-serine/glycine site on NMDARs is saturated byglycine or D-serine at physiological conditions remains somewhatcontroversial, though most evidence indicates that the D-serine/glycine site is not saturated in several brain areas. Elevations inglycine and/or D-serine concentrations evokes NMDAR-mediatedresponses in the prefrontal cortex, neocortex, hippocampus, andbrainstem slices in vitro (Chen et al., 2003; Thomson et al., 1989;Wilcox et al., 1996; Depoortere et al., 2005; Berger and Isaacson,1999) and in the prefrontal cortex, hippocampus, and thalamus in

vivo (Chen et al., 2003; Panizzutti et al., 2005; Kinney et al., 2003;Thiels et al., 1992; Salt, 1989), signifying that the NMDAR glycinemay not be fully saturated at synapses in these brain regions.Incomplete saturation of the D-serine/glycine site suggests thatagonists of the D-serine/glycine site are capable of regulatingNMDAR-mediated neurotransmission.

3.1. Glycine

Although glycine is an abundant amino acid found throughoutthe brain, its synaptic concentrations are tightly regulated byglycine transporters. At NMDAR-expressing synapses, extracellu-lar glycine concentrations are primarily derived from astroglialcells and clearance is mediated by glycine transporter 1 (GlyT-1)(Kinney et al., 2003; Lim et al., 2004). GlyT-1s are closely associatedto NMDARs (Cubelos et al., 2005) and there at least 6 glial andneuronal subtypes (GlyT-1a–f) (Chen et al., 2004b; Hanley et al.,2000). GlyT-1 effectively maintains low, subsaturating levels ofglycine, as GlyT-1 blockers like ALX-5407 are capable of enhancingNMDAR activity (Chen et al., 2003; Bergeron et al., 1998; Martinaet al., 2004). Spillover from glycinergic neurons also contributes asmall amount of glycine to NMDARs (Ahmadi et al., 2003);although distant diffusion from glycinergic neurons is limited bythe high affinity glycine transporter 2 (GlyT-2) that is responsiblefor glycine reuptake near strychnine-sensitive glycine A receptors(Poyatos et al., 1997). In addition, System A-family transporters(SNAT) on astrocytes and neurons transport a range of smallneutral amino acids including glycine, and by modulating glycineuptake and release may also contribute to the dynamic regulationof extracellular glycine (Mackenzie and Erickson, 2004; Javitt et al.,2005).

Biosynthesis of glycine occurs through the conversion of L-serine to glycine by the enzyme serine hydroxymethyltransferase(Appaji Rao et al., 2003). This enzyme is also capable of functioningin a reverse direction, thereby eliminating glycine (Appaji Raoet al., 2003). Additionally, the glycine cleavage system in astrocytesefficiently degrades glycine and generates the concentrationgradient between the cytosol and extracellular space that allowsglycine transporters to remove glycine from the synaptic cleft(Sakata et al., 2001; Oda et al., 2007).

3.2. D-Serine

The discovery of D-serine in the brain revolutionized the long-standing belief that only L-isomers of amino acids existed inmammalian tissues. D-Serine was found to be a highly selectiveendogenous activator of the NMDAR D-serine/glycine site (Mothet


et al., 2000; Shleper et al., 2005; Stevens et al., 2003). The origin ofD-serine in mammals was puzzling until the identification of theenzyme serine racemase (SRR), which directly converts L-serine toD-serine (Wolosker et al., 1999) in the presence of the co-factorspyridoxal 50phosphate, magnesium, and ATP (De Miranda et al.,2002). SRR is also able to convert D-serine into L-serine with a muchlower affinity and possesses a,b-elimination activity capable oftransforming L- or D-serine into pyruvate and ammonia (Foltynet al., 2005; De Miranda et al., 2002). Like D-serine, SRR is present inboth astrocytes and neurons of the brain (Williams et al., 2006b;Wolosker et al., 1999; Yoshikawa et al., 2006; Miya et al., 2008;Kartvelishvily et al., 2006), with a regional distribution that closelyresembles that of NMDARs (Schell et al., 1997). Though enriched inthe brain, SRR protein has also been detected in the murine liverand the human liver, kidney, and heart (Wolosker et al., 1999; Xiaet al., 2004). Similarly, D-serine levels are much higher in the CNSthan in peripheral tissues (Hashimoto et al., 1995). In the adulthuman and rodent brain, D-serine and SRR are predominantlylocalized to the forebrain, with high levels in the cerebral cortexand hippocampus, and minimal levels in the cerebellum andbrainstem (Xia et al., 2004; Schell et al., 1995). The low D-serineconcentrations in caudal brain areas coincide with the emergenceof D-amino acid oxidase (DAO), the D-serine catabolic enzyme(Wang and Zhu, 2003). Prior to the appearance of DAO, D-serinelevels are relatively high in caudal regions of the developing brain,where D-serine released from Bergman glia mediates NMDAR-dependent neuronal migration in the cerebellum (Kim et al., 2005).

DAO is highly selective for D-serine degradation at physiologicalpH, where it catalyzes the oxidative deamination of D-serine toproduce an a-keto acid, ammonia, and hydrogen peroxide(D’Aniello et al., 1993; Pollegioni et al., 1992). The braindistribution of DAO is inversely related to that of endogenous D-serine concentrations, with the highest levels of DAO in astrocytes,Golgi-Bergmann glia, and tanycytes of the hindbrain andcerebellum (Moreno et al., 1999). Lower levels of DAO have beendetected in the neurons of the prefrontal cortex, hippocampus, andsubstantia nigra (Verrall et al., 2007; Moreno et al., 1999). In theperiphery, DAO is most highly expressed in the kidneys and liver(Katagiri et al., 1991).

Modulating DAO function is G72 (also known as LG72 or DAOactivator), a gene unique to primates (Chumakov et al., 2002).Initially, G72 was reported to be an activator of DAO (Chumakovet al., 2002); however a recent study indicates that G72 mayinstead repress DAO activity (Sacchi et al., 2008). The function ofG72 remains controversial, as in mammalian cell lines and ratprimary hippocampal neurons, G72 was described to have analternate role, acting as a mitochrondrial protein that promotedmitochrondrial fragmentation and dendritic arborization (Kvajoet al., 2008). Though G72 mRNA has been detected in the humandorsolateral prefrontal cortex (Korostishevsky et al., 2004),difficulties in detecting native G72 protein have been noted(Benzel et al., 2008).

Recent studies have begun to elucidate the regulatory pathwayof D-serine and serine racemase (Fig. 2). Activation of astrocyticAMPA receptors stimulates D-serine release, an effect that isdependant on the rise of intracellular calcium and the soluble N-ethyl-malemide-sensitive factor attachment protein receptor(SNARE) (Mothet et al., 2005). Calcium has been shown to bindand activate SRR (Cook et al., 2002), indicating that SRR activity isregulated by neuronal depolarization and the related calciumentry. The binding of glutamate-receptor-interacting-protein(GRIP) to SRR enhances the formation of D-serine (Baumgartet al., 2007). It has been proposed that either GRIP disassociatesfrom phosphorylated AMPA receptors and activates SRR in thecytosol or GRIP mediates translocation of SRR to the proximity ofAMPA receptors for the formation of a ternary complex between

the GluR2 subunit of the AMPA receptor, SRR, and GRIP (Baumgartet al., 2007). Activation of metabotropic glutamate receptors(mGluR5) on astrocytes have also recently been shown to enhanceD-serine production and SRR activity by inducing phospholipase C(PLC) that eliminates phosphatidylinositol (4,5)-bisphosphate(PIP2) inhibition of SRR (Mustafa et al., 2009). Additionally,protein-interacting with kinase C (PICK1) has been shown to bindto the C-terminus of SRR (Fujii et al., 2006), possibly to directlymodulate its activity. Alternatively or in addition, PICK1 may escortprotein kinase C (PKC) to SRR, leading SRR phosphorylation andaltered SRR activity (Fujii et al., 2006). Following synthesis, D-serine undergoes vesicular storage and release from astrocytes(Martineau et al., 2008; Mothet et al., 2005; Williams et al., 2006b)or is released through a nonvesicular pathway from neurons(Kartvelishvily et al., 2006) and possibly astrocytes (Ribeiro et al.,2002). Synaptic D-serine facilitates NMDAR activation, althoughactive NMDARs are in turn capable limiting D-serine availabilitythrough translocation of cytosolic SRR to the plasma membranewhich reduces its capacity to form D-serine (Balan et al., 2009) and/or by promoting release of nitric oxide that enhances DAO functionand suppresses SRR activity (Shoji et al., 2006a,b; Mustafa et al.,2007). NMDAR-mediated feedback regulation of D-serine avail-ability may be a means of preventing receptor over-excitation.

Intracellular levels of D-serine are regulated by SRR and DAO;however clearance of D-serine from the synaptic space is assuredby various sodium-dependent and sodium-independent transpor-ters expressed on neurons and glia (Ribeiro et al., 2002; O’Brienet al., 2005; Helboe et al., 2003; Javitt et al., 2002). Among thetransporters, the alanine-serine-cysteine transporter 1 (Asc-1)mediates the majority of D-serine reuptake in the brain (Rutteret al., 2007). Asc-1 is located on the presynaptic terminal,dendrites, and cell body of neurons (Helboe et al., 2003) and isthe only transporter that exhibits a high D-serine affinity (Rutteret al., 2007).

