NAAG peptidase inhibition reduces locomotor activity and some

stereotypes in the PCP model of schizophrenia via group II mGluR

Rafal T. Olszewski,*,1 Noreen Bukhari,* Jia Zhou,� Alan P. Kozikowski,�,2 Jarda T. Wroblewski,�Susan Shamimi-Noori,* Barbara Wroblewska,* Tomasz Bzdega,* Stefano Vicini,§ Franca B.

Barton¶ and Joseph H. Neale*

*Department of Biology, Georgetown University, Washington, D.C., USA

�Department of Pharmacology, Georgetown University, Washington, D.C., USA

§Department of Physiology, Georgetown University, Washington, D.C., USA

�Department of Drug Discovery Program, Georgetown University, Washington, D.C., USA

¶Center for New Designs in Learning and Scholarship, Georgetown University, Washington, D.C., USA

Abstract

Phencyclidine (PCP) administration elicits positive and neg-

ative symptoms that resemble those of schizophrenia and is

widely accepted as a model for the study of this human dis-

order. Group II metabotropic glutamate receptor (mGluR)

agonists have been reported to reduce the behavioral and

neurochemical effects of PCP. The peptide neurotransmitter,

N-acetylaspartylglutamate (NAAG), is a selective group II

agonist. We synthesized and characterized a urea-based

NAAG analogue, ZJ43. This novel compound is a potent

inhibitor of enzymes, glutamate carboxypeptidase II

(Ki ¼ 0.8 nM) and III (Ki ¼ 23 nM) that deactivate NAAG fol-

lowing synaptic release. ZJ43 (100 lM) does not directly

interact with NMDA receptors or metabotropic glutamate

receptors. Administration of ZJ43 significantly reduced PCP-

induced motor activation, falling while walking, stereotypic

circling behavior, and head movements. To test the hypo-

thesis that this effect of ZJ43 was mediated by increasing the

activation of mGluR3 via increased levels of extracellular

NAAG, the group II mGluR selective antagonist LY341495

was co-administered with ZJ43 prior to PCP treatment. This

antagonist completely reversed the effects of ZJ43. Addition-

ally, LY341495 alone increased PCP-induced motor activity

and head movements suggesting that normal levels of NAAG

act to moderate the effect of PCP on motor activation via a

group II mGluR. These data support the view that NAAG

peptidase inhibitors may represent a new therapeutic

approach to some of the components of schizophrenia that

are modeled by PCP.

Keywords: mGluR3, N-acetylaspartylglutamate, NMDA

receptors, phencyclidine, schizophrenia.

J. Neurochem. (2004) 89, 876–885.

Schizophrenia is a chronic brain disorder that affects multiple

cognitive and behavioral elements (reviewed by Lewis and

Lieberman 2000). Genetic data clearly support the hypothesis

that polymorphisms in the human genome influence suscep-

tibility to schizophrenia (Pulver et al. 2000). Given the long-

standing roles of the dopamine D2 antagonists, haloperidol

and chlorpromazine, in the treatment of this disorder,

dysfunctional dopaminergic neurotransmission has long been

seen as central to the pathophysiology of schizophrenia

(Carlsson 1988). However, a number of observations have

given impetus to the hypothesis that glutamatergic transmis-

sion may play a central role in the expression of schizophre-

nic symptoms if not the etiology of the disorder (reviewed by

Greene 2001; Tsai and Coyle 2002). Support for this

hypothesis was provided by observation of the effects

of the dissociative anesthetics phencyclidine (PCP) and

ketamine (Javitt and Zukin 1991; Krystal et al. 1994; Krystal

Received October 16, 2003; revised manuscript received December 18,

2003; accepted December 22, 2003.

Address correspondence and reprint requests to J. H. Neale, Depart-

ment of Biology, Georgetown University, Washington, D.C. 20057,

USA. E-mail: nealej@georgetown.edu1On leave from Institute of Genetics and Animal Breeding, Polish

Academy of Sciences, Jastrzebiec, Poland.2Current address: Department of Medicinal Chemistry and Pharmacog-

nosy, University of Illinois at Chicago, 833 S. Wood St., Chicago, IL

60612–7230, USA.

Abbreviations used: CHO, Chinese hamster ovary; NAAG, N-ace-

tylaspartylglutamate; mGluR3, group II metabotropic glutamate recep-

tor; PCP, phencyclidine.

Journal of Neurochemistry, 2004, 89, 876–885 doi:10.1111/j.1471-4159.2004.02358.x

876 � 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 876–885

et al. 1999). These drugs act as noncompetitive NMDA

receptor antagonists and cause the cognitive impairments,

positive symptoms, and negative symptoms of schizophrenia.

Similarly, mice in which NMDA receptor expression has

been suppressed by 95% display schizophrenia-like symp-

toms (Mohn et al. 1999).

