Expression of D-serine and glycine transporters in the prefrontal cortex and cerebellum in schizophrenia

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Schizophrenia Research 102 (2008) 283 – 294 www.elsevier.com/locate/schres

Expression of D-serine and glycine transporters in the prefrontal cortex and cerebellum in schizophrenia P.W.J. Burnet a,⁎, L. Hutchinson a , M. von Hesling a , E.-J. Gilbert a , N.J. Brandon b,1 , A.R. Rutter b,2 , P.H. Hutson b,3 , P.J. Harrison a a

Department of Psychiatry, University of Oxford, Neurosciences Building, Warneford Hospital, Oxford OX3 7JX, United Kingdom b Merck Sharp and Dome, The Neuroscience Research Centre, Harlow, Essex, CM20 2QR United Kingdom Received 4 December 2007; received in revised form 6 February 2008; accepted 19 February 2008 Available online 8 April 2008

Abstract The NMDA receptor co-agonists D-serine and glycine are thought to contribute to glutamatergic dysfunction in schizophrenia. They are removed from the synapse by specific neuronal and glial transporters, the status of which is clearly relevant to theories of D-serine and glycine function in the disorder. D-serine is primarily transported by Asc-1, and glycine by GlyT1 but maybe also by SNAT2. As a first step to addressing this issue, we studied Asc-1, GlyT1 and SNAT2 expression in dorsolateral prefrontal cortex and cerebellum of 18 subjects with schizophrenia and 20 controls, using immunoblotting and in situ hybridization. Asc-1 protein and SNAT2 mRNA were decreased in schizophrenia in both regions. GlyT1 mRNA and protein, and Asc-1 mRNA, were not altered. Antipsychotic administration for 14 days did not alter expression of the genes in rat brain. Unchanged GlyT1 suggests that glycine transport is not markedly affected in schizophrenia, and therefore that increased synaptic removal is not the basis for the putative deficit in glycine modulation of NMDA receptors in the disorder. Lowered Asc-1 in schizophrenia implies that D-serine reuptake is reduced, perhaps as a response to decreased synaptic D-serine availability. However, this interpretation remains speculative. Further investigations will be valuable in the evaluation of these transporters as potential therapeutic targets in psychosis. © 2008 Elsevier B.V. All rights reserved. Keywords: D-serine; d-amino acid oxidase; Glycine; mRNA; NMDA receptor; Antipsychotic; Transporter

1. Introduction

⁎ Corresponding author. Tel.: +44 1865 223621; fax: +44 1865 251076. E-mail address: [email protected] (P.W.J. Burnet). 1 Current address: Wyeth Discovery Neuroscience, 865 Ridge Rd, Monmouth Junction, NJ 08550 USA. 2 Current address: Biopolis at One North, 11 Biopolis Way, The Helios #03-01/02, Singapore 138667. 3 Current address: Merck Sharp and Dome inc., 770 Sunnytown Pike, West Point, PA, 19446, USA. 0920-9964/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2008.02.009

Functioning of the N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) requires binding of an allosteric modulator to the strychnine-insensitive glycine binding site (Danysz and Parsons, 1998; Schell, 2004). In addition to glycine itself (Thomson et al., 1989; Danysz and Parsons, 1998), D-serine is a potent endogenous coagonist at this site (Matsui et al., 1995; Mothet et al., 2000; Miller, 2004; Panatier et al., 2006).