4. The predominant physiological co-agonist of theNMDAR D-serine/glycine site

Growing evidence indicates that D-serine, rather than glycine, isthe dominant endogenous ligand for the D-serine/glycine site ofmost NMDARs. Depletion of D-serine by treatment with DAO hasbeen shown to attenuate NMDAR activity, as measured bybiochemical and electrophysiological approaches in cerebellarslices, hippocampal slices, hippocampal cell cultures, and retinapreparations (Mothet et al., 2000; Yang et al., 2003; Gustafsonet al., 2007). Moreover, in hypothalamic slices, NMDAR currentsare substantially reduced following elimination of D-serine by DAO,while a loss of glycine by a glycine oxidase enzyme does notproduce an effect (Panatier et al., 2006). Similarly, removal of D-serine with a recombinant D-serine deaminase enzyme suppressedNMDAR-mediated light-evoked responses in retinal cells andNMDAR-induced neurotoxicity in organotypic hippocampal slices(Gustafson et al., 2007; Shleper et al., 2005). In a senescence-accelerated mouse strain, deficient NMDAR-dependent LTP in thehippocampus was associated with a diminished production of D-serine, but not lowered levels of glycine (Mothet et al., 2006). Theeffects of diminished NMDAR-mediated neurotransmission inthese experiments could be fully reversed by the application ofexogenous D-serine (Mothet et al., 2000, 2006; Yang et al., 2003;Panatier et al., 2006; Gustafson et al., 2007). Together, theseexperiments favor D-serine as the predominant physiological co-agonist for the NMDAR D-serine/glycine site.

Increases in D-serine are also capable of further enhancingNMDAR signaling. This has been demonstrated several in vitro

studies examining NMDAR-evoked excitatory responses in theprefrontal cortex, hippocampus, striatum, and in hippocampal

Fig. 2. Schematic of putative D-serine pathway. Serine racemase (SRR) synthesizes D-serine (~) from L-serine (!), while D-amino acid oxidase (DAO) is responsible for D-serine

degradation. Stimulation of AMPA receptors (AMPAR) and association of glutamate-receptor-interacting-protein (GRIP) with SRR increases the production of D-serine.

Activation of metabotropic glutamate receptors (mGluR) also enhances D-serine synthesis by stimulating phospholipase C (PLC) that degrades phosphatidylinositol (4,5)-

bisphosphate (PIP2), thereby eliminating PIP2 inhibition of SRR. Protein-interacting with kinase C (PICK1) binds to SRR, possibly to directly regulate its activity or to bring

protein kinase C into the vicinity of SRR. D-Serine is released via vesicular and non-vesicular pathways into the synapse, where it then binds to the NMDA receptor (NMDAR) D-

serine/glycine site. Sodium-dependent and -independent transporters, such as Asc-1, mediate D-serine clearance from the extracellular space.


cultures (Chen et al., 2003; Martina et al., 2003; Yang et al., 2003;Chapman et al., 2003). In mice that lack DAO activity, the resultingelevation in D-serine potentiates NMDAR-mediated currents inspinal cord neurons (Wake et al., 2001). Mice that lack the neuronaltransporter Asc-1 also display NMDAR-dependent hyperexcitability(Xie et al., 2005), presumably from the elevations in extracellular D-serine. Finally, exogenous administration of D-serine has beenshown to elevate hippocampal responses in vivo, as measured bychanges in relative cerebral blood volume in a functional magneticresonance imaging (fMRI) study (Panizzutti et al., 2005).

Thus, these findings demonstrate a capacity for D-serine and itsmodulatory enzymes to dynamically regulate NMDAR activity, anddisturbances in this pathway could conceivably contribute topsychopathologies associated with abnormal NMDAR-mediatedneurotransmission.

5. Dysfunction of the NMDAR in schizophrenia

A large body of evidence supports a central role for aberrantNMDAR function in the pathophysiology of schizophrenia. Inaddition to the induction of psychotomimetic effects, subanes-thetic doses of a non-competitive NMDAR antagonist increaseamphetamine-induced dopamine release in the striatum to anextent that mimics the exaggerated responses seen in schizo-phrenic subjects (Kegeles et al., 2000). Chronic exposure to NMDARinhibitors in rodents and primates also lowers dopamine levels inthe prefrontal cortex and affects dopamine receptor binding(Tsukada et al., 2005; Jentsch et al., 1997), consistent with findingsin patients with schizophrenia (Abi-Dargham et al., 2002; Daviset al., 1991; Narendran et al., 2005). This suggests that aberrant

dopaminergic transmission in schizophrenia may be the conse-quence of a defect in the regulatory glutamatergic neuronalpathway. In addition, antagonists of the NMDAR disrupt activity inthe prefrontal cortex, affecting the efficiency of neuronal firing andsynchronization (Jackson et al., 2004; Kargieman et al., 2007),which may contribute to disturbances in cortical processing andcognitive function observed in schizophrenia. Furthermore,genetic association studies have identified a number of suscept-ibility genes that influence NMDAR function (Ross et al., 2006;Harrison and Weinberger, 2005). In drug-naıve schizophreniapatients, decreased in vivo hippocampal NMDAR binding andreduced plasma levels of endogenous NMDAR agonists have beenreported (Pilowsky et al., 2006; Hashimoto et al., 2003; Yamadaet al., 2005). Postmortem studies have found numerous alterationsin NMDAR receptor binding, transcript levels, and subunit proteinexpression in the cortex, hippocampus, and thalamus of schizo-phrenic individuals (Kristiansen et al., 2007). Reductions inparvalbumin-immunoreactive cells (GABAergic interneurons)and diminished expression of GAD67, the GABA synthesis enzyme,are also frequently observed in the postmortem hippocampus andprefrontal cortex (Reynolds et al., 2004; Torrey et al., 2005).Administration of NMDAR antagonists can replicate the loss ofparvalbumin and GAD67 (Keilhoff et al., 2004; Kinney et al., 2006),alter GABA-mediated inhibitory control of cortical neurons(Homayoun and Moghaddam, 2007), and disrupt the developmentof GABAergic neurons (Abekawa et al., 2007). Thus, NMDAhypofunction could contribute to the abnormalities in severalgenes and neurotransmitter systems implicated in the biologicalmechanism underlying schizophrenia. The indication of aberrantNMDAR function in schizophrenia pathogenesis prompts a need to


further understand how NMDAR hypofunction may arise in thisdisease and predicts that this system could be useful for thedevelopment of novel therapeutics. Genetic, clinical, postmortem,and pharmacological studies indicate that the NMDAR D-serine/glycine site and its regulators may be involved in the pathophy-siology and treatment of schizophrenia.

6. Genetic studies: G72, DAO, and SRR

Archival family, twin, and adoption studies indicate thatschizophrenia is highly heritable, but no single gene exhibits astrong effect. Instead, accumulating evidence indicates thatschizophrenia has a heterogeneous etiology involving a complexinterplay of multiple genes, epigenetics, and environmental factors(Harrison and Weinberger, 2005; Ross et al., 2006). Several of thegenes associated with schizophrenia risk are modulators ofNMDAR D-serine/glycine site activation. In particular, genesinvolved in D-serine catabolism and synthesis have been identified.

G72 was initially identified by Chumakov et al. (2002), whoexamined markers within a 5-Mb segment from chromosome13q33, a region that had previously been linked to schizophrenia inearlier linkage analyses. A significant association between G72 andschizophrenia was identified in French Canadian and Russianpopulations (Chumakov et al., 2002). In a yeast two-hybrid screen,G72 strongly associated with DAO and in vitro assays confirmed aregulatory effect of G72 on DAO activity (Chumakov et al., 2002;Sacchi et al., 2008). The positive association of G72 withschizophrenia susceptibility has since been replicated in numerousstudies (Shinkai et al., 2007; Schumacher et al., 2004; Fallin et al.,2005; Korostishevsky et al., 2004; Opgen-Rhein et al., 2008) andcontinues to be significant following meta-analyses (Shi et al.,2008). Additionally, G72 is one of the best supported locus forbipolar disorder (Prata et al., 2008; Schumacher et al., 2004; Fallinet al., 2005; Williams et al., 2006a). Furthermore, some evidenceindicates an association with major depression and panic disorder(Rietschel et al., 2008; Schumacher et al., 2005), with one largestudy of 2831 individuals suggesting that G72 may influencepredisposition to episodes of mood disorder across traditionalbipolar and schizophrenia categories (Williams et al., 2006a).Despite the number of positive associations, these studies havedemonstrated considerable allelic heterogeneity, with few studiesreporting association with the same allele at a SNP marker. Thelimited allelic compatibility along with the difficulties inidentifying endogenous G72 protein (Benzel et al., 2008) havemade it difficult to understand how G72 is dysregulated inschizophrenia and other psychiatric illnesses. However, Koros-tishevsky et al. (2004) did amplify G72 mRNA showing that it isoverexpressed in the dorsolateral prefrontal cortex of postmortemschizophrenia patients.

Interestingly, recent genetic classification and fMRI investiga-tions in healthy and schizophrenic populations report that geneticvariation in the G72 gene may influence cognitive function (Jansenet al., 2009; Hall et al., 2008; Opgen-Rhein et al., 2008; Goldberget al., 2006). Carriers of G72 risk variants differed in theirperformance during tests of working memory, verbal initiation,attention, and semantic fluency, and displayed differentialrecruitment of brain regions relevant to cognitive ability, includingthe hippocampal complex and prefrontal cortex (Jansen et al.,2009; Hall et al., 2008; Opgen-Rhein et al., 2008; Goldberg et al.,2006). By modulating DAO activity, G72 could contribute to theregulation of NMDAR-mediated cognitive function in the healthybrain and impact an array of diseases characterized by cognitiveimpairment.

The Chumakov et al. (2002) study also demonstrated that SNPmarkers within the DAO gene may confer an increased vulner-ability to schizophrenia, as surveyed in a French Canadian

population. This has been independently replicated in a numberof subsequent genetic investigations in German, Han Chinese, Irish,American schizophrenia samples (Schumacher et al., 2004; Liuet al., 2004; Corvin et al., 2007; Wood et al., 2007). Furthermore,some studies have indicated epistasis between DAO and G72,where the combined effect of polymorphisms in these genesresults in a greater risk of schizophrenia (Chumakov et al., 2002;Corvin et al., 2007). However, negative studies examining DAO

have been reported (Shinkai et al., 2007; Yamada et al., 2005), andthe evidence for an association between DAO and schizophreniasusceptibility is not as prevalent as that for G72. Similar to G72,functional variants in DAO have yet to be identified.