Major impetus to the study of a new therapeutic approach

to this disorder was provided by the report that some

neurochemical and behavioral responses to PCP are reduced

by treatment with group II selective metabotropic glutamate

receptor (mGluR) agonists (Moghaddam and Adams 1998;

Cartmell et al. 1999; Cartmell et al. 2000). The role of

glutamate release in PCP and LSD-induced halucinogenesis

has been further strengthened by data demonstrating the

interactions of group II mGluR agonists and antagonists with

discriminative behavior that was conditioned using these

drugs (Winter et al. 2003). The intersection of catecholam-

ine- and glutamate-mediated neurotransmission is highligh-

ted by the finding that group II mGluR agonists and

antagonists influence dopamine and noradrenaline release

in the prefrontal cortex (Swanson et al. 2004). The presence,

in a broad spectrum of mammalian brain circuits, of a group

II selective peptide transmitter, N-acetylaspartylglutamate

(NAAG; reviewed by Neale et al. 2000) provides opportun-

ity for a novel mGluR-based therapeutic approach to

schizophrenia.

Following synaptic release, NAAG is inactivated by

membrane bound extracellular peptidase activity (Riveros

and Orrego 1984; Robinson et al. 1987) that appears to result

from the expression of at least two different genes (Bacich

et al. 2002; Bzdega et al. 2004). With the aim of enhancing

the actions of synaptically released NAAG, we synthesized

and characterized a series of compounds based on the

structure of NAAG. Many of these compounds are potent

(low nanomolar) inhibitors of the extracellular peptidase

activity that inactivates the peptide following synaptic release

(Nan et al. 2000; Kozikowski et al. 2001; Kozikowski et al.

2004). In this report, we describe the characterization of a

new peptidase inhibitor and the application of this compound

to test the hypothesis that increasing the duration of synaptic

availability of extracellular NAAG will increase activation of

group II mGluRs and decrease some behavioral effects of

PCP administration.

Materials and methods

Synthesis of 221

A mixture of L-leucine (1.32 g, 10.0 mmol), p-TsOH (3.0 g,

15.8 mmol) and benzyl alcohol (6.0 g, 59.4 mmol) in

benzene (50 mL) was refluxed with a Dean-Stark to collect

water overnight. The solution was cooled and a white solid

precipitated, which was filtered, washed with ethyl ether and

dried to give a salt 221 (3.0 g, 76.3%) (Fig. 1).

Synthesis of urea 574

To a mixture of tosylate of L-glutamate dibenzylester

(600 mg, 1.20 mmol) and triphosgene (120 mg, 0.40 mmol)

in CH2Cl2 (15 mL), cooled to )78�C, was slowly addedEt3N (0.56 mL, 4.0 mmol) in CH2Cl2 (4.0 mL). The reaction

mixture was stirred at )78�C for 1 h, then allowed to warmto room temperature for 30 min. A mixture of compound 221

(430 mg, 1.09 mmol) and Et3N (0.14 mL, 1.0 mmol) in

CH2Cl2 (5.0 mL) was added to the above isocyanate

solution. The mixture was stirred at room temperature

overnight. The mixture was washed with 1 M HCl and brine,

dried over anhydrous Na2SO4 and evaporated. The crude

product was purified by flash chromatography (SiO2, 1 : 20

CHCl3/MeOH) to give product 574 (400 mg, 64.0%).1H

NMR (CDCl3) d 7.30 (s, 15H), 5.72 (d, 1H, J ¼ 8.1 Hz),

5.58 (d, 1H, J ¼ 8.7 Hz), 5.17–5.05 (m, 6H), 4.64–4.51 (m,

2H), 2.50–2.32 (m, 2H), 2.18 (m, 1H), 1.97 (m, 1H), 1.69–

1.41 (m, 3H), 0.88 (d, 3H, J ¼ 5.7 Hz), 0.86 (d, 3H, J ¼ 5.7

Hz). 13C NMR (CDCl3) d 174.16, 172.93, 172.67, 156.87,128.42, 128.37, 128.22, 128.11, 128.04, 127.99, 127.95,

67.07, 66.78, 66.26, 52.29, 51.52, 41.89, 30.09, 27.93,

24.55, 22.63, 21.85 (Fig. 1).

Synthesis of compound ZJ43

To a solution of compound 574 (130 mg, 0.23 mmol) in t-

BuOH (15 mL) was added 20% Pd(OH)2/C (50 mg). The

reaction mixture was hydrogenated with H2 at 1 atm for

30 min. Filtration, concentration and lyophilization afforded

white solid ZJ43 (65 mg, 94.3%). [a]D )0.33� (c 0.60,MeOH); 1H NMR (D2O) d 4.27–4.18 (m, 2H), 2.50 (t, 2H,J ¼ 7.2 Hz), 2.15 (m, 1H), 1.96 (m, 1H), 1.74–1.58 (m, 3H),

0.92 (d, 3H, J ¼ 6.3 Hz), 0.88 (d, 3H, J ¼ 6.3 Hz); 13C

NMR (D2O) d 178.28, 177.60, 176.64, 159.68, 52.90, 52.23,40.11, 30.35, 26.51, 24.66, 22.48, 20.87. Analysis

(C12H22N2O9Æ1.2H2O) calculated: C, 44.22; H, 6.93; N,8.60. Found: C, 44.21; H, 7.02; N, 8.25 (Fig. 1).

