Changes in NMDA receptor subunits and interacting PSD ... - Nature

Molecular Psychiatry (2006) 11, 737–747 & 2006 Nature Publishing Group All rights reserved 1359-4184/06 $30.00 www.nature.com/mp

ORIGINAL ARTICLE

Changes in NMDA receptor subunits and interacting PSD proteins in dorsolateral prefrontal and anterior cingulate cortex indicate abnormal regional expression in schizophrenia LV Kristiansen1, M Beneyto1, V Haroutunian2 and JH Meador-Woodruff1 1 2

Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Birmingham, AL, USA and Department of Psychiatry, Mount Sinai School of Medicine, New York, NY, USA Abnormal expression of the N-Methyl-D-Aspartate (NMDA) receptor and its interacting molecules of the postsynaptic density (PSD) are thought to be involved in the pathophysiology of schizophrenia. Frontal regions of neocortex including dorsolateral prefrontal (DLPFC) and anterior cingulate cortex (ACC) are essential for cognitive and behavioral functions that are affected in schizophrenia. In this study, we have measured protein expression of two 0 alternatively spliced isoforms of the NR1 subunit (NR1C2 and NR1C2 ) as well as expression of the NR2A–D subunits of the NMDA receptor in DLPFC and ACC in post-mortem samples from elderly schizophrenic0 patients and a comparison group. We found significantly increased expression of NR1C2 but not of NR1C2 in ACC, suggesting altered NMDA receptor cell membrane expression in this cortical area. We did not find significant changes in the expression of either of the NR1 isoforms in DLPFC. We did not detect changes of any of the NR2 subunits studied in either cortical area. In addition, we studied expression of the NMDAinteracting PSD molecules NF-L, SAP102, PSD-95 and PSD-93 in ACC and DLPFC at both transcriptional and translational levels. We found significant changes in the expression of NFL in DLPFC, and PSD-95 and PSD-93 in ACC; increased transcript expression was associated with decreased protein expression, suggesting abnormal translation and/or accelerated protein degradation of these molecules in schizophrenia. Our findings suggest abnormal regional processing of the NMDA receptor and its associated PSD molecules, possibly involving transcription, translation, trafficking and protein stability in cortical areas in schizophrenia. Molecular Psychiatry (2006) 11, 737–747. doi:10.1038/sj.mp.4001844; published online 16 May 2006 Keywords: glutamate; translation; in situ hybridization; postmortem; Western blotting; cortex

Introduction Schizophrenia is a devastating psychiatric illness that affects approximately 1% of the population. Based on clinical and molecular observations, abnormal glutamatergic neurotransmission has been proposed, in concert with other neurotransmitter systems, to be involved in the pathophysiology of this illness.1,2 The heteromeric N-Methyl-D-Aspartate (NMDA) receptor is an ionotropic membrane-bound glutamate receptor, which is assembled from four or five subunits with an obligatory NR1 subunit associated with different combinations of NR2A–D subunits. The NR1 subunit is encoded by a single gene, which, by means of alternative splicing of one N-terminal Correspondence: Dr LV Kristiansen, Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, SC 560, 1530 3rd Avenue South, Birmingham, AL 35294-0017, USA. E-mail: [email protected] Received 13 October 2005; accepted 17 April 2006; published online 16 May 2006

(N1), and two C-terminal (C1 and C2) exons, generates eight different NR1 isoforms.3 Contrary to the expression of the NR2 subunits, the NR1 subunit is normally expressed in excess in the endoplasmatic reticulum (ER) where it, when unassembled, is degraded by an activity-dependent process known as ER-associated degradation (ERAD).4,5 Assembly of NR1 isoforms with different NR2 subunits determines essential functional attributes, including synaptic targeting, modulation of channel gating and receptor interaction with intracellular signaling proteins.6–11 Inclusion of different alternative splice variants of the NR1 subunit confers different intracellular interactions that affect receptor signaling and transport. For example, the presence of the C1 exon in the NR1 subunit modulates an interaction between the NMDA receptor and neurofilament-light (NF-L), a member of the intermediate cytoskeletal filaments, causing stabilization of the receptor in the cell membrane by preventing its ubiquitination and subsequent degradation.12,13 Additionally, exclusion of the C2 exon causes a potential stop codon of the transcript to be

NMDA transcript and protein expression in schizophrenia LV Kristiansen et al

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lost, resulting in an alternate splice variant containing a shorter, unique C-terminus, termed C20 .3 Alternative splicing of the C2 cassette regulates cellular trafficking and cell surface expression of the NMDA receptor.14–16 NMDA receptors are predominantly expressed in the postsynaptic membrane where they interact with an intracellularly organized multiprotein complex termed the postsynaptic density (PSD).17,18 Proteins of the membrane-associated guanylate-kinase family (MAGUKs), a group of PSD-associated proteins characterized by the presence of guanylate kinase, PDZ and Src homology 3 (SH3) protein-interacting domains, function as scaffolding and organizer proteins of the PSD.19 Proteins such as SAP 90/PSD95, PSD-93/Chapsyn 110 and SAP102 are prominent examples of MAGUK proteins that interact with the NMDA receptor through their PDZ domains, binding specifically to C-terminal sequences of the NR2 subunit.20–23 These interactions are involved in receptor clustering at the synaptic membrane, downstream signaling including modulation of kinase and phosphatase activity, as well as regulated synaptic trafficking of the NMDA receptor.24–30 In addition, binding of newly synthesized NMDA receptor subunits