Preliminary studies have indicated that genetic variation in theSRR gene could contribute to schizophrenia. Investigationsexamining variants in the promoter and 50-terminus of SRR havedemonstrated significant associations with schizophrenia (Goltsovet al., 2006; Morita et al., 2006; Labrie et al., 2009), contrary topolymorphisms in the central and 30 region of SRR (Strohmaieret al., 2007; Labrie et al., 2009). Consequently, it is possible that the50 end of SRR is of importance in mediating abnormal SRR functionin schizophrenia. Accordingly, a significantly associated variantwas found to induce a 60% reduction in SRR promoter function(Morita et al., 2006). Furthermore, all SRR markers demonstrating apositive association are in close proximity to exon 1b, the majorSRR isoform in the brain (Yamada et al., 2005), suggesting apotential modulation of SRR transcription in the brain.

PICK1, an interacting partner of SRR, has also been described toconfer susceptibility to schizophrenia, particularly to the dis-organized subtype (Fujii et al., 2006; Hong et al., 2004). The PICK1

gene is found on chromosome 22q13.1, which a genetic locus thathas been often linked to schizophrenia (Hong et al., 2004). PICK1 isa scaffolding protein that regulates the subcellular localization andsurface expression of a number of binding partners (Dev andHenley, 2006), many of which are relevant to schizophrenia,including glutamate receptors, dopamine transporters, neuregulin,and ErbB tyrosine kinase receptors (Dev and Henley, 2006). PICK1has also been demonstrated to have an important role in NMDAR-dependent forms of synaptic plasticity (Terashima et al., 2008).

7. Evidence for abnormal modulation of the NMDAR D-serine/glycine site in patients with schizophrenia

Kynurenic acid (KYNA) is the only known endogenous NMDARD-serine/glycine site antagonist (Erhardt et al., 2009). It alsofunctions as a non-competitive inhibitor of a-7 nicotinic acet-ylcholine receptors (Hilmas et al., 2001). Elevations in kynurenicacid have been found in the CSF and postmortem brain ofschizophrenia patients (Erhardt et al., 2001; Schwarcz et al., 2001).Additionally, increased activity of kynurenine aminotransferase-1(KAT-1), a synthesis enzyme for KYNA, was reported in schizo-phrenic individuals (Table 1) (Kapoor et al., 2006).

D-Serine levels were found to be reduced in the CSF of drug-naıve patients with schizophrenia (Hashimoto et al., 2005b;Bendikov et al., 2007). Serum analysis has also indicateddiminished D-serine, along with a concomitant elevation in L-serine, suggesting a dysfunction of SRR activity (Hashimoto et al.,2003). Indeed, changes in SRR protein expression have beenreported in the postmortem hippocampus and cortex of schizo-phrenic individuals, with some studies indicating a decrease(Bendikov et al., 2007), while others an increase (Table 1) (Steffeket al., 2006; Verrall et al., 2007). Postmortem findings have alsodemonstrated an elevation in DAO protein in the hippocampus andcerebellum of patients with schizophrenia (Verrall et al., 2007;Bendikov et al., 2007), as well as an increase in cortical DAO activity(Table 1) (Madeira et al., 2008; Kapoor et al., 2006). Further supportfor abnormalities in the D-serine pathway in schizophrenia is

Table 1Changes in the expression of genes affecting the NMDAR D-serine/glycine site in postmortem schizophrenic brain.

Gene Brain region Refs.

Prefrontal cortex Cortex Hippocampus Cerebellum

Serine racemase (SRR) mRNA: NS;

protein: ", NS

mRNA: NS;

protein: #, NS

Protein: #, " mRNA: NS;

protein: NS

Bendikov et al. (2007),

Verrall et al. (2007),

Steffek et al. (2006),

Kapoor et al. (2006)

D-amino acid oxidase (DAO) mRNA: NS mRNA: NS;

protein: NS;

activity: "

Protein: " mRNA: ", ";protein: t";activity: "

Bendikov et al. (2007),

Verrall et al. (2007),

Madeira et al. (2008),

Kapoor et al. (2006)

Alanine-serine-cysteine transporter (Asc-1) mRNA: NS;

protein: #mRNA: NS;

protein: #Burnet et al. (2008)

Glycine transporter 1 (GlyT-1) mRNA: NS;

protein: NS

mRNA: NS;

protein: NS

Burnet et al. (2008)

System A amino acid transporter 2 (SNAT2) mRNA: # mRNA: # Burnet et al. (2008)

Kynurenine aminotransferase-1 (KAT-1) mRNA: NS;

activity: NS

mRNA: NS;

activity: "Kapoor et al. (2006)

NS, no significant change; ", increase; #, decrease; t", trend for increase (near significance).


indicated by reductions in PICK1 mRNA (Beneyto and Meador-Woodruff, 2006) and Asc-1 protein (Burnet et al., 2008) in theprefrontal cortex. It has been speculated that lower levels of Asc-1may be a response to diminished D-serine availability (Burnet et al.,2008), though further investigation is required. Additionally,postmortem findings will benefit from replication with morebrain series and regions to determine the extent of the abnormalexpression and function of NMDAR D-serine/glycine site mod-ulators in schizophrenia. Use of biopsied olfactory epithelium fromliving patients (Sawa and Cascella, 2009) may also revealmolecular changes associated with the NMDAR D-serine/glycinesite, while limiting potential confounds that include the effects oflong-term exposure to medications, agonal state, and postmorteminterval.

Although conventional antipsychotics do not directly alter D-serine levels, improvement of schizophrenia symptoms arecorrelated with an elevation in D-serine (Ohnuma et al., 2008). Asimilar effect has been observed for glycine. In medication-freeschizophrenia patients, circulating levels of glycine have beenfound to be reduced and glycine availability inversely correlateswith the severity of negative symptoms (Sumiyoshi et al., 2004;Neeman et al., 2005). Treatment efficacy to ameliorate thesenegative symptoms correlated with a rise in plasma glycine levels(Sumiyoshi et al., 2005). Together, these studies suggest anabnormality in available glycine and/or D-serine in schizophreniaand predict the potential therapeutic utility of these compounds.

8. Pharmacological treatments targeting the NMDAR D-serine/glycine site

Since NMDAR hypoactivity potentiates schizophrenia-likesymptoms and is implicated in the pathophysiology of schizo-phrenia, it may follow that NMDAR activation could alleviatesymptoms of this disorder. The NMDAR D-serine/glycine site hasbeen proposed as a potential therapeutic target, as increasing itsactivation offers a safer alternative to elevations in glutamatelevels that can promote neurotoxicity (Olney, 1994; Coyle, 2006).To date, clinical trials have been conducted with the partial agonistD-cycloserine, the full agonists glycine and D-serine, and the GlyT-1inhibitor sarcosine (Table 2). Supporting the therapeutic effec-tiveness of correct modulation of disrupted glutamatergic path-ways are clinical reports that activation of mGluR2/3 receptorsprovides symptomatic improvements in schizophrenia patientswithout major adverse effects (Patil et al., 2007).

An initial study assessing the clinical efficacy of D-cycloserinein conjunction with conventional medications observed a U-shaped dose response curve (Goff et al., 1995), since as a partial

agonist D-cycloserine can function as an agonist or antagonistdepending on the degree of occupancy at the D-serine/glycine site(Sheinin et al., 2001). In this study of only 9 patients with dosesescalating every 2 weeks, improvements in negative and cognitivedeficits were found at an optimal dose (50 mg/day) (Goff et al.,1995). Although the capacity of D-cycloserine to improve negativesymptoms has been replicated in some larger placebo-controlledstudies (Heresco-Levy et al., 2002; Goff et al., 1999b), evidencesupporting the effectiveness of D-cycloserine is weak. D-Cyclo-serine was not found to be beneficial as adjunctive treatment in a6-month placebo-controlled trial (Goff et al., 2005), in a largemulticenter 16-week trial with a placebo comparison (Buchananet al., 2007), nor in a systematic review of the literature and meta-analysis (Tuominen et al., 2005; Tuominen et al., 2006). Amidst allthese negative findings, one study shows that D-cycloserineenhances temporal lobe activation in schizophrenia patientsduring a memory task and that this response is correlated with asignificant decrease in negative symptoms (Yurgelun-Todd et al.,2005). However, the overall disparity in findings indicates alimited therapeutic effect of D-cycloserine in the general patientpopulation.

Clinical trials examining the effects of a high dose of glycine(0.8 g/kg/day) administered as adjuvant treatment have demon-strated promising results, particularly in the amelioration ofprimary negative symptoms in patients with chronic schizophre-nia (Javitt et al., 1994, 2001; Leiderman et al., 1996; Tuominenet al., 2006). Some studies have indicated that glycine may alsoimprove cognitive and positive symptoms (Heresco-Levy et al.,1999, 2004), although these additional benefits are not supportedin a meta-analysis (Tuominen et al., 2005, 2006). In contrast, alarge multicenter study found that glycine administration did notameliorate negative or cognitive symptoms compared to placebotreatment (Buchanan et al., 2007). The lack of improvement in thistrial may be related to the higher percentage of patients treatedwith second-generation antipsychotics rather than conventionalantipsychotics. Additionally, serum levels of glycine in this trialwere lower compared to those in some of the previous positivestudies. Though glycine is generally well-tolerated with minimalserious side effects, glycine treatment has been associated withreoccurrent gastrointestinal upset (Heresco-Levy et al., 2004; Diazet al., 2005). In addition, glycine can be converted to L-serine, whichcan subsequently lead to the biosynthesis of D-serine. Conse-quently, a rise in D-serine may contribute to the therapeutic effectsof large doses of glycine. Accordingly, clinical trials administeringglycine treatments to schizophrenia patients have reported anelevation serum serine levels (Heresco-Levy et al., 1999, 2004;Javitt et al., 2001).

Table 2Modulators of the NMDAR D-serine/glycine site in clinical trials with schizophrenia patients.