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The actions of these NMDAR modulators are terminated by reuptake from the synapse and, in the case of D-serine, via degradation by d-amino acid oxidase (DAO, DAAO; Nagata et al., 1989; Mothet et al., 2000) and possibly other enzymes (Martineau et al., 2006). Several transporters mediate the synaptic reuptake. For D-serine, the key transporter is Asc-1 (sodiumindependent alanine–serine–cysteine transporter 1, encoded by SLC7A10 [Fukasawa et al., 2000; Nakauchi et al., 2000; Helboe et al., 2003; Matsuo et al., 2004; Rutter et al., 2007)]), although at least one other likely exists (Javitt et al., 2002; Ribeiro et al., 2002; Martineau et al., 2006). In brain, glycine is removed from the synapse primarily via glycine transporter 1 (GlyT1, encoded by SLC6A9; Smith et al., 1992; Kim et al., 1994; Gadea and Lopéz-Colomé, 2001; Tsai et al., 2004); in addition, glycine can be transported by Asc-1 (Nakauchi et al., 2000; Helboe et al., 2003; Matsuo et al., 2004), and potentially by small neutral amino acid transporter 2 (SNAT2, also known as SAT2, ATA2, SA1, or SLC38A2), (Sugawara et al., 2000; Yao et al., 2000; Mackenzie and Erickson, 2004). The significance of Asc-1 and GlyT1 for NMDAR function is shown by the altered glutamate signalling and other abnormalities that occur following genetic (Tsai et al., 2004; Gabernet et al., 2005; Xie et al., 2005; Yee et al., 2006; Rutter et al., 2007) or pharmacological (Martina et al., 2004, 2005) manipulation. Based on the psychotogenic effects of NMDAR antagonists such as phencyclidine (Javitt and Zukin, 1991), NMDAR hypofunction has become a prominent pathophysiological hypothesis of schizophrenia (Olney and Farber, 1995; Coyle, 1996: Goff and Coyle, 2001; Krystal et al., 2003) and has led to a range of therapeutic strategies to treat cognitive and negative symptoms (Coyle and Tsai, 2004; Millan, 2005; Javitt, 2008). The evidence for NMDAR hypofunction in the disorder is diverse, albeit indirect. For example, serum and cerebrospinal fluid D-serine concentrations are decreased (Hashimoto et al., 2003, 2005; Bendikov et al., 2007), perhaps because DAO activity is increased (Kapoor et al., 2006; Burnet et al., unpublished data), whereas levels of a negative NMDAR modulator, kynurenic acid, are increased (Schwarcz et al., 2001). A possible aetiological role for NMDAR hypofunction in schizophrenia is now implicated by genetic associations of the disorder with DAO and several other genes that influence NMDAR signalling (Chumakov et al., 2002; Harrison and Owen, 2003; Moghaddam, 2003; Harrison and Weinberger, 2005; Detera-Wadleigh and McMahon, 2006). Despite the various lines of evidence, there are many unanswered questions. First, the relative con-

tributions of glycine and D-serine to NMDAR modulation are unclear; they may be differentially important in forebrain and hindbrain (Schell, 2004; Martineau et al., 2006). Second, it is not known whether glycine, Dserine, or both, are affected in schizophrenia. Third, the regulation of synaptic D-serine and glycine is not well understood, but is controlled at least in part by the transporters mentioned (Berger et al., 1998; Bergeron et al., 1998; Betz et al., 2006; Rutter et al., 2007); in the case of D-serine, one must also consider the relationship between its synaptic reuptake and its subsequent intracellular degradation by DAO. At present, there is no information about synaptic Dserine and glycine transport in schizophrenia, but it is an important issue relevant to understanding the involvement of these co-agonists in the disorder. Direct biochemical measurements of synaptic reuptake are problematic in post mortem human brain. Instead, expression of the transporters provides a surrogate means to assess the issue. We have studied GlyT1, Asc1 and SNAT2 expression in dorsolateral prefrontal cortex (DPFC) and cerebellum in control subjects and those with schizophrenia. The cerebellum was chosen, along with the DPFC, because of emerging evidence that it is involved in the NMDAR hypofunction of schizophrenia (e.g. Avila et al., 2002; Kapoor et al., 2006; Verrall et al., 2007). We also examined the effect of haloperidol and clozapine on these gene products in rat brain, to address the possibility of medication confounds. Some of the data have been presented in an abstract (Burnet et al., 2006). 2. Experimental procedures 2.1. Subjects and tissues studied Frozen tissue was taken at autopsy from DPFC (Brodmann area 9/46) and cerebellar cortex of 18 subjects meeting DSM-IIIR criteria for schizophrenia and 20 comparison subjects with no history of psychiatric or neurological disorder (‘controls’). As shown in Table 1, the cohort comprises two brain series, detailed previously (Eastwood et al., 2000; 2001b). The schizophrenia subtypes were paranoid (n = 8), disorganised (n = 5), or unknown (n = 5). One patient had a history of comorbid alcohol misuse. All had been treated longterm with conventional antipsychotic drugs and were being prescribed medication at the time of death. Two patients died by suicide. Neuropathological examination revealed no abnormalities other than age-related changes within normal limits. Frozen sections (14 μm) were cut from each block and slide mounted for in situ

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Table 1 Demographic details of subjects studied Oxford series

Number Sex (M, F) Age (years) Brain pH Post mortem interval Age at onset (years) Duration of illness (years)

London series

Combined series

Controls

Schizophrenics

Controls

Schizophrenics

Controls

Schizophrenics

10 4,6 60.9 (6.4) 6.62 (0.14) 36.9 (4.6) NA NA

9 6,3 57.9 (5.6) 6.35 (0.09) 40.4 (5.9) 28.4 (3.4) 29.4 (4.8)