Assay of ZJ43 inhibition of NAAG peptidase activity

The inhibitory activity of ZJ43 on cloned human GCPII was

determined using a fluorescent assay of NAAG peptidase

activity. The enzyme was obtained from a Chinese hamster

Fig. 1 Preparation of ZJ43.

NAAG peptidase inhibition and schizophrenia 877

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 876–885

ovary (CHO) cell line transfected with the cloned human

cDNA for GCPII using the calcium phosphate method (Chen

and Okayama 1987). Cells grown to confluency, were

harvested in 50 mM Tris-HCl, pH 7.4 and subjected to

freezing and thawing. Cell membranes were washed three

times by centrifugation, and the final aliquots of membranes

were adjusted to a protein concentration of 20 mg/mL and

stored at )80�C. The GPCPII activity was assayed in a two-step process. In the first step, aliquots of membranes (4 lg)were incubated for 2 h at 37�C in 50 mM Tris-HCl, pH 7.4 inthe presence of 4 lM NAAG and the given inhibitor, in a

volume of 100 lL, to allow for the accumulation of glutamateproduced in the GPCII reaction. In the second step, the

amount of accumulated glutamate was measured using the

Amplex Red Glutamic Acid Assay Kit (Molecular Probes),

and the fluorescence was detected with a Dynex fluorescent

plate reader using excitation at 530 nm and emission at

590 nm. Dose–response curves were used to calculate IC50values by nonlinear regression using the Sigma-Plot software.

In each assay, a concentration curve of NAAG was used to

determine the Km. The final Ki values for each inhibitor were

calculated from the respective IC50 and Km values using the

Cheng and Prusoff equation (Cheng and Prusoff 1973).

For GCPIII assays, transfected cells were harvested in

50 mM Tris-HCl buffer, pH 7.5, frozen at )80�C, thawed andsonicated. Membranes were sedimented in a refrigerated

microcentrifuge for 15 min, washed once, and resuspended in

the same buffer. Protein concentrations were determined by

the bicinchoninic acid method (Pierce). NAAG peptidase

activity was determined using 4 lM NAAG as a substrate. Forradiochemical assays, trace amounts of 3H-NAAG (Perkin

Elmer/NEN) were added to the reaction, and substrate and

product were separated by ion exchange chromatography as

previously described (Fuhrman et al. 1994).

Test of interactions of ZJ43 with mGluRs and other

receptors

The potential of ZJ-43 to interact as agonist or antagonist

with mGluRs was determined using membranes from cell

lines expressing cloned metabotropic glutamate receptors of

group I (mGluR1 and mGluR5), group II (mGluR2 and

mGluR3) and group III (mGluR4 and mGluR6). Changes in

the signal transduction responses (increase in PI hydrolysis

or decrease in cAMP) were measured after the exposure to

100 lM concentrations of the ZJ compounds as has been

described previously (Wroblewska et al. 1997; Nan et al.

2000). Potential interactions of 100 lM ZJ43 with the

MK801 and PCP/TCP binding sites of the NMDA receptor

were assayed via the NIMH Psychoactive Drug Screening

Program using [3H]-MK801 and [3H]-TCP. The interaction

of 100 lM ZJ43 with the assays for the following receptorsand transporters were assayed via the NIMH Psychoactive

Drug Screening Program: (serotonin) 5-HT1A, r5-HT1B,

5-HT1D. 5-HT1E, 5-HT2B, r5-HT2C, 5-HT3, 5-HT5a,

5HT6 (dopamine) D1, rD2, rD3, rD4, D5 (opioid), mu,

delta, kappa (acetylcholine) M1-5 (adrenergic) alpha1a,

alpha1b, alpha2A, rBeta1, rBeta2 (GABA) rGABAa, rBZP

(histamine) hH1, rH2, hH4 (peptide) V1-3 (prostanoid) EP3

(canabiniod) CB1 (transporters) SERT, NET and bDAT.

Assay of zj43 as agonist or antagonist at NMDA receptors

in vitro

NMDA receptor-mediated currents were examined in cul-

tured cerebellar granule cells as described by Losi et al.

2003. Primary cultures of rat cerebellar granule neurons

(CGC) were prepared from postnatal day 7 (P7) Sprague–

Dawley rat cerebella. Cerebella were dissected and dissoci-

ated as described by Gallo et al. (1987). Cells were dispersed

with trypsin (0.25 mg/mL, Sigma, St. Louis, MO, USA) and

plated at a density of 1.1 · 106 cells/mL on glass coverslips(Fisher Scientific) coated with poly-L-lysine (10 mg/mL;

Sigma) in 35 mm Nunc dishes. Cells were cultured in basal

Eagle’s medium supplemented with 10% bovine calf serum,

2 mM glutamine, and 100 mg/mL gentamycin (all from

Invitrogen Corporation Carlsbad, CA, USA), and maintained

at 37�C in 6% CO2. The final concentration of KCl in the

culture medium was adjusted to 25 mM potassium (high K+).