Clinical trial Design Daily dose Sample

sizea

Treatment

duration

Patient sample Antipsychotics Therapeutic effect

D-Cycloserine

Cascella et al. (1994) Open label 250 mg 7 6 weeks Chronic Typical #: +, �,

gen psychopath

Goff et al. (1995) Single blind 5–250 mg 9 10 weeks

(2 weeks/dose)

Chronic Typical ": � at 50 mg

Rosse et al. (1996) Double-blind,

placebo-controlled

10, 30 mg 13 4 weeks Chronic Molindone NS

van Berckel et al. (1996) Single blind 15–250 mg 7 24 days

(4 days/dose)

Not specified Drug-free ": � at 100 mg

Heresco-Levy et al. (1998) Double-blind,

placebo-controlled

50 mg 9 6 weeks Chronic,

treatment-resistant

Typical/atypical ": �

Goff et al. (1999b) Double-blind,

placebo-controlled

50 mg 38 8 weeks PDS Typical ": �

van Berckel et al. (1999) Double-blind,

placebo-controlled

100 mg 25 8 weeks Chronic, prominent

negative symptoms

Typical #: +, gen

psychopath


placebo-controlled

50 mg 16 6 weeks Treatment-resistant Typical/atypical ": �, gen

psychopath

Duncan et al. (2004a) Double-blind,

placebo-controlled

50 mg 22 4 weeks Prominent negative

symptoms, PDS

Typical NS

Goff et al. (2005) Double-blind,

placebo-controlled

50 mg 26 6 months Prominent negative

symptoms

Typical NS

Buchanan et al. (2007) Double-blind,

placebo-controlled

50 mg 133 16 weeks Prominent negative

symptoms

Typical/atypical NS

Goff et al. (2008) Double-blind,

placebo-controlled

50 mg

once/week

33 8 weeks Chronic Typical/atypical ": �, cognitive

Glycine

Waziri (1988) Open label,

naturalistic

5–25 g 11 8–9 months Chronic Drug-free ": 4 patients

Rosse et al. (1989) Open label 10.8 g 6 4 days–8 weeks Chronic Typical ": 2 patients;

#: 2 patients

Costa et al. (1990) Open label 15 g 6 5 weeks Treatment-resistant Typical ": 2 patients

Javitt et al. (1994) Double-blind,

placebo-controlled

30 g 14 8 weeks Chronic Typical ": �

Leiderman et al. (1996) Open label 60 g 5 8 weeks Chronic Typical/atypical ": �Heresco-Levy et al. (1996) Double-blind,

placebo-controlled

60 g 11 6 weeks Chronic,

treatment-resistant

Typical/atypical ": �, cognitive,

gen psychopath


placebo-controlled

60 g 19 6 weeks Treatment-resistant Typical/atypical ": �, cognitive

Javitt et al. (2001) Double-blind,

placebo-controlled

60 g 12 6 weeks Chronic Typical/atypical ": �, cognitive


placebo-controlled

60 g 14 6 weeks Chronic,

treatment-resistant

Olanzapine/

risperidone

": +, �, cognitive

Buchanan et al. (2007) Double-blind,

placebo-controlled

60 g 133 16 weeks Prominent negative

symptoms

Typical/atypical NS

D-Serine

Tsai et al. (1998) Double-blind,

placebo-controlled

30 mg/kg 28 6 weeks Prominent negative

symptoms, PDS,

treatment-resistant

Typical/atypical/

drug-free


Lane et al. (2005) Double-blind,

placebo-controlled

2 g 57 6 weeks Acutely ill Risperidone NS


placebo-controlled

30 mg/kg 38 6 weeks Treatment-resistant Risperidone/

olanzapine


Sarcosine

Tsai et al. (2004a) Double-blind,

placebo-controlled

2 g 36 6 weeks Chronic Typical/

risperidone/

drug-free

": all


placebo-controlled

2 g 57 6 weeks Acutely ill Risperidone ": �, cognitive,

gen psychopath

Lane et al. (2008) Double-blind 1, 2 g 16 6 weeks Acutely ill Drug-free/

drug-naıve

Modest ": +/�at 2 g in drug

naıve patients

", improvement; #, exacerbation; NS, no significant change.

Abbreviations: +, positive symptoms;�, negative symptoms; gen psychopath, general psychopathology; all, +/�/cognitive symptoms/general psychopathology; PDS, primary

deficit syndrome.a Number of patients that completed the trial.


In a preliminary study, D-serine (30 mg/kg) in combination withantipsychotic drugs was found to be therapeutically beneficial, as itconsiderably reduced positive, negative, and cognitive symptomsof schizophrenia (Tsai et al., 1998). These ameliorative effects wereconfirmed in a subsequent clinical trial in which D-serine was

added to risperidone or olanzapine, and improvements in positive,negative, cognitive, and depressive symptoms were found intreatment-resistant patients (Heresco-Levy et al., 2005). However,in patients with an acute exacerbation of psychosis, D-serine didnot produce any benefits beyond risperidone monotherapy (Lane


et al., 2005). The lack of beneficial effects could be related todifferential treatment responses in acutely ill patients compared totreatment-resistant individuals and indicates that D-serine may beless effective in treating psychotic symptoms. A meta-analysis ofclinical trials with D-serine and glycine indicated that thesecompounds improved negative symptoms of schizophrenia whenadministered as adjuvant therapies, but determined that theseeffects were not fully consistent and required further replication(Tuominen et al., 2006). Overall, D-serine was well-tolerated anddevoid of significant side effects (Tsai et al., 1998; Lane et al., 2005;Heresco-Levy et al., 2005). One concern with D-serine treatmentshas been renal toxicity, since large doses of D-serine have beenfound to cause reversible acute tubular necrosis in rats (Caroneet al., 1985). The nephrotoxic effects of D-serine were not observedin mice, guinea pigs, rabbits, dogs, and gerbils (Kaltenbach et al.,1979) and analysis of kidney function parameters did not revealany abnormalities in the clinical trials (Tsai et al., 1998; Lane et al.,2005; Heresco-Levy et al., 2005).

As an alternative to directly activating the NMDAR D-serine/glycine site, clinical investigations also examined sarcosine, aninhibitor of GlyT-1 that effectively raises synaptic levels of glycine(Bergeron et al., 1998). Sarcosine (2 g/day) cotreatment withconventional medications or risperidone significantly reducedpositive, negative, cognitive, and general psychopathology symp-toms in patients with stable chronic schizophrenia and in patientswith acute exacerbation of schizophrenia (Tsai et al., 2004a; Laneet al., 2005). Recently, the effectiveness of sarcosine was examinedin a drug-free cohort displaying acute psychotic symptoms; thefirst clinical trial examining a glycine reuptake inhibitor in absenceof other medications (Lane et al., 2008). Though symptomaticamelioration was observed, it was limited to a small patientsubgroup with no previous antipsychotic exposure and no placebocontrol was conducted (Lane et al., 2008). Larger studies withplacebo or active comparators will be necessary to determine thetherapeutic benefits of NMDAR D-serine/glycine site agonists asfirst-line antipsychotics.

A peculiarity of NMDAR D-serine/glycine site agonists is thatalthough ameliorative effects are found in combination withsecond-generation antipsychotics, such as risperidone and olan-zapine, these treatments are ineffective when administered inconjunction with clozapine (Table 3) (Tsai et al., 1999; Lane et al.,2006; Goff et al., 1996). Clozapine is a commonly used atypical

Table 3Clinical trials with modulators of the NMDAR D-serine/glycine site added to clozapine

Clinical trial Design Daily dose Sample sizea

D-Cycloserine

Goff et al. (1996) Single blind 5–250 mg 10

Goff et al. (1999a) Double-blind,

placebo-controlled

50 mg 11

Glycine

Potkin et al. (1999) Double-blind,

placebo-controlled

30 g 19

Evins et al. (2000) Double-blind,

placebo-controlled

60 g 27

Diaz et al. (2005) Double-blind,

placebo-controlled

60 g 12

D-Serine

Tsai et al. (1999) Double-blind,

placebo-controlled

30 mg/kg 20

Sarcosine


placebo-controlled

2 g 20

#, exacerbation; NS, no significant change; �, negative symptoms; PDS, primary deficita Number of patients that completed the trial.

antipsychotic with a broad pharmacological profile that includesaffinity for D1- and D2-like dopamine receptors and 5-HT2

receptors (Ross et al., 2006). Also, clozapine has been shown toincrease extracellular levels of glutamate (Melone et al., 2001),potentiate NMDAR-mediated synaptic transmission (Arvanovet al., 1997), reverse antagonist blockade of NMDAR channels in

vivo (Bressan et al., 2005), normalize PCP-induced neuronalhyperactivity in the prefrontal cortex (Kargieman et al., 2008),and attenuate behavioral deficits induced by NMDAR inhibitors(Gaisler-Salomon and Weiner, 2003; Lipina et al., 2005). Thus,clozapine may augment NMDAR activity to an extent that impedesfurther enhancements with D-serine/glycine site agonists. Addi-tionally, clozapine may augment synaptic levels of glycine byinhibiting glycine reuptake into neurons and glia (Javitt et al.,2005; Williams et al., 2004).

Overall, clinical trials with D-serine/glycine site activators haveindicated therapeutic potential, particularly D-serine and sarco-sine. However, thus far, studies are generally conducted with arelatively low number of participants (<30) over a 6 week period(Tables 2 and 3). Consequently, longer studies investigating agreater number of patients are required to ascertain the benefits ofD-serine/glycine site agonists. Also, since the majority of studiesexamine treatment-resistant patients with stable chronic symp-toms, assessment of these proposed antipsychotics in a morediverse patient population may reveal additional improvements.Regardless, clinical trials to date demonstrate promising ameli-orative effects and warrant further investigation with agonists ofthe NMDAR D-serine/glycine site. Compounds targeting the D-serine/glycine site may offer a novel and well-tolerated alternativefor the treatment of schizophrenia.

In addition, investigations that examine how direct and indirectagonists of the NMDAR D-serine/glycine site affect the tonic andphasic components of NMDAR activation will be important indetermining the consequences and safety of long-term adminis-tration of these compounds. Exogenous D-serine or glycineadministration could potentially increase tonic extracellular D-serine/glycine levels. Alternatively, exogenous D-serine or glycinecould be rapidly cleared from the synaptic space by reuptaketransporters, leading to increased intracellular stores andenhanced agonist release during phasic stimulation. Furthermore,inhibition of GlyT-1 may facilitate tonic NMDAR activation,although this would be dependent on the rate of synaptic efflux

treatment.