10 6,4 63.3 (6.7) 6.38 (0.12) 37.8 (5.2) NA NA

9 5,4 62.4 (6.4) 6.36 (0.08) 48.8 (8.2) 26.8 (1.8) 37.2 (8.4)

20 10, 10 62.1 (4.5) 6.50 (0.09) 37.3 (3.4) NA NA

18 11,7 60.2 (4.2) 6.36 (0.06) 44.6 (5.0) 27.8 (2.1) a 32.5 (4.3) a

Values are mean (SEM). NA: not applicable. a Known for 15 subjects.

hybridization or homogenised for protein extraction. The research was approved by the Oxfordshire Psychiatric Research Ethics Committee and Oxfordshire NHS Research Ethics Committee B (O02.040).

0.5× SSC at room temp for 10 min. Slides were rinsed in ddH2O, dried and apposed to X-ray film (Kodak, Biomax MS) for 14–28 days. 2.3. Generation of antisera to human Asc-1

2.2. In situ hybridization Amplified human GlyT1 (bases 1501–2364, a region common to GlyT1a, -1b and -1c, accession # NM_210649), Asc-1 (bases 601–1440, accession # NM_019849) and SNAT2 (bases 841–1680, accession # NM_018976) cDNAs were sub-cloned into pGEM-T Easy Vector (Promega, Southampton, UK) following manufacturer's recommendations: cDNAs were incubated with the pGEMT Easy plasmid at a 3:1 molar ratio in the presence of T4 DNA ligase (1 unit) for 1 h at room temperature. Ligated DNA (60 ng) was then transformed into JM109 competent cells, and the selection, amplification and extraction of clones performed using established methods. Protocols for riboprobe production and in situ hybridization have been previously described (Burnet et al., 1994). Briefly, riboprobes were produced by linearising the pGEM-T constructs with appropriate restriction endonucleases and transcribing using either SP6 or T7 promoter sites in the presence of [35S]UTP (1000 Ci/mmol). The probe was diluted with hybridization buffer (75% formamide, 3× standard saline citrate [SSC], 1× Denhardt's solution, 50 mM sodium phosphate buffer, pH =7.4, 10% dextran sulphate, 0.1 mg/ ml sheared salmon sperm DNA and 20 mM dithiothreitol) to a final activity of 1.2× 104 cpm/ml. The diluted probe (200 μl) was added to each slide of human tissue sections and covered with a glass coverslip. All slides were incubated over night at 50 °C in lidded Perspex trays lined with filter paper soaked with 3× SSC/75% formamide. For post-hybridization washing, slides were first rinsed in 2× SSC, incubated in RNase A (20 µg/ml) for 30 min at room temp, and immersed in 2× SSC at 55 °C for 10 min, 0.5× SSC at 55 °C for 60 min and finally

The rabbit polyclonal antibody against human Asc-1 (peptide CPPSLLPATDKPSKPQ) was produced and affinity-purified by Bio-Mol International (Exeter, UK), and provided by Drs Nick Brandon, Richard Rutter and Peter Hutson, formerly at Merck, Sharp and Dohme, Harlow, Essex, UK. The concentration of anti-Asc-1 antibody was 0.15 mg/ml. Specificity of Asc-1 was tested by western blotting using overexpressed human Asc-1 in HEK293 cells, and by pre-absorption of the antisera with the antigenic peptide. 2.4. Western blotting Frozen tissue sections were homogenised in suspension buffer as described (Eastwood et al., 2001a). Immediately before electrophoresis samples were mixed 4:1 with 5× loading buffer (final concentration 0.1 M Tris–Chloride, 2% sodium dodecyl sulphate [SDS], 10% glycerol, 1% β-mercaptoethanol, 0.02% Bromophenol blue) and boiled for 10 min. Pilot studies demonstrated that 20 μg total protein loaded onto gels gave linear determination of target immunoreactivity. Equal protein concentrations and molecular weight markers (GE Healthcare, Buckinghamshire, UK or BioRad, Hertfordshire, UK) were fractionated in duplicate by electrophoresis on a 12% SDS/polyacrylamide gel and transferred onto a polyvinyl difluoride membrane. Membranes were immersed in blocking buffer (2% non-fat milk, 0.1% Tween20 in PBS) for 1 h followed by a 2 h incubation with primary antibody (Asc-1, 1:100 dilution; GlyT1, sc16701 Santa Cruz,1:500 dilution) in PBS containing 0.1% Tween20 (PBS-T), and 1% BSA. Membranes were then