To study NAAG effect on NMDA receptor-mediated minia-

ture excitatory postsynaptic currents (NMDA-mEPSCs), the

medium was replaced at 4 days in vitro with 5 mM potassium

(low K+) medium (MEM supplemented with 5 mg/mL

glucose, 0.1 mg/mL transferrin, 0.025 mg/mL insulin,

2 mM glutamine and 20 mg/mL gentamycin from Invitrogen)

as previously described, in order to achieve functional

synapse formation (Prybylowski et al. 2002). Recordings

were made from CGC on day 7 in vitro. Details of

electrophysiology are described in Losi et al. (2002) and

Prybylowski et al. (2002). Briefly, whole-cell recordings of

CGCs were performed with a patch-clamp amplifier (Axo-

patch 200, Axon Instrument, Foster City, CA, USA). These

neurons were voltage clamped at )60 mV and access

resistance was monitored throughout the recordings. The

recording chamber was continuously perfused at 5 mL/min

with an extracellular medium composed (in mM) of: NaCl

(145), KCl (5), MgCl2 (1), CaCl2 (1), HEPES (5), glucose

(5), sucrose (25), phenol red (0.25 mg/L) (all from Sigma) at

pH 7.4 with NaOH. The solution in the recoding electrode

contained (in mM): potassium gluconate (145), HEPES (10),

ATP Mg (5), GTP Na (0.2), and BAPTA (10), adjusted to

pH 7.2 with KOH. NMDA-mEPSCs were recorded at

)60 mV in a similar extracellular solution as above but

without Mg2+ and in the presence of 1 mM TTX, D-serine

(5 lM) and 50 mM BMI (Sigma). NMDA 200 lM and ZJ43(100 lM) solutions locally perfused by means of a Y tube.

Animals

The experimental protocols used in this research were

approved by the Georgetown University Animal Care and

878 R. T. Olszewski et al.

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 876–885

Use Committee consistent with guidelines of the US National

Institutes of Health.

Male Sprague-Dawley rats (Taconic, Germantown, MD,

USA) weighing 280–330 g were housed in groups of three

and maintained on a 12 : 12 h light–dark cycle. Food and

water were available ad libitum. Animals were habituated to

the test room 24 h prior to observation. Behavioral testing

was performed between 09.00 h and 18.00 h. Each animal

was used once for this study to avoid habituation to the

testing chamber.

Drug administration

On the day of testing, the rats were first habituated to a

plastic activity chamber and a data acquisition system

measured their baseline activity for 20 min. Rats were then

removed, injected with ZJ43 (150 mg/kg, i.p.), or saline

(0.9% NaCl) with or without the antagonist LY341495

(1 mg/kg, i.p.) and returned to the activity chamber. After

measuring the rat’s activities for 20 min, phencyclidine

(10 mg/kg, i.p.), or saline was administered and the animals

were returned to the activity chamber. Activities were then

recorded continuously for an additional 160 min (Moghad-

dam and Adams 1998).

Behavioral assessment

A data acquisition system (Medical Associates, St. Albans,

VT, USA) consists of evenly spaced photocells in a plastic

chamber with nonconsecutive photodetectors evenly spaced

over 43 · 43 cm. Locomotion was determined as beam

breaks outside a designated region around the animal indica-

ting the animal’s body had relocated (Cartmell et al. 1999).

An observer, blind to the treatment, also recorded certain

stereotypical behaviors over the 200 min observation period

using a time-sampling procedure. Forty-one-minute obser-

vations were performed every 5 min during the total

observation period. The observer counted the frequency of

each stereotypical behavior during each one-minute obser-

vation. Stereotypical behaviors were categorized as follows:

‘still’ consisted of completely still or sleeping; ‘sniffing’

consisted of head up sniffing, head down sniffing, top or

bottom of cage sniffing for more than 3 s; ‘mouth move-

ments’ consisted of licking or gnawing any part of cage, jaw

opening and closing, tongue flicks or gaping not directed to

any stimulus; ‘head movements’ consisted of bobbing head

up and down or sideways, ‘falling’ consisted of falling,

‘tremors’ consisted of body shaking and ‘circles’ consisted of

walking in circles (Kelley and Delfs 1994).

Statistical methods

Combinations of one, two or all three treatments were

administered to independent samples of 5–12 rats. For

analysis of motor activity, activity data were collected and

summed over successive 5-minute intervals. Motor activity

data from 80 to 180 min were used for the statistical analysis.

Each treatment combination group was evaluated statistically

against the others without regard to the factored component

treatments. The outcomes of motor measurements were

subjected to generalized estimating equations analysis using

PROC GENMOD in SAS Version 9.0. The effects included

in the model were time (modeled with a linear and a

quadratic component) and treatment group. Interaction terms

were included in preliminary models, and removed if not

significant (p > 0.05). Differences could be detected between

groups at any of the times. GEE models adjust for

autocorrelation, namely, the tendency for the sequential

readings of each rat to be correlated to each other. Ninety-

nine percent confidence intervals of the estimated regression

models were generated to compare the entire curve as well as

separate portions of curves to isolate intervals with the largest

differences among groups.