Duration Patient sample Therapeutic effect

10 weeks

(2 weeks/dose)

Primary deficit syndrome #: � at 50 mg

6 weeks Prominent negative symptoms #: �

12 weeks Chronic, treatment-resistant NS

8 weeks Prominent negative symptoms NS

12 weeks Treatment-resistant NS

6 weeks Prominent negative symptoms,

PDS, treatment-resistant

NS

6 weeks PDS, treatment-resistant NS

syndrome.


and influx of glycine originating from astroglial and neuronal cells.Passive diffusion of glycine from the CSF could also contribute toincreased tonic NMDAR stimulation in the presence of an GlyT-1antagonist (Supplisson and Bergman, 1997). Prolonged NMDARstimulation can potentiate neuronal death (Hardingham andBading, 2003) and studies have demonstrated that excessive D-serine/glycine site activation may contribute to excitotoxicity andneuroinflammation in several neurodegenerative diseases (Sasabeet al., 2007; Wu et al., 2004; Wolosker et al., 2008). However, thepossibility that chronic exposure to D-serine/glycine site agonistsproduce such effects in patients with schizophrenia (that areproposed to have limited D-serine/glycine site occupancy) ispresently unknown.

Enhanced activity of the NMDAR D-serine/glycine site may alsobe beneficial for the treatment of other psychiatric disorders,including several anxiety syndromes and drug addiction, byaugmenting the extinction of learned behaviors (Davis et al.,2006). Extinction is considered to be a form of inhibitory learning,whereby acquired behavioral responses are suppressed followingthe repetitive exposure to a conditioned stimulus in the absence ofa reinforcing (unconditioned) stimulus (Davis et al., 2006). Studiesof fear extinction in rats found that administration of D-cycloserineaccelerated the effects of extinction and that this accelerationcould be blocked by an NMDAR D-serine/glycine site antagonist(Walker et al., 2002). Since fear extinction in rodents is similar toexposure-based psychotherapy in the humans, translationalresearch from preclinical to clinical studies was initiated toexamine the therapeutic effects of D-cycloserine (Norberg et al.,2008). The initial study examined individuals with a fear of heights(acrophobia) and found that compared to placebo, D-cycloserineenhanced the effects of virtual reality exposure therapy, resultingin a larger reduction of acrophobic symptoms at 1 week and 3months following treatment (Ressler et al., 2004). D-Cycloserinewas effective when given as single doses prior to the exposuresessions (Ressler et al., 2004), consistent with findings in animalstudies demonstrating that D-cycloserine is better able to enhanceextinction learning when administered acutely (Parnas et al., 2005;Davis et al., 2006). Subsequent placebo-controlled studies inpatients with obsessive-compulsive disorder (Kushner et al., 2007;Wilhelm et al., 2008) and social phobia (Hofmann et al., 2006;Guastella et al., 2008) have confirmed the ability of D-cycloserine toaugment the effects of exposure therapy. Additionally, extinctionof the preference for a cocaine-associated environment wasfacilitated by D-cycloserine (Botreau et al., 2006), suggesting thatthis partial agonist of the NMDAR D-serine/glycine site aids inextinguishing craving behaviors related to drug addiction. Fromthese studies with D-cycloserine, it is not clear whether fearextinction and exposure therapy are facilitated by the augmenta-tion of NMDAR responses during extinction or by the reduction ofNMDAR activity during the memory consolidation process.

9. Animal models based on diminished NMDAR function

Consistent with the psychotomimetic effects of NMDARantagonists in humans, non-competitive inhibition of the NMDARin animals produces a range of behavioral impairments that arereminiscent of the symptoms of schizophrenia (Bubenıkova-Valesova et al., 2008). Behavioral abnormalities following acute orchronic treatment with an NMDAR antagonist in rodents includelocomotor hyperactivity, information-processing disturbances,social approach impairments, and deficits in reversal learning andworking memory (Nilsson et al., 1997; Yee et al., 2004; van derMeulen et al., 2003; Ellenbroek and Cools, 2000; Bubenıkova-Valesova et al., 2008). Similarly, administration of PCP in non-human primates produces impairments in prepulse inhibition(Linn et al., 2003), social behaviors (Mao et al., 2008), and working

memory (Thompson et al., 1987). Chronic PCP treatment followedby a withdrawal period has also been shown to induce enduringcognitive deficits, along with reduced dopaminergic utilization inthe prefrontal cortex (Jentsch et al., 1997). Furthermore, repeatedexposure to PCP has been shown to induce neurodegenerationthroughout the brain (Ellison and Switzer, 1993), which mayresemble some of the neuroanatomical changes associated withschizophrenia (Beckmann, 1999; Harrison and Weinberger,2005).

To address neurodevelopmental aspects of schizophrenia,models that involve perinatal treatment with NMDAR antagonistshave been developed (Bubenıkova-Valesova et al., 2008). Thesemodels test the hypothesis that viral or environmental insultsoccurring during the late second trimester of pregnancy, a periodimportant to the development of the fetal CNS, subsequentlyincreases the likelihood of developing schizophrenia in adulthood(Beckmann, 1999). In rats, the corresponding period is in the first 2weeks of postnatal life (Clancy et al., 2001), and administration ofNMDAR antagonists during this time results in an increase intransient neuronal apoptosis (Ikonomidou et al., 1999). Earlyexposure to NMDAR inhibitors leads to the emergence of severalschizophrenia-related behaviors in adult animals, includingdeficits in information-processing (Wedzony et al., 2008),enhanced sensitivity to NMDAR blockers and dopamine-releasingstimulants (Abekawa et al., 2007; Wedzony et al., 2005), andimpairments in working and reference memory (Andersen andPouzet, 2004). Additionally, perinatal administration of NMDARantagonists induces enduring alterations in hippocampal NR1subunit expression (Harris et al., 2003), mesolimbic dopaminereceptor binding (du Bois et al., 2008), synaptogenesis in thehippocampus (Bellinger et al., 2002), and decreases the number ofcortical parvalbumin-positive GABAergic neurons in adult rats(Abekawa et al., 2007). Recently, oligodendrocyte differentiationand myelination have also been found to be affected by PCPtreatment in developing rats (Lindahl et al., 2008), consistent withthe white matter abnormalities that have been observed inschizophrenia (Harrison and Weinberger, 2005). Thus, earlychanges in NMDAR function produce lasting changes relevant toschizophrenia pathophysiology.

Since schizophrenia is a heritable disease proposed to involveabnormalities in the glutamatergic system, genetic animal modelsof aberrant NMDAR function have been established. These modelsalso have the advantage of reproducing the chronic and develop-mental nature of NMDAR hypofunction theorized to occur inschizophrenia. Mice with complete and global loss of the NMDA-NR1 subunit die neonatally (Forrest et al., 1994). However,targeted ablation of the NR1 subunit in the dentate gyrus of thehippocampus results in spatial working memory impairments(Niewoehner et al., 2007), while loss of NR1 in hippocampal CA3pyramidal cells results in deficient associative memory recall inadult mice (Table 4) (Nakazawa et al., 2002). Mice with a 95%reduction in normal levels of NR1 show abnormalities in CNSdevelopment, disrupted sensorimotor gating, impaired social andsexual interactions, and increased spontaneous locomotor activity,stereotypy, and sensitivity to amphetamine (Table 4) (Elberger andDeng, 2003; Mohn et al., 1999; Moy et al., 2006). Alternatively,overexpression of the NMDA receptor composed of NR1-NR2Bsubunits in the mouse forebrain enhances NMDAR-dependentsynaptic potentiation and produces improvements in learning andmemory (Tang et al., 1999).

Although these studies support that disturbances in theNMDAR produce phenotypes that are potentially relevant to thesymptoms of schizophrenia, they do not directly examine theeffects of modulating the NMDAR D-serine/glycine site. Consider-ing the growing evidence indicating abnormal D-serine availabilityand NMDAR D-serine/glycine site function in the pathophysiology

Table 4Behavioral phenotypes in genetic mouse models targeting the NMDAR-NR1 subunit.

Mutant mouse model Behavioral phenotypes relevant to psychiatric disease Refs.

NR1 knockdown (90–95% loss) Deficits in social and sexual interactions that are reversible by

clozapine; hyperactivity and increased stereotypy; resistance to

hyperlocomotion and stereotypy induced by NMDAR antagonists;

increased amphetamine-induced stereotypy; reduced aggression;

PPI deficit that is reversible by antipsychotics; increased startle

reactivity; deficits in selective attention

Mohn et al. (1999),

Duncan et al. (2004b, 2006),

Miyamoto et al. (2004),

Bickel et al. (2007)

NR1+/N598Q Impaired maternal behaviors; increased mortality Single et al. (2000)

iCA1-KO of NR1 (inducible, reversible,

and CA1-specific NR1 KO)

Deficits in spatial and nonspatial learning and memory, spatial

memory consolidation, and retention of contextual fear associations

Shimizu et al. (2000),

Rondi-Reig et al. (2001)

CA3-NR1 KO (NR1 deletion in

hippocampal CA3 pyramidal cells)

Deficits in associative memory recall (memory retrieval deficits upon

partial removal of spatial cues); impairments in spatial working

memory and adaptive nonspatial learning and extinction

Nakazawa et al. (2002, 2003),

Kishimoto et al. (2006)

CA3 NR1 knockdown with AAV-Cre

(10–30% loss of NR1 in dorsal

hippocampal CA3 region)

Impaired associative learning Rajji et al. (2006)

DG-NR1 KO (deletion of NR1 in dendate

gyrus granule cells of the hippocampus)

Impairments in context discrimination (decreased ability to

distinguish two similar contexts); deficits in spatial working memory

McHugh et al. (2007),

Niewoehner et al. (2007)

iFB-KO of NR1 (inducible, reversible,

and forebrain-specific NR1 KO)

Deficits in contextual and cued fear memories; impaired

nonspatial memory retention and consolidation

Cui et al. (2004, 2005)

Abbreviations: KO, knockout; PPI, prepulse inhibition. Not listed are several genetic models demonstrating that aberrant NR1 function affects behaviors relevant to drug

addiction and nociception.


of schizophrenia (as described earlier), animal models investigat-ing the effects of decreased occupancy and activation of theNMDAR D-serine/glycine site may be more proximal to the neuralchanges proposed to occur in schizophrenia. Furthermore,examination of such animal models may uncover novel targetsfor the treatment of this disease.