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washed three times in PBS-T, incubated for 1 h with HRPconjugated secondary antibody in blocking buffer (antigoat immunoglobulin 1:5000, DAKO, Cambridgeshire, UK, for GlyT1; anti-rabbit immunoglobulin 1:5000, Chemicon, Hampshire, UK, for Asc-1; and anti-mouse immunoglobulin 1:5000 [DAKO] for β-actin), followed by washing three times in PBS-T. Immunoreactive bands were visualized on X-ray film using the ‘ECL Plus’ kit (GE Healthcare) on Kodak BioMax X-Omat AR film (Sigma-Aldrich, UK). For quantitative measurements, blots were stripped (Re-Blot Plus western blot recycling kit, Chemicon) and re-probed with anti-β−actin (1:10,000; Chemicon). Bands were analyzed by densitometry and optical density values for the target immunoreactive bands normalized to those of the β-actin. Specificity for all antibodies was assessed in control experiments where the primary antibody was omitted.

Fig. 2. Western blots of GlyT1 and Asc-1 in human brain. A) GlyT1 immunoreactivity migrates as a diffuse band of ~100 kDa (kDa) in extracts of cerebellum (CB) and dorsolateral prefrontal cortex (DPFC). B) Asc-1 immunnoreactivity migrates as a single band at ~40 kDa. C) Preabsorption of Asc-1 antiserum.

2.5. Statistical analysis Previous studies (Eastwood et al., 2000; East et al., 2002) showed that the abundance of some (but not all) mRNAs and proteins differs between the two sources of tissue (Table 1). The reasons are unknown, but may relate to variation in how the tissue was collected and processed. To allow the primary analysis to be performed on data from the two series combined, we computed Z scores (Eastwood et al., 2000). Diagnostic groups were compared using the Z scores, by ANOVA with age, pH and post mortem interval as covariates. To complement this statistical approach, we also analysed the ‘raw’ data using ANOVAwith tissue origin (Oxford or London) as a between-subjects factor. Correlations between variables and demographic factors were assessed using the Spearman coefficient and partial correlations. 2.6. Antipsychotic administration to rats

Fig. 1. Distribution of Asc-1, GlyT1 and SNAT2 mRNAs in human brain. A: Asc-1 mRNA, cerebellum. B: Asc-1 mRNA, dorsolateral prefrontal cortex. C: GlyT1 mRNA, cerebellum. D: GlyT1 mRNA, dorsolateral prefrontal cortex. E: SNAT2 mRNA, cerebellum. F: SNAT2 mRNA, dorsolateral prefrontal cortex.

Adult male Sprague–Dawley rats (Harlan-Olac, Bicester, UK) weighing 225–250 g were treated with haloperidol (1 mg/kg), clozapine (25 mg/kg), or the same volume of saline by intra-peritoneal injection once-daily for 14 days, sacrificed and brain tissue processed as described (Law et al., 2004). All procedures were carried out in accordance with UK Home Office regulations. Protein was extracted from frontal cortex

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Table 2 GlyT1, Asc-1 and SNAT2 expression in schizophrenia⁎ Oxford series

Combined series⁎

London series

Controls

Schizophrenics

Controls

Schizophrenics

Controls

Schizophrenics

(n = 5–8)

(n = 8–10)

(n = 6–10)

(n = 7–9)

(n = 13–18)

(n = 15–18)

0.84 (0.03) 1.53 (0.03)

0.95 (0.05)c 1.53 (0.04)

0.75 (0.01) 1.27 (0.02)

0.77 (0.02) 1.29 (0.02)

0.79 (0.02) 1.38 (0.04)

0.85 (0.03) 1.40 (0.04)

GlyT1 mRNA (nCi/g tissue) Prefrontal cortex 164 (10) Cerebellum 534 (27)

209 (29) 566 (49)

121 (9) 438 (26)

109 (3) 423 (18)

146 (9) 480 (22)

162 (20) 490 (30)

Asc-1 (OD units) Prefrontal cortex Cerebellum

0.82 (0.02)b 1.26 (0.05)b

0.99 (0.04) 1.42 (0.02)

0.86 (0.03)b 1.37 (0.03)

0.94 (0.03) 1.44 (0.04)

0.84 (0.02) 1.32 (0.03)

Asc-1 mRNA (nCi/g tissue) Prefrontal cortex 121 (5) Cerebellum 236 (14)

123 (2)c 262 (20)c

132 (8) 307 (21)

142 (6) 289 (23)

127 (5) 274 (16)

132 (4) 275 (15)

SNAT2 mRNA (nCi/g tissue) Prefrontal cortex 323 (21) Cerebellum 716 (59)

244 (20)a 551 (56)c

208 (4) 535 (46)

192 (7)c 416 (34)c

251 (16) 605 (43)

217 (12) 483 (36)

GlyT1 (OD units) Prefrontal cortex Cerebellum

0.87 (0.02) 1.47 (0.09)

⁎For primary analysis (the two series combined), see Figs. 2–4. Values are mean (SEM). ap b 0.02, bp b 0.05, cp b 0.1, unpaired t-tests, 2-tailed.

and used for quantitative western blots with GlyT1, Asc1 and β-actin antisera as described above. Sections of frontal cortex were used for SNAT2 mRNA in situ hybridization.