Manually recorded stereotype data were analyzed by one-

way ANOVA using SPSS software 11.0 with significant

differences noted with p < 0.05.

Results

ZJ43 (Fig. 2) potently inhibited the ability of the cloned

human enzyme, glutamate carboxypeptidase II, to hydrolyze

NAAG with an IC50 of 2.4 nM and a Ki of 0.8 nM. In this

assay system, 2-PMPA had an IC50 of 4.1 nM and a Ki of

1.4 nM. This level of inhibition was similar to that obtained

with other NAAG analogues containing the urea core and

that previously reported for 2-PMPA (Slusher et al. 1999).

When tested against cloned mouse GCP III (Bzdega et al.

2004), ZJ43 had a Ki of 23 nM.

In assays of second messenger production via activation of

cloned mGluRs1, 2, 3, 4, 5 and 7, ZJ43 (100 lM) failed to actas an agonist or antagonist. In the NIMH Psychoactive Drug

Screen, 100 lM ZJ43 failed to significantly interact with a

series of receptors including the MK801 and PCP binding

sites on the NMDA receptor or with the series of transmitter

receptors and transporters identified in Methods. When ZJ43

(100 lM) was applied to cerebellar granule cells in cultureunder whole cell voltage clamp conditions, this compound

failed to elicit any significant membrane current (three cells,

each tested 3–6 times) under conditions where NMDA

elicited very substantial currents. Additionally, no significant

effects of coapplication of 100 lM ZJ43 (three CGCs) were

HOOC COOH

COOH

HNH

NH H

O

Fig. 2 Structure of the NAAG peptidase inhibitor, ZJ43.

NAAG peptidase inhibition and schizophrenia 879

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 876–885

detected on currents elicited by 200 lM NMDA. Analysis ofthe peak currents and kinetics of the spontaneous miniature

spontaneous excitatory postsynaptic currents in these cultures

(three cells) revealed no significant change induced by local

application of 100 lM ZJ43. These currents are primarily

mediated by NMDA receptors. These data support the

conclusion that the peptidase inhibitor is not a physiologi-

cally relevant NMDA agonist or antagonist.

PCP (10 mg/kg) induced a dramatic increase in motor

activity in rats immediately following intraperitoneal

injection as determined by distance moved (Fig. 3a,b).

ZJ43 significantly decreased this motor effect of PCP. The

group II antagonist LY341495 (LY) was used to determine if

the effect of ZJ43 was mediated via NAAG activation of

group II mGluRs. When injected prior to PCP, the antagonist

enhanced the PCP-induced motor activity, presumably by

blocking the normal NAAG and glutamate activation of

group II mGluRs. The administration of ZJ43 significantly

reduced the activity induced by LY + PCP while the

reduction in PCP-induced movement obtained by ZJ treat-

ment was eliminated by the group II antagonist.

Two normal rat behaviors were affected by PCP treatment.

Grooming behavior was modestly reduced by both PCP and

LY. ZJ43 significantly increased the behavior and this action

was blocked by LY (data not shown). Rats injected with

DISTANCE - ALL GROUPS

Time (min)

0 50 100 150 200

Dis

tanc

e (c

m)

0

500

1000

1500

2000

2500

s-pcpzj-pcply-pcply-zj-pcps-s

DISTANCE - TOTAL

Dis

tanc

e (c

m)

0

10000

20000

30000

40000

50000

s-pcps-s

ly-zj-

pcp

zj-s

ly-s

zj-pc

p

ly-pc

p

(a)

(b)

Fig. 3 Locomotor activity data following PCP injection. Rats were

placed in observation chamber at zero time, injected with saline, ZJ43

with and without LY341495 at 20 min and with PCP at 40 min. Rats

were habituated to the data acquisition system from 0 to 40 min with

no significant difference in locomotor activity between the groups

during this time. (a) Average distance traveled by 6–12 rats in each

treatment group was summed over 5-min intervals for 200 min. Data

were analyzed using GENMOD procedure. There was a significant

difference between s-pcp and zj-pcp (z ¼ 0.0069) and no significant

difference between ly-pcp and ly-zj-pcp (z ¼ 0.0193) between 80 and

180 min. (b) Total distance traveled between 80 and 180 min for five

groups of rats (saline + saline; saline + PCP; ZJ43 + PCP;

LY341495 + PCP; ZJ43 + LY341495 + PCP). Data were analyzed

using one-way ANOVA in SPSS software. There was a significant dif-

ference between s-pcp and zj-pcp (p ¼ 0.021) and ly-pcp and ly-zj-pcp

(p ¼ 0.040).