10. Animal models relevant to the NMDAR D-serine/glycine site

Several genetic animal models examining the effects of aberrantfunction of the NMDAR D-serine/glycine site and its modulatoryenzymes have been developed (Table 5). Grin1D481N mutant micewith a five-fold reduction in NMDAR glycine affinity displaybehavioral abnormalities that include persistent latent inhibition(LI) and impairments in sociability, spatial recognition, learning, andmemory (Labrie et al., 2008a; Duffy et al., 2008b). LI persistence isalso seen in mice administered the highly selective NMDAR D-serine/glycine site antagonist L-701,324 (Labrie et al., 2008a) and theNMDAR channel blocker MK-801 (Lipina et al., 2005). Prolonged LIhas been associated with the deficits in information-processing andcognitive flexibility observed in schizophrenia patients (Weiner,2003; Moser et al., 2000) and has been correlated with the severity ofnegative symptoms (Rascle et al., 2001; Cohen et al., 2004).Furthermore, sociability deficits in animals, as seen in the Grin1D481N

mice, resembles the social withdrawal aspect of the negativesymptoms of schizophrenia (Ellenbroek and Cools, 2000). Impairedsociability is also seen in mice carrying an N-nitroso-N-ethylurea(ENU)-induced point mutation in the Srr gene that results in a loss ofSRR activity and lowered D-serine levels (Labrie et al., 2009). Inaddition, the SrrY269* mice demonstrate a deficit in sensorimotorgating, a reflexive and pre-attentive form of information-processingreliant on brainstem circuits (Fendt et al., 2001) that is commonlydisrupted in patients with schizophrenia (Braff et al., 1992). Like theGrin1D481N mice, animals with the SrrY269* mutation displaydysfunctions in cognitive ability that include deficits in spatialobject discrimination and long-term memory (Labrie et al., 2009;Basu et al., 2009). Thus, these genetic models of reduced NMDAR D-serine/glycine site occupancy demonstrate disturbances related toschizophrenia, particularly to the negative and cognitive symptoms,suggesting that the D-serine/glycine site and its modulators may berelevant to the pathophysiology of these symptoms. An important

limitation of these models is the apparent of lack of behavioralphenotypes recapitulating the psychotic features of schizophreniaand the influence of the D-serine/glycine site on other neurotrans-mitter systems implicated in schizophrenia requires furtherinvestigation. In contrast, Grin1D481N/K483Q mice that have two pointmutations in the NMDAR D-serine/glycine site and more severereductions in D-serine/glycine site occupancy demonstrate abnorm-alities related to psychosis, including locomotor hyperactivity,enhanced stereotypy, and striatal dopaminergic and serotonergichyperfunction (Ballard et al., 2002). However, these animals alsodemonstrate a resistance to antipsychotics and behavioral deficitsthat are so profound (Ballard et al., 2002) that their relevance toschizophrenia symptomatology is limited.

Animal models with enhanced activation of the NMDAR D-serine/glycine site demonstrate a resistance to schizophrenia-likesymptoms and improvements in cognitive performance. Dao1G181R

mice lack functional DAO, resulting in an elevation in brain D-serine levels (Labrie et al., 2008b; Hashimoto et al., 1993) and an in

vivo augmentation of NMDAR function (Almond et al., 2006). Thesemice have been reported to be resistant to the psychotomimeticeffects of MK-801 (Almond et al., 2006; Hashimoto et al., 2005a)and methamphetamine (Hashimoto et al., 2008), as indicated by anattenuation of drug-induced hyperactivity and stereotypy. Like-wise, pharmacological administration of D-serine has been shownto reverse behavioral deficits induced by MK-801 or geneticdiminution of NMDAR D-serine/glycine site activation (Lipina et al.,2005; Labrie et al., 2008a). For example, D-serine normalized MK-801-induced hypermotility, persistent LI, and novelty discrimina-tion deficits in wild-type mice (Nilsson et al., 1997; Lipina et al.,2005; Karasawa et al., 2008) and reversed the impairments insociability, sensorimotor gating, spatial recognition and memory inGrin1D481N and SrrY269* mutant mice (Labrie et al., 2008a, 2009).Furthermore, Dao1G181R mutant animals display enhancements inspatial reversal learning and increased extinction rates that aresimilarly observed in C57BL/6J mice given exogenous treatmentsof D-serine (Labrie et al., 2008b; Duffy et al., 2008b). Consequently,DAO inactivation and augmented D-serine concentrations maynormalize NMDAR hypofunction and cognitive inflexibility inschizophrenia. Moreover, the improvements in reversal learningand extinction imply that DAO inhibition and D-serine may bebeneficial for the treatment of many other psychopathologies

Table 5Behavioral phenotypes of genetically modified mice affecting NMDAR D-serine/glycine site function.

Gene Function Mutant mouse model Behavioral phenotypes Refs.

NR1 NMDAR subunit

with D-serine/

glycine binding

site

Grin1D481N (5-fold reduction

in glycine affinity)

Persistent LI; impairments in sociability,

spatial object recognition, spatial reference

learning and memory; behavioral deficits

reversible by D-serine or clozapine; reduced

anxiety; increased startle reactivity

Labrie et al. (2008a),

Kew et al. (2000)

Grin1D481N/K483Q (biphasic

NMDAR glycine affinity:

6 and 90-fold reductions)

Hyperactivity and increased stereotypy that

is resistant to antipsychotics and zolpidem;

resistance to MK-801-induced hyperactivity;

increased startle reactivity; impaired nest

building and performance in motor tests and

in a visible platform learning task; striatal

dopaminergic and serotonergic hyperfunction

Ballard et al. (2002)

Serine racemase (SRR) D-serine

synthesis

Srr�/�(exon 1 KO; loss of

SRR and �89% reduction

in D-serine)

Males show open-field hyperactivity and

impaired spatial reference memory; females

show increased anxiety and startle reactivity

Basu et al. (2009)

SrrY269* (loss of SRR activity

and �95% reduction

in D-serine)

Deficits in PPI, sociability, spatial object

recognition, spatial reference memory;

behavioral deficits reversible by D-serine or

clozapine; increased sensitivity to

MK-801-induced hyperactivity and PPI deficits

Labrie et al. (2009)

D-amino acid oxidase

(DAO)

D-serine

catabolism

Dao1G181R (loss of DAO

activity)

Improved spatial reversal learning and

extinction (C57BL/6J background)

Labrie et al. (2008b)

Enhanced spatial reference learning and memory;

resistance to hyperactivity, stereotypy, and

ataxia induced by NMDAR antagonists;

resistance to methamphetamine-induced

stereotypy; increased motor coordination

and hypoactivity; elevated startle reactivity;

enhanced nociception (ddY background)

Maekawa et al. (2005a),

Almond et al. (2006),

Hashimoto et al.

(2005a, 2008)

G72 Regulator

of D-amino

acid oxidase

G72tg (expression of

primate-specific LG72

protein in mouse)

Impaired PPI that is reversible by haloperidol;

deficits in motor coordination and olfactory

discrimination; increased compulsive behaviors

(more grooming and females exhibit increased

digging behavior); decreased aggression in males;

increased sensitivity to PCP-induced hyperactivity

Otte et al. (2009)

Glycine transporter 1

(GlyT-1)

Glycine

reuptake

transporter

GlyT1(+/�) (50% loss of

GlyT-1 function and

glycine uptake)

Enhanced spatial reference memory; resistance

to amphetamine-induced PPI deficit; increased

sensitivity to MK-801-induced PPI disruption

Tsai et al. (2004b)

CamKIIaCre:Glyt1tm1.2fl/fl

(GlyT-1 KO in forebrain neurons)

Improved associative learning, LI, spatial and

object recognition memory; resistance to

PCP-induced hyperactivity

Yee et al. (2006),

Singer et al. (2007)

Alanine-serine-cysteine

transporter (Asc-1)

D-serine reuptake Asc-1(�/�) (Asc-1 KO) Tremors, ataxia, seizures, and early postnatal death Xie et al. (2005)

Abbreviations: KO, knockout; PPI, prepulse inhibition; LI, latent inhibition.


involving persistent maladaptive behaviors, such as obsessive-compulsive disorder and post-traumatic stress syndrome.

Though D-serine and similar compounds have demonstratedpromising effects in clinical trials with medicated schizophreniapatients (Heresco-Levy et al., 2005; Tsai et al., 1998; Coyle, 2006),there are issues regarding the administration of these compounds, aslarge doses are required for therapeutic effect. In contrast, DAOinhibitors cross the blood-brain-barrier readily (Adage et al., 2008;Hashimoto et al., 2009). To date several compounds that inhibit DAOactivity have been developed, including AS057278, CBIO, compound8, and compound 27 (Adage et al., 2008; Hashimoto et al., 2009;Smith et al., 2008; Duplantier et al., 2009). DAO inhibitors arecapable of regulating brain D-serine levels, as AS057278 was shownto elevate D-serine in the rat cortex and midbrain (Adage et al., 2008),while compound 27 augmented D-serine in the cerebellum of mice(Duplantier et al., 2009). In rodents, AS057278 was found tonormalize PCP-induced sensorimotor gating deficits and hyperlo-comotion (Adage et al., 2008). Conversely, compound 8 was notfound to be effacious in reversing the behavioral effects ofpsychostimulants, although this may be attributed to the inabilityof compound 8 to significantly elevate cortical D-serine levels (Smith

et al., 2008). An alternative approach is to enhance the efficacy ofDAO inhibitors with a low dose of D-serine. CBIO administered inconjunction with D-serine (30 mg/kg), but not either compoundalone, was shown to attenuate MK-801-induced disruptions insensorimotor gating (Hashimoto et al., 2009). Interestingly, thetypical antipsychotic chlorpromazine has been shown to be a potentin vitro and in vivo inhibitor of DAO activity, in addition to itsestablished action as a D2 receptor antagonist (Yagi et al., 1956).Together, these findings support that inhibition of DAO function mayhave therapeutically potential. However, these investigations arestill at a preclinical stage and whether DAO inhibitors are capable ofalleviating disease symptoms in patients has yet to be examined.