3. Results All three transcripts were expressed in the cerebellum (Fig. 1A, C, E) and DPFC (Fig. 1B, D, F), with the

Fig. 3. GlyT1 expression in schizophrenia. A) GlyT1 immunoreactivity, DPFC. B) GlyT1 immunoreactivity, cerebellum. C) GlyT1 mRNA, DPFC. D) GlyT1 mRNA, cerebellum. Data are Z scores, showing control subjects (circles) and subjects with schizophrenia (triangles). No significant differences between groups.

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Fig. 4. Asc-1 expression in schizophrenia. A) Asc-1 immunoreactivity, DPFC. B) Asc-1 immunoreactivity, cerebellum. C) Asc-1 mRNA, DPFC. D) Asc-1 mRNA, cerebellum. Data are Z scores, showing control subjects (circles) and subjects with schizophrenia (triangles). ⁎p = 0.028, ⁎⁎p = 0.011.

strongest signal in both regions seen for SNAT2 mRNA. Sense riboprobes produced minimal or no signal (data not shown). In cerebellum, all three transcripts were concentrated in the granule cell layer and. In DPFC, signal was present across layers II–VI, with enhancement in layer II for SNAT 2 mRNA (Fig. 1F). Specific signal (i.e. greater than background) was seen in the white matter of both regions for SNAT2 mRNA, but not for Asc-1 or GlyT1 mRNAs. The GlyT1 antibody detected a diffuse band of ~ 100 kDa (Fig. 2A). The Asc-1 antibody detected a single band of 40 kDa (Fig. 2B), which was abolished after pre-absorption with the peptide to which it was raised (Fig. 2C). Specificity was confirmed in extracts of HEK 293 cells transfected with human Asc-1 (data not shown), which also showed a single 40 kDa band. As anticipated (see Section 2.5), some effects of tissue origin were seen (Table 2). GlyT1 mRNA, GlyT1, and SNAT2 mRNA levels were higher in the Oxford than London brains in both regions (all p b 0.01); the opposite was true for Asc-1 mRNA, and for Asc-1 in DPFC (all p b 0.05).

3.2. Asc-1 and Asc-1 mRNA in schizophrenia Asc-1 immunoreactivity was decreased in schizophrenia in DPFC (F1,30 = 7.429, p = 0.011; Fig. 4A) and

3.1. GlyT1 and GlyT1 mRNA in schizophrenia There was no change in GlyT1 or GlyT1 mRNA in schizophrenia in either DPFC or cerebellum (Fig. 3 and Table 2; All F b 1.8; 0.11 b p b 0.97).

Fig. 5. SNAT2 mRNA in schizophrenia. A) DPFC. B) Cerebellum. Data are Z scores, showing control subjects (circles) and subjects with schizophrenia (triangles). ⁎⁎⁎p b 0.005.

P.W.J. Burnet et al. / Schizophrenia Research 102 (2008) 283–294 Table 3 Asc-1, GlyT1 and SNAT2 expression in rat frontal cortex following antipsychotic administration Saline

Haloperidol Clozapine

(n = 6)

(n = 7)

Asc-1 protein (OD units) 1.01 (0.06) 0.96 (0.06) GlyT1 protein (OD units) 0.87 (0.05) 0.92 (0.03) SNAT2 mRNA (nCi/g tissue) 85.1 (5.1) 90.1 (4.8)

(n = 5) 1.03 (0.05) 0.85 (0.02) 80.6 (7.4)

Values are mean (SEM).