COMPLETELY STILL

Drugs

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0

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20

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s-s s-pcp zj-pcp ly-s ly-pcpzj-s ly-zj-pcp

SNIFFING

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Fre

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0

10

20

30

40

50

60

s-s s-pcp zj-pcp ly-s ly-pcpzj-s ly-zj-pcp

(a)

(b)

Fig. 4 Frequencies of staying in place without noticeable body

movements (still) (a) and sniffing (b) are presented. In Figs 3–6 data

were obtained in 32 1-min observations taken every 5 min beginning

immediately after PCP injection at minute 40. The treatment groups

were saline + saline (s-s) n ¼ 6; saline + phencyclidine (s-pcp) n ¼ 9;

ZJ43 + saline (zj-s) n ¼ 6; ZJ43 + phencyclidine (zj-pcp) n ¼ 12;

LY341495 + saline (ly-s) n ¼ 5; LY341495 + phencyclidine (ly-pcp)

n ¼ 12; LY341495 + ZJ43 + phencyclidine (ly-zj-pcp) n ¼ 12. Data

was analyzed using one-way ANOVA in SPSS software. (a) With

respect to being still, the S-PCP group was significantly different from

the ZJ-PCP group (p ¼ 0.015) and different from other groups at

p < 0.001. (b) With respect to sniffing, the S-PCP was not significantly

different from ZJ-PCP at p < 0.05 and was different from all other

groups at p < 0.02.

880 R. T. Olszewski et al.

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 876–885

saline with or without ZJ43 or LY spent a substantial amount

of time without moving in the activity test chamber (Fig. 4a).

PCP administration dramatically decreased this ‘still’ time.

ZJ43 significantly reduces this PCP-induced behavior and the

group II antagonist completely blocks the effects of LZJ43.

Sniffing (Fig. 4b) was increased by both PCP and

ZJ43 + PCP. LY completely blocked both of these increases.

The PCP induces a series of discrete stereotypic behav-

iors in which normal rats spontaneously engage only at

very low frequency. These included stereotypic mouth

movements (Fig. 5a), falling while walking (Fig. 5b),

tremors (Fig. 6a), walking in circles (Fig. 6b), and head

movements (Fig. 7a) including head bobbing (Fig. 7b) and

sideways head movements (Fig. 7c). In each of these

behaviors, injection of ZJ43 produced a statistically signi-

ficant decrease in the PCP-induced behavior (falling,

circling movements, total head movement, head bobbing)

or a substantial, but not statistically significant (p > 0.05),

trend toward a decrease (mouth movements, tremors, and

sideways head movement). In all of these stereotypic

behaviors, the ZJ43-PCP treatment group had a significantly

lower frequency of the behavior than did the group injected

with PCP + ZJ43 + LY341495.

Discussion

Group II mGluR agonists have been shown to be effective in

reducing some of the behavioral and neurochemical effects of

PCP administration in rats (Moghaddam and Adams 1998;

Cartmell et al. 1999). This action is blocked by the selective

group II antagonist LY341495 (Cartmell et al. 2000). We

previously demonstrated that NAAG is a selective agonist for

mGluR3 (Wroblewska et al. 1997) while its efficacy at

mGluR2 is at least 15-fold less (Cartmell et al. 1998;

Schweitzer et al. 2000). The data presented here support the

hypothesis that following injection of the NAAG peptidase

inhibitor, ZJ43, the levels of this peptide transmitter are

sufficient to activate group II receptors and decrease some of

the behavioral effects of PCP. Beyond this, the group II

antagonist significantly increased the overall movement and

stereotypic sideways head movement induced by PCP. This

result would support the hypothesis that in the absence of

MOUTH MOVEMENTS

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s-s s-pcp zj-pcp ly-s ly-pcpzj-s ly-zj-pcp

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s-s s-pcp zj-pcp ly-s ly-pcpzj-s ly-zj-pcp

(a)

(b)

Fig. 5 Frequencies of stereotypic mouth movements (a) and falling

(b) are presented. Sampling method and treatment groups are as in

Fig. 4.S-PCP is not statistically different from ZJ-PCP (p ¼ 0.25), nor

is it different from LY-PCP or ZJ-LY-PCP. However, ZJ-PCP is sta-

tistically different from LY-PCP (p ¼ 0.001) and ZJ-LY-PCP

(p ¼ 0.002).ZJ-PCP is significantly different from S-PCP (p ¼ 0.02),

from LY-PCP and ZJ-LY-PCP (p < 0.01).

TREMORS

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Fig. 6 Frequencies of stereotypic tremors (a) and circling movements

(b) are presented. Sampling method and treatment groups are as in

Fig. 4.S-PCP is not statistically different from ZJ-PCP (p ¼ 0.12), nor

is it different from LY-PCP or ZJ-LY-PCP. However, ZJ-PCP is sta-

tistically different from LY-PCP (p ¼ 0.006) and ZJ-LY-PCP

(p ¼ 0.02). ZJ-PCP is significantly different from S-PCP, LY-PCP and

ZJ-LY-PCP (p £ 0.001).

NAAG peptidase inhibition and schizophrenia 881

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 876–885

peptidase inhibition, natural activation of the group II

mGluRs by NAAG or glutamate reduces some of the

schizophrenia-like effects of PCP. Consistent with the

view that schizophrenia may involve hypoactivity of

the NAAG-mGluR system, Nudmamud et al. (2003) repor-

ted a significant and selective decrease in NAAG levels in the

superior temporal cortex of schizophrenics relative to brain

tissue from a matched group of normal controls and

individuals with affective disorders.