Recently, transgenic mice carrying the human G72 genomicregion have been developed (Otte et al., 2009). In the brains ofthese mice, G72 transcripts were most abundant in the cerebellum,hippocampus, cortex, and olfactory bulb, while in the peripheryG72 was elevated in the heart and spleen (Otte et al., 2009). Severalphenotypes relevant to psychiatric disease were displayed in theG72-expressing mice, including sensorimotor gating disruption,enhanced sensitivity to PCP, and an increase in compulsivebehaviors (Otte et al., 2009). Further analysis of these mice may


uncover the molecular and cellular functions of G72 in vivo andclarify the mechanism by which G72 induces behavioral dis-turbances in the mouse.

Homozygous deletion of all GlyT-1 subtypes in mice results insevere motor and respiratory deficits leading to death on the firstpostnatal day (Gomeza et al., 2003). However, heterozygote GlyT-1knockout mice are fully viable (Tsai et al., 2004b), as are mice witha selective elimination of GlyT-1 in the forebrain (Yee et al., 2006).Reduced GlyT-1 expression led to a corresponding increase inglycine availability and augmented NMDAR-evoked excitatorypostsynaptic currents in hippocampal slices (Yee et al., 2006; Tsaiet al., 2004b). Lower levels of GlyT-1 also enhanced cognitiveperformance in several behavioral tasks. Heterozygote GlyT-1knockout mice demonstrated greater spatial memory retentionand a decreased sensitivity to amphetamine-induced disruption ofsensorimotor gating (Tsai et al., 2004b). Additionally, mice with aloss of forebrain GlyT-1 showed improvements in LI, spatial andobject recognition memory, and a resistance to PCP-inducedhyperactivity (Yee et al., 2006; Singer et al., 2007). The procognitiveeffects of diminished GlyT-1 activity have also been reported inpharmacological studies. Treatment with GlyT-1 antagonistspotentiates LI in rodents under parametric conditions that reduceLI in controls and reverses LI abnormalities, spatial memorydeficits, and hyperlocomotion induced by MK-801 or ampheta-mine (Lipina et al., 2005; Black et al., 2009; Manahan-Vaughanet al., 2008; Boulay et al., 2008; Harsing et al., 2003). Inneurodevelopmental models of schizophrenia, inhibition ofGlyT-1 normalizes impairments in sensorimotor gating, LI, andsocial recognition memory in adult rats (Le Pen et al., 2003; Blacket al., 2009; Boulay et al., 2008). However, usage of GlyT-1inhibitors in clinical settings may merit caution, as animal studieshave found that high doses of a GlyT-1 antagonist, such as ALX-5407 or LY2365109, induces compulsive over-activity (akathisia)(Harsing et al., 2003; Lipina et al., 2005), which may be related tospill-over onto inhibitory glycine A receptors (Perry et al., 2008).Rodents given a high dose (10 mg/kg) of ALX-5407 weredemonstrated to have high glycine levels in caudal brain regionsalong with respiratory and motor impairments (Perry et al., 2008),suggesting an increased modulation of strychnine-sensitiveglycine A receptors. Thus, modulation of D-serine and its regulatoryenzymes may be advantageous over altering glycine levels, as theNMDAR D-serine/glycine site is the only known binding site of d-serine and side effects related to the activation of strychinine-sensitive glycine receptors are averted.

11. Comparison of animal models examining genes related tothe NMDAR D-serine/glycine site and other genetic modelsbased on schizophrenia susceptibility genes

Since schizophrenia is a complex disorder involving both geneticand environmental factors, manipulation of one genetic orneurochemical pathway is unlikely to replicate all aspects of thedisease. Instead, targeting specific pathways may reveal theirimportance in specific clinical features associated with schizo-phrenia. Overall, marked reduction in the function of the NMDAR D-serine/glycine site and in the activity of D-serine modulatoryenzymes appear to consistently recapitulate some of the neurocog-nitive abnormalities and negative symptoms characteristic ofschizophrenia. Deficits in spatial learning and memory as well associal interactions are particularly apparent, although this mayreflect the choice in assessed behavior and the limitations in validbehavioral paradigms. Models affecting NMDAR D-serine/glycinesite activity also demonstrate an altered sensitivity to psychotomi-metic agents and are responsive to antipsychotic treatment.However, consistent with being a genetically heterogeneous disease,many other candidate susceptibility genes have been implicated in

the pathophysiology of schizophrenia (Harrison and Weinberger,2005; Ross et al., 2006), suggesting the involvement of a synergisticinteraction of several risk genes and neurotransmitter systems.

Discoveries in human genetic studies have fueled the devel-opment of several genetic mouse models based on schizophreniasusceptibility genes. Among the candidate risk genes, catechol-O-methyltransferase (COMT) is the only one known associate afunctional mutation with increased vulnerability for schizophre-nia. COMT is involved in dopamine metabolism and the val158metpolymorphism in COMT affects enzymatic activity (Lachman et al.,1996; Chen et al., 2004a). Carriers for the Met allele have the lowestCOMT activity, resulting in reduced dopamine degradation (Chenet al., 2004a). In addition, the Met allele has been associated with amore efficient activation of the prefrontal cortex and improvedcognitive performance in tasks dependent on prefrontal corticalfunction in healthy and schizophrenic individuals (Sheldrick et al.,2008; Heinz and Smolka, 2006). Mice that overexpress the humanCOMT-Val variant demonstrated deficits in attentional set-shiftingand impairments in object recognition and working memory, alongwith diminished stress responses and pain sensitivity (Papaleoet al., 2008). In contrast, COMT knockout mice have an increase indopamine in the frontal cortex and improved working memory,but elevated stress responses and pain sensitivity (Papaleo et al.,2008; Gogos et al., 1998). Consequently, these findings indicatethat the COMT val158met polymorphism may be involved inabnormal cognitive processing and stress reactivity in diverseclinical disorders and may influence natural variation in cognitionand stress in healthy populations.

The COMT gene is located on the 22q11 locus, which is a regionthat has been linked to greater schizophrenia risk (Ross et al., 2006;Harrison and Weinberger, 2005). Deletions in this chromosomalregion are associated with velocardiofacial syndrome, a diseaseaccompanied with an increased prevalence of psychotic symptoms(Ross et al., 2006; Harrison and Weinberger, 2005). The prolinedehydrogenase (PRODH) gene is also located in the 22q11 region.Studies have identified a significant association of PRODH withschizophrenia vulnerability (Li et al., 2004; Kempf et al., 2008) andthe presence of missense mutations in this gene in patients withschizophrenia (Jacquet et al., 2002). PRODH is involved in thecatabolism of proline, which in turn affects glutamate availabilityand acetylcholine function in the cortex (Delwing et al., 2007, 2003).Mice with a deficiency in PRODH exhibit aberrant glutamatergictransmission, deficits in sensorimotor gating and associativememory, and exaggerated responses to stress and amphetamine(Paterlini et al., 2005; Gogos et al., 1999). Additionally, an epistaticinteraction between the PRODH and COMT genes was demonstratedin these mice, as reductions in PRODH resulted in an upregulation ofCOMT mRNA in the frontal cortex and altered behavioral responsesto a COMT inhibitor (Paterlini et al., 2005).

Neuregulin-1 (NRG1) was originally identified as a candidategene following fine-mapping of a locus on chromosome 8p, aregion that has been frequently linked to schizophrenia (Stefans-son et al., 2002). Numerous studies have since confirmed theassociation of NRG1 with greater schizophrenia risk (Harrison andWeinberger, 2005) and a variant in the NRG1 promoter region hasbeen associated with decreased cortical activation, the develop-ment of psychotic symptoms, and reduced premorbid IQ (Hallet al., 2006). NRG1 has a range of roles in the development andfunction of the nervous system, affecting neuronal migration anddifferentiation, synapse formation, glial proliferation, synapticplasticity, and neurotransmitter receptor expression and function(Esper et al., 2006). Furthermore, the NRG1 receptor ErbB4 iscritically involve in glutamatergic synapse maturation andsignaling (Li et al., 2007a). Mice with heterozygous deletions inselective domains of NRG1 demonstrate locomotor hyperactivity,altered exploratory behaviors, disrupted sensorimotor gating and


LI, and are responsive to antipsychotics (Stefansson et al., 2002;Duffy et al., 2008a; Rimer et al., 2005). In rodents, locomotorhyperactivity and disrupted LI are considered to be modelsrelevant to the positive symptoms of schizophrenia, correspondingrespectively to the psychomotor agitation and loss of LI observed inpatients displaying acute psychotic symptoms (Bubenıkova-Valesova et al., 2008; Weiner, 2003). Reductions in the ErbB4receptor similarly induce hyperactivity in mice, as well asimpairments in prepulse inhibition and spatial learning andmemory (Stefansson et al., 2002; Golub et al., 2004). Together,these results suggest an important role for NRG1 and ErbB4 in thebiological mechanism underlying the positive and cognitivesymptoms of schizophrenia. Interestingly, a recent study indicatesthat NRG1-ErbB4 signaling may contribute to NMDAR hypofunc-tion in schizophrenia, as NRG1-induced activation of ErbB4 wasfound to be elevated in the prefrontal cortex of schizophreniapatients and increased NRG1 stimulation led to the suppression ofNMDAR activation in human cortical tissue (Hahn et al., 2006).