cerebellum (F1,25 = 5.456, p = 0.028; Fig. 4B). Asc-1 mRNA was unchanged (both p N 0.4; Fig. 4C and D). Similar trends were seen in the two brain series considered separately (Table 2). Repeating the diagnostic group comparisons using the actual Asc-1 data with tissue origin added as a between-subjects factor confirmed the results seen using the Z scores (data not shown). 3.3. SNAT2 mRNA in schizophrenia SNAT2 mRNA was reduced in schizophrenia, both in DPFC (F1,28 = 9.172, p = 0.005; Fig. 5A) and cerebellum (F1,24 = 10.96, p = 0.003; Fig. 5B). The reductions were also significant when the raw data were analysed with tissue origin as a between-subjects factor (data not shown). 3.4. Correlational analyses In DPFC, there were inverse correlations between GlyT1 and Asc-1 (r = − 0.494, n = 32, p = 0.004), and between GlyT1 mRNA and Asc-1 mRNA (r = − 0.436, n = 25, p = 0.018). No correlations were seen between the expression of any of the transporters and age at death, age at onset, or duration of illness (all p N 0.1; data not shown). There were no male–female differences nor diagnosis × sex interactions (data not shown). 3.5. Antipsychotic effects on GlyT1, Asc-1 and SNAT2 Western blots from frontal cortex of antipsychotic treated rats revealed GlyT1 and Asc-1 immunoreactivity migrating at ~ 100 kDa and 40 kDa respectively (data not shown), as predicted from previous reports and the observations in human tissue. Neither haloperidol nor clozapine affected GlyT1 or Asc-1 immunoreactivity, or SNAT2 mRNA (Table 3). 4. Discussion NMDAR hypofunction in schizophrenia is thought to be at least partly attributable to impairment in the

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functioning of the endogenous NMDAR modulators glycine and D-serine (Coyle and Tsai, 2004; Millan, 2005; Javitt, 2008). One factor influencing the synaptic availability of D-serine and glycine is the status of their reuptake transporters. Thus, the distribution of these transporters in human brain, and their possible altered abundance in schizophrenia, is required as part of the evaluation of this system in both the pathophysiology and pharmacotherapy of the disorder. As these issues have not hitherto been addressed, we studied the major glycine and D-serine transporters, GlyT1 and Asc-1 respectively, as well as SNAT2, in two brain areas (DPFC and cerebellum) and compared their mRNA and protein expression between patients with schizophrenia and control subjects. The main findings are that: (1) all three genes are expressed robustly in both areas; (2) expression of GlyT1 was unaltered in schizophrenia, but Asc-1 was decreased, as was SNAT2 mRNA; (3) antipsychotic administration did not affect expression of the genes in rat brain. The findings suggest that in schizophrenia the synaptic transport system for D-serine is down-regulated, but that for glycine is not markedly affected. 4.1. Expression of GlyT1, Asc-1 and SNAT2 in human brain The distribution of GlyT1 and Asc-1 in human DPFC and cerebellum has not been reported, to our knowledge, and there are only limited data for SNAT2. GlyT1 mRNA (Fig. 1C, D) and immunoreactivity (Fig. 2A) were present in the DPFC and abundant in the cerebellum, consistent with findings in rodents (Smith et al., 1992; Zafra et al., 1995a,b). Although it was beyond the scope of this study to examine cellular or subcellular expression profiles of each gene, prior studies reveal GlyT1 expression by both neurons and glia. In cerebral cortex, GlyT1 mRNA is concentrated in pyramidal neurons and astrocytes; in cerebellum, it is in Bergmann glia and possibly in Purkinje cells (Guastella et al., 1992; Smith et al., 1992; Zafra et al., 1995a). Initial immunocytochemical studies revealed GlyT1 immunoreactivity limited to astrocytes and Bergmann glia (Zafra et al., 1995b), but use of additional antibodies has shown GlyT1 also present in neurons, both in presynaptic terminals and in some dendrites (Cubelos et al., 2005). Neuronal GlyT1 is limited to glutamatergic neurons, at least in the forebrain (Cubelos et al., 2005). The relative contributions of neuronal and glial GlyT1 to glycine reuptake remains unclear, and may vary between forebrain and hindbrain (Cubelos et al., 2005); recent data from a conditional GlyT1 mutant mouse