The total distance moved by rats in the test chamber

increased dramatically following PCP injection and this

increase was significantly reduced by ZJ43 (Fig. 3b). While

the total distance moved by the LY + PCP group is not

significantly greater than that of the saline + PCP group at

p < 0.05, there is a trend in this direction. Given this and the

significant decrease in distance moved relative to LY + PCP

when ZJ43 is combined with LY + PCP, it is difficult to

conclude with certainty that this ZJ43 effect is blocked by the

group II antagonist. The ability of ZJ43 to attenuate the

locomotor activity induced by PCP is not likely to be a

universal action on drug-induced increases in locomotor

activity inasmuch as group II agonists are reported to

attenuate PCP- but not d-amthetamine-induced locomotor

activity (Cartmell et al. 1999). In mice, 1 mg/kg of

LY341495 resulted in no change in basal locomotor activity,

consistent with the data presented here. However, higher

doses increased this activity (O’Neill et al. 2003).

The use of ‘still time’ as an index of PCP effects on overall

motor activity (Fig. 4a) presents a clearer picture of the

action of ZJ43 as do falling (Fig. 5b), circling (Fig. 6b), total

head movement (Fig. 7a), and head bobbing (Fig. 7b). These

PCP-induced behaviors were clearly inhibited by ZJ43 and

this effect was blocked by LY. In none of these behaviors was

the effect of ZJ43 + LY + PCP significantly different from

LY + PCP and both groups were dramatically different from

ZJ43 + PCP.

PCP induced a significant increase in sniffing behavior

and ZJ43 not only failed to reduce this behavior but showed

a tendency (p ¼ 0.17) to increase the behavior. Strikingly,

the group II antagonist completely blocked the PCP effect

and the PCP + ZJ43 effect. These data suggest that the

induction of sniffing by PCP is mediated via group II

receptors and for this reason, elevation of NAAG levels

may increase this response. Both NAAG and group II

mGluRs are found in the olfactory bulb. This result

emphasizes the diversity of behaviors induced by PCP

and the likely complexity of neuronal circuits and receptors

associated with each. These data would suggest that while

NAAG peptidase inhibition will reduce some of the PCP-

induced behaviors, it may fail to affect others and may

increase some, such as the sniffing behavior. PCP produces

other relevant behavior effects, including sensorimotor

gating and cognitive deficits that remain to be explored

with NAAG peptidase inhibition. Important to considering

the potential therapeutic value of NAAG peptidase inhib-

itors is the relevance of the various PCP-induced conditions

to the spectrum of elements that constitute the human

schizophrenic condition.

HEAD MOVEMENTS

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BOBBING HEAD

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SIDEWAYS HEAD MOVEMENT

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s-s s-pcp zj-pcp ly-s ly-pcpzj-s ly-zj-pcp

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Fig. 7 Frequencies of total stereotypic head movements (a) and head

bobbing (b), and sideways head movements (c) are presented.

Sampling method and treatment groups are as in Fig. 3. Total stere-

otypic movement in the ZJ-PCP group was significantly less than that

in the S-PCP group (p ¼ 0.005) while the LY-PCP and the ZJ-LY-PCP

groups presented significantly higher frequency of head stereotypic

head movements than the S-PCP group. Stereotypic bobbing move-

ment in the ZJ-PCP group was significantly less than that in the S-PCP

group (p ¼ 0.004) while the LY-PCP and the ZJ-LY-PCP groups

presented significantly higher frequency of head stereotypic head

movements than the S-PCP group. Stereotypic sideways head

movement in the ZJ-PCP group was not lower than the S-PCP group

at a statistically significant level (p ¼ 0.09). S-PCP and ZJ-PCP were

significantly different from LY-PCP and ZJ-LY-PCP (p < 0.001).

882 R. T. Olszewski et al.

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 876–885

In some behavioral assays (Fig. 7), LY 341495 increased

PCP-induced behaviors or there was a tendency in this

direction. This could be interpreted as the antagonist

blocking the normal effects of endogenous NAAG that

occur in the absence of peptidase inhibition. The question

may then arise, why does the coadministration of ZJ43 with

PCP and LY342495 not block the actions of PCP and the

antagonist. It seems likely that this is due to the greater

ability of the systemically administered antagonist to enter

the brain and antagonize the receptor relative to the efficacy

of systemically applied ZJ43 to vs. inhibit the enzyme and

increase NAAG. LY341495 has proven to be highly

bioavailable in other studies in vivo (O’Neill et al. 2003;

Winter et al. 2003; Swanson et al. 2004). Alternatively, this

action of the antagonist may be mediated by mGluR2 while

NAAG is 15-fold more potent at mGluR3.