Dysbindin (dystrobrevin binding protein 1) is another suscept-ibility gene that was initially associated with schizophreniathrough linkage to chromosome 6p (Straub et al., 2002). Thepositive association of this locus with schizophrenia has beenreplicated in several subsequent independent studies, some ofwhich show that genetic variation in the dysbindin gene influencesgeneral cognitive ability and prefrontal brain function in healthypopulations, as well as negative symptoms and cognitive decline inschizophrenia patients (Burdick et al., 2006; Fallgatter et al., 2006;Fanous et al., 2005; Burdick et al., 2007). The functions of dysbindinare not well understood, although growing evidence supports arole in neurotransmitter release, affecting the kinetics, amount,and probability of presynaptic vesicular release and the morphol-ogy of vesicles (Chen et al., 2008). In primary neuronal culturesdysbindin has been shown affect extracellular glutamate releaseand promote neuronal viability (Numakawa et al., 2004).Additionally, dysbindin binds to b-dystrobrevin, a member ofthe dystrophin protein complex that affects synaptic structure andsignaling (Benson et al., 2001). Studies of the homozygous sandymice that have a loss in dysbindin protein show behavioraldisturbances that include impairments in object recognitionmemory, social interactions, long-term and spatial workingmemory (Feng et al., 2008; Takao et al., 2008; Bhardwaj et al.,2009). Pharmacological responses to amphetamine and painsensitivity are also abnormal in the sandy mice (Bhardwaj et al.,2009). Thus, results demonstrating social withdrawal and cogni-tive deficits in mice lacking dysbindin protein are consistent withassociation studies indicating that aberrant dysbindin activity maycontribute to the negative symptoms and cognitive dysfunction inschizophrenia. Furthermore, the behavioral abnormalities in thesandy mice are surprisingly similar to mice with aberrant NMDARD-serine/glycine site function and this may reflect convergingfunctions of these genes. Indeed, epistatic effects and commonprotein–protein interactions have been reported for schizophreniasusceptibility genes, suggesting some overlap of common biolo-gical processes (Nicodemus et al., 2007; Camargo et al., 2007;Paterlini et al., 2005).

Disrupted-in-schizophrenia 1 (DISC1) was first identified at thebreakpoint of a balanced chromosomal translocation t(1;11)(q42.1; q14.3) which cosegregated in a large Scottish family withschizophrenia and other major psychiatric disorders (Millar et al.,2000). Multiple independent studies have since shown associationbetween polymorphisms in DISC1 and schizophrenia susceptibilityin diverse population samples (Chubb et al., 2008), along withseveral reports indicating that allelic variation in DISC1 isassociated with abnormal cortical and hippocampal gray mattervolume and function, positive symptoms, cognitive impairments,and social anhedonia (Di Giorgio et al., 2008; Szeszko et al., 2008; Li

et al., 2007b; Tomppo et al., 2009). DISC1 plays an important role inCNS development and neuronal functions that includes involve-ment in neuronal migration and maturation, neurite outgrowth,cytoskeletal function, synaptic transmission, and plasticity (Chubbet al., 2008; Ross et al., 2006). Mice with altered DISC1 functiondisplay phenotypes relevant to schizophrenia and mood disorders(Clapcote et al., 2007; Hikida et al., 2007; Pletnikov et al., 2008;Shen et al., 2008; Li et al., 2007b). Examination of mice with ENU-induced missense mutations in exon 2 of the DISC1 gene revealedthat a mutation at amino acid position 100 (L100P) resulted inschizophrenia-like behavioral abnormalities that were normalizedby typical and atypical antipsychotics, while a mutation at aminoacid position 31 (Q31L) produced depressive-like phenotypes thatwere reversible by antidepressant treatment (Clapcote et al.,2007). Both the L100P and Q31L mutant mice demonstrateddisrupted LI, working memory impairments, and a decrease inbrain volume; however, the L100P mutant mice also demonstratedlocomotor hyperactivity and severe sensorimotor gating deficits,while the Q31L showed additional phenotypes related to depres-sion, anhedonia, and social withdrawal (Clapcote et al., 2007).Expression of truncated DISC1 protein on a background ofendogenous mouse DISC1 protein led to numerous structural,cellular, and behavioral perturbations (Hikida et al., 2007;Pletnikov et al., 2008; Shen et al., 2008). These included anenlargement of the lateral ventricles, reductions in corticalthickness, neurite outgrowth, parvalbumin GABAergic neuronsin the hippocampus and cortex, as well as hyperlocomotion,sensorimotor gating deficits, spatial memory impairments, andaugmented depressive-like behaviors (Hikida et al., 2007; Pletni-kov et al., 2008; Shen et al., 2008). Furthermore, expression of a C-terminal portion of DISC1 in mice at postnatal day 7, but not inadulthood, produced social impairments, depressive-like beha-viors, spatial working memory deficits, and decreased dendriticbranching in the hippocampus (Li et al., 2007b). Overall, studies ingenetic mouse models investigating the effects of altered DISC1function indicate a broad spectrum of abnormalities pertinent toschizophrenia and affective disorders, suggesting that DISC1 is amajor component in a multidimensional risk pathway forpsychiatric illness.

Several other candidate genes involved in schizophrenia riskhave been identified and described in recent reviews (Harrison andWeinberger, 2005; Ross et al., 2006). For some genes, preliminarycharacterization of genetic mouse models have indicated pheno-types that are relevant to schizophrenia and other psychiatricconditions. In contrast, a lack of overt behavioral phenotypes hasbeen observed in some candidate gene mouse models. Forexample, regulator of G-protein signaling 4 (RGS4) has beenassociated with schizophrenia in multiple studies (Harrison andWeinberger, 2005) and decreased RGS4 transcripts have beenreported in a number of cortical regions in schizophrenia patients(Mirnics et al., 2001). However, mice with a RGS4 deletion showintact locomotor activity, prepulse inhibition, and workingmemory (Grillet et al., 2005).

Absence of behavioral abnormalities in the RGS4 null mice andother candidate gene mouse models may be related to develop-mental compensatory effects, the obscuring of phenotypes by amixed genetic background, procedural limitations, or a lack ofinvolvement of the gene in the assessed behaviors or schizo-phrenia. Hence, it is crucial that behavioral phenotyping strategiestake into account the influences of sex and background strain, inaddition to undertaking a comprehensive approach that assessesmultiple endophenotypes related to psychiatric illness andcontrols for sensory, motor, visual, olfactory, and other functionsthat may influence behavioral performance. Since conventionalknockout and certain transgenic technologies induce gene muta-tions from conception, the lifelong absence of a gene will often


induce compensatory processes and developmental defects thatcontribute to the adult phenotype in tandem with the targetedmutation. Consequently, mutations that have regional andtemporal specificity may be of interest, as this strategy maynotably limit widespread compensatory changes. Inducible andregion-specific mutations will also allow the study of alterationsthat more closely reflect the subtle perturbations found inschizophrenia and other psychiatric disorders. Furthermore,assessment of mice with single nucleotide substitutions similarto those reported in schizophrenia will be required to study theeffects of variations at risk alleles. Many of the polymorphismsrelated to schizophrenia affect regulatory elements of genes andexamination of animal models with these genetic abnormalitieswill help clarify the effects these mutations have on geneprocessing and regulation, as well as their role in the morecomplex molecular processes involved in schizophrenia. Anextension to this approach is to eliminate the mouse gene ofinterest and replace it with the human ortholog (knock-in)containing the risk variant, in an effort to better predict theeffects of the polymorphism in humans. Since schizophrenia likelyarises from the simultaneous disruption of several genes,compound mutant mice in which several candidate genes aretargeted may offer the greatest potential for future geneticallymodified mice. Partial gain- or loss-of-function mutations inseveral risk genes within the same animal would most closelyreplicate the etiological mechanism of schizophrenia and would behighly valuable for the development of novel therapeuticinterventions.

12. The future of NMDAR D-serine/glycine site modulators inthe treatment of schizophrenia

Overall findings from preclinical and clinical studies demon-strate apparent beneficial effects of NMDAR D-serine/glycine siteactivators, particularly on the negative and cognitive symptomsdomains. Though administration of direct agonists, such as D-serine and glycine, are of therapeutic value, such treatmentsrequire large doses and exhibit difficulties in penetrating theblood–brain-barrier. Consequently, agents targeting proteinsinvolved in D-serine metabolism may be a more effective strategy,providing selective modulation of the NMDAR D-serine/glycinesite. Inhibition of DAO activity in the brain is of particular interestas it would circumvent any nephrotoxicity associated with thecatabolism of high levels of systemic D-serine (Maekawa et al.,2005b). Development of DAO antagonists has recently begun(Adage et al., 2008; Hashimoto et al., 2009; Smith et al., 2008).Intravenous injections of the DAO inhibitor AS057278 were foundto readily cross the blood-brain-barrier and enhance D-serinecontents in the rat brain (Adage et al., 2008). Chronic adminis-tration of AS057278 in mice was shown to normalize PCP-induceddeficits in behavioral tasks relevant to the cognitive and positivesymptoms of schizophrenia (Adage et al., 2008). However,elevations in D-serine following DAO inhibition are considerablylower than with exogenous D-serine treatments (Smith et al., 2008;Ferraris et al., 2008) and since DAO is predominantly located incaudal brain regions (Moreno et al., 1999) it remains to bedetermined if DAO inhibition can effectively elevate D-serine infrontal brain areas. This limitation may be overcome by combiningDAO inhibition with a low dose of exogenous D-serine. Indeed,coadministration of D-serine and a DAO antagonist were shown toproduce greater ameliorative effects in animals treated with anNMDAR antagonist than either compound applied alone (Hashi-moto et al., 2009). Thus, DAO inhibition in combination with D-serine administration may be a valuable therapeutic approach forthe treatment of schizophrenia. Activators of SRR function mayalso provide beneficial effects, however such compounds have yet

to be developed. Targeting SRR function may be a highly effectivemeans of modulating synaptic D-serine availability due to theprevalence of SRR in the forebrain (Xia et al., 2004; Schell et al.,1995), its colocalization with NMDARs (Schell et al., 1997), and itsprominent capacity to regulate D-serine abundance (Woloskeret al., 1999; Foltyn et al., 2005; Strısovsky et al., 2005). In sum,agents targeting the NMDAR D-serine/glycine site are examples offundamentally novel therapies that may deliver symptomaticimprovement with substantially fewer side effects than currentantipsychotics.

Acknowledgements

V.L. is supported by a Natural Sciences and EngineeringResearch Council (NSERC, Canada) studentship. J.C.R. is a CanadaResearch Council (CRC) chair. The author’s studies are supportedby the CIHR (GMH-79044).

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