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suggest that astrocytic GlyT1 is relatively unimportant with regard to NMDAR modulation (Yee et al., 2006). Asc-1 mRNA (Fig. 1A, B) and protein (Fig. 2B) were both readily detected in grey but not white matter of DPFC and cerebellum, largely in agreement with findings in rat and mouse brain. In those species, Asc-1 expression is exclusively neuronal, and concentrated in cortical pyramidal neurons and in Purkinje cells (Helboe et al., 2003; Matsuo et al., 2004). With regard to the latter observation, our autoradiographs show Asc1 mRNA also clearly present over the granule cell layer (Fig. 1A); this suggests there may be a more widespread expression in human than rodent cerebellum, although this remains to be corroborated immunocytochemically. The subcellular distribution of Asc-1 is unclear. Helboe et al. (2003) reported a predominantly presynaptic distribution, but Matsuo et al. (2004), using additional antibodies, found a dendritic localization. The lack of glial Asc-1 suggests that it is not responsible for the glial transport of D-serine (Rutter et al., 2007). Notably, there appears to be a cellular discrepancy between the solely neuronal reuptake of Dserine by Asc-1, and the glial localization of the Dserine metabolizing enzyme DAO, at least in the cerebellum (Verrall et al., 2007). Hence the glial Dserine transporter(s) need to be identified (Javitt et al., 2002; Ribeiro et al., 2002; Martineau et al., 2006), and then studied in schizophrenia. Our finding of abundant SNAT2 mRNA in cerebellum (Fig. 1E), and of a clear but lesser signal in the DPFC (Fig. 1F), is consistent with in situ hybridization (Yao et al., 2000) and immunohistochemical (Gonzales-Gonzales et al., 2005) data in rodent brain. Within these regions, SNAT2 immunoreactivity is observed over neurons and astrocytes. In the cerebellum, Bergmann glia, astrocytes, and granule cells express SNAT2, but Purkinje cells do not (Yao et al., 2000; Gonzales-Gonzales et al., 2005). Most neuronal immunostaining for SNAT2 is perikaryal and dendritic (Gonzales-Gonzales et al., 2005; Melone et al., 2006). The phenotype of SNAT2-expressing neurons is unclear. Gonzales-Gonzales et al. (2005) found expression limited to presumed glutamatergic neurons in rat brain, whereas Melone et al. (2006), who studied human as well as rat cerebral cortex, also identified SNAT2 in a significant proportion of GABAergic neurons. In summary, our regional distribution data, in concert with previous findings, suggests that GlyT1, Asc-1 and SNAT2 are expressed by one or more neuronal types in DPFC and cerebellum. GlyT1 and SNAT2 are also expressed by glia but Asc-1 is not (Fig. 6).

Fig. 6. Synaptic localization of Asc-1, GlyT1 and SNAT2 in human brain. A schematic figure summarising the known localizations of these transporters (see Section 4.1 for details and citations). D-serine is removed from the synapse by Asc-1, which is expressed by neurons, pre-synaptically and also post-synaptically. There is also likely at least one glial transporter for D-serine. Glycine is transported by GlyT1, located in glia and also neuronally in synaptic terminals and dendrites; the relative importance of these sites is unclear. SNAT2 is expressed by glia and neurons; in the latter, it appears mainly post-synaptic.

4.2. Expression of glycine and D-serine transporters in schizophrenia NMDAR modulation by glycine and/or D-serine is thought to be reduced in schizophrenia as envisaged by theories of NMDAR hypofunction. The fact that none of the transporters were increased in abundance in schizophrenia implies that any co-agonist deficiency that might exist in the synapse is not due to excessive reuptake. Rather, we found one transporter unchanged (GlyT1) and two reduced (Asc-1 and SNAT2). Considering first the unaltered level of GlyT1, this finding suggests that any synaptic deficiency of glycine does not produce a homeostatic response (down-regulation) in transporter expression; the ‘intact’ level of GlyT1 is relevant when considering it as a therapeutic target. The decreased Asc-1 abundance in schizophrenia in both DPFC and cerebellum (Fig. 4) is noteworthy, but its interpretation is not straightforward. It does not appear to be secondary to antipsychotic medication, at least as indicated by the rat data (Table 3). Our preferred hypothesis is that the reduction is secondary to a reduced synaptic concentration of D-serine, as hypothesised to occur in schizophrenia because of increased DAO activity and other factors. A reduction of this kind would be consistent with the response of many synaptic transporters to changes in their substrate concentration (Bernstein and Quick, 1999; Munir et al., 2000; Zahniser and Doolen, 2001; Lopez-Bayghen et al., 2003). Whether this presumed down-regulation is