The mechanism by which activation of a group II mGluR

by NAAG or an exogenous agonist might reduce PCP-

induced behaviors is not clear. It has been demonstrated that

NAAG acts on presynaptic group II mGluRs to reduce

synaptic release of GABA and glutamate (Zhao et al. 2001;

Xi et al. 2002; Garrido Sanabria et al. 2003). Indeed,

Moghaddam and Adams (1998) reported that treatment with

a group II mGluR agonist also reduced PCP-induced increase

in extracellular levels of glutamate in the prefrontal cortex. If

one action of NAAG peptidase inhibition is a decrease in

glutamate release, it could be argued that decreasing

glutamate would enhance the action of PCP since this

dissociative anesthetic is an NMDA receptor antagonist. One

solution to this paradox may be that NMDA antagonists

evoke a compensatory increase in glutamate release or that

NMDA activity in some circuits reduces activation inhibitory

circuits affecting glutamate and dopamine release. Group II

receptors have been identified at both pre- and postsynaptic

neuronal sites and mGluR3 is also highly expressed by

astrocytes (Ohishi et al. 1994; Tamaru et al. 2001). NAAG

activation of postsynaptic group II receptors on neurons and

glia reduces cAMP levels via Gi. While group II receptor

activation has been shown to affect a spectrum of neuronal

events (Pin and Acher 2002), including transcriptional

regulation of GABAA receptor subunit expression (Ghose

et al. 1997), the potential consequences of this activation are

likely to vary among cell types and circuits.

While NAAG is considerably more potent as an agonist at

mGluR3 than mGluR2 (Wroblewska et al. 1997; Cartmell

et al. 1998; Schweitzer et al. 2000), it is not possible to

conclusively determine from the antagonist actions of

LY341495 that the peptide is acting via mGluR3. One

group II agonist, LY314582 (a racemic mixture of

LY379268), is reported to reduce the effects of PCP in

wild-type but not mGluR2 knockout mice (Spooren et al.

2000). Unfortunately, this agonist also significantly reduces

basal locomotor activity and thus its apparent suppression

of PCP-induced locomotor behavior may be due to a

nonspecific suppression effect. b-NAAG, a nonhydrolyzableanalogue of NAAG in which the peptide bond is formed via

the b carboxyl of aspartate, is a selective mGluR3 antagonist(Lea et al. 2001) and NAAG peptidase inhibitor. It may be

possible to use this compound to test the hypothesis that

mGluR3 activation mediates relief from PCP-induced be-

haviors.

An alternative interpretation of these data is that ZJ43 is

acting directly as a group II agonist based on its structural

relationship to NAAG. In order to test this, we examined

the ability of 100 lM ZJ43 to act as an agonist or antagonistat NMDA or mGluRs and found that it did not have an

effect. Additionally, some studies have been interpreted to

suggest that NAAG acts as an NMDA antagonist and this

has led to speculation that increases in NAAG levels would

thus contribute to the expression of schizophrenia (Greene

2001; Tsai and Coyle 2002; Flores and Coyle 2003).

However, only one study has directly tested the efficacy of

NAAG as an NMDA receptor antagonist (Losi et al. 2003)

and 200 lM peptide was found to have no significant

antagonist actions when tested against 10 lM NMDA nor

did the peptide significantly affect NMDA receptor medi-

ated spontaneous excitatory synaptic currents. Beyond this,

the antagonist theory of NAAG in schizophrenia (Tsai and

Coyle 2002; Flores and Coyle 2003) posited that decreased

peptidase activity and increased levels of NAAG levels

contributed to the expression of schizophrenia, while the

data presented in this paper demonstrate the contrary result.

Similarly inconsistent with the data presented here and with

data on group II mGluR activation reducing some PCP-

induced behavior and neurochemical changes (Moghaddam

and Adams 1998; Cartmell et al. 1999; Cartmell et al.

2000) is the report of Flores and Coyle (2003) that chronic

treatment with neuroleptics, haloperidol and clozapine,

increase the total NAAG peptidase activity in the prefrontal

cortex.

NAAG peptidase inhibitors represent a potentially

important new therapeutic approach to increase synaptic

levels of a selective endogenous agonist at group II

mGluRs. These inhibitors have been shown to be effective

in models of excitotoxicity including anoxia, inflammatory

pain, allodynia, diabetic neuropathy and ALS (Slusher et al.

1999; Yamamoto et al. 2001 submitted; Zhang et al. 2002;

Ghadge et al. 2003). In these systems, it is speculated that

efficacy is obtained as NAAG activates presynaptic recep-

tors to inhibit glutamate release (Garrido Sanabria et al.

2003) and/or acts via glia (Bruno et al. 1998). The data

presented here militate in favor of a role for NAAG and

NAAG peptidase inhibitors in expression of some of the

behavioral effects of PCP. More detailed analyses of this

and other animal models of schizophrenia will be required

to determine if NAAG peptidase inhibition represents a

significant new therapeutic approach to the treatment of the

human disorder.

NAAG peptidase inhibition and schizophrenia 883

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 876–885

Acknowledgements

This research is supported by NIH grants NS 38080 (JN) and NS

42672 (JN, AK and JW) and by a grant from Howard Hughes

Medical Institute.

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