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successful in restoring synaptic levels of D-serine towards normal in schizophrenia is not known. There are, in any case, problems with the interpretation. Not all synaptic transporters always down-regulate in response to decreased substrate concentration (Gegelashvilli and Schousboe, 1997; Zahniser and Doolen, 2001; Zahniser and Sorkin, 2004; Mandela and Ordway, 2006), and so decreased Asc-1 does not necessarily indicate lowered synaptic D-serine levels. Indeed, it is not even certain that the latter occurs in schizophrenia: in contrast to the plasma and cerebrospinal fluid reductions (Hashimoto et al., 2003, 2005; Bendikov et al., 2007), D-serine concentrations in brain tissue are unaltered (Kumashiro et al., 1995; Bendikov et al., 2007; Hashimoto et al., 2007). As a further consideration, in mice that lack DAO activity and have elevated D-serine levels, Asc-1 expression is unaltered (Almond et al., 2006). In total, further studies are needed to establish the causes and consequences of reduced Asc-1 in schizophrenia. In terms of mediating molecular mechanisms, the fact that Asc-1 mRNA was unchanged indicates that the lower Asc-1 immunoreactivity in schizophrenia arises translationally or post-translationally. We found a strong inverse correlation in DPFC between Asc1 and GlyT1 expression, present for both mRNA and protein, suggesting a co-ordinated, reciprocal regulation of expression of the two transporters, which in turn may indicate that a change in level of either D-serine or glycine is compensated for by a reciprocal change in the other modulator. This proposal is supported by the recent demonstration of a similarly strong inverse correlation between glycine and D-serine levels in the mouse brain (Hashimoto et al., 2007). The significance of the SNAT2 reduction seen in schizophrenia is unclear, especially as only its mRNAwas measured and an effect on the protein remains to be shown. SNAT2 is not thought to play a major role in glycine reuptake, although a decrease in SNAT2 expression might be significant in synaptic or cell populations that lack GlyT1. Alternatively, since glutamine is an important SNAT2 substrate (Mackenzie and Erickson, 2004), there could be a link between the SNAT2 reduction and the lower glutamine levels reported in chronic schizophrenia in several brain regions (Abbott and Bustillo, 2006). Speculatively, as glutamine–glutamate cycling contributes to synthesis of the glutamate neurotransmitter pool (Bak et al., 2006), altered glutamine transport might have downstream effects on glutamatergic signalling and NMDAR function. Finally, SNAT2 is a key regulator of cell volume, raising the possibility that decreased SNAT2 expression has broader pathophysiological implications (Franchi-Gazzola et al., 2006).

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4.3. Limitations and conclusions This study provides information about the distribution of glycine and D-serine transporters in the human brain, and the first evidence for their differential involvement in schizophrenia. Further studies of several kinds are now needed. For example, replication in additional brain series and regions to establish the generalizability of the findings, especially given the variability between the two cohorts studied here, and to investigate more clearly the possible effect of demographic, clinical and treatment variables. Also, it is the functional state of the transporters that directly impacts on the synaptic availability of D-serine and glycine, and this can be regulated in many ways (e.g. by membrane trafficking, ubiquitinylation); altered expression is neither necessary nor sufficient for their activity to be affected (e.g. Zahniser and Doolen, 2001; Aragon and Lopez-Corcuera, 2003). Functional studies are problematic in post mortem tissue; animal and in vitro models will be required to determine the molecular processes that link transporter expression with function, and that in turn modify the regulation of synaptic D-serine and glycine. Ultimately, integration of the data with findings in schizophrenia concerning other molecules that influence D-serine and glycine metabolism, such as serine racemase and DAO, as well as the status of other NMDAR modulators, will be needed for a complete picture to emerge. This will be a worthwhile though difficult task since the results will aid in the rational development of therapeutic interventions targeting NMDAR co-agonists and their synaptic transporters. Role of funding source Work supported by a research grant from the UK Medical Research Council (G0500180), and a Centre Award from the Stanley Medical Research Institute. The funding bodies had no role in study design, in the collection, analysis and interpretation of the data, in the writing of the report, or in the decision to submit the paper for publication.

Contributors PWJB designed the study, carried out or supervised the experiments, performed the analyses, did literature searches and co-wrote the paper. LH, MvH and EJG assisted with the experiments. PJH co-designed the study, did literature searches and wrote the first draft of the paper. All authors contributed to and have approved the final manuscript.

Conflict of interest PJH and PWJB have received unrestricted educational grants from GlaxoSmithKline and Merck. PJH has received honoraria for giving educational lectures or chairing scientific meetings from BMS, GSK, Janssen, Lilly, Merck, Sanofi and Servier pharmaceutical companies, and has been a consultant to Curidium, Janssen, Organon, and Wyeth.

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Acknowledgements We thank Nick Brandon, Richard Rutter and Peter Hutson (formerly at Merck, Sharp and Dohme, Harlow, UK) for the Asc-1 antibody, and Margaret Esiri, Robert Kerwin, and the MRC Brain Bank, Institute of Psychiatry, London for tissue provision and neuropathological assessments. Valerie West assisted with preparing the reference list. We dedicate this paper to Robert Kerwin, who made seminal contributions to the study of glutamate in schizophrenia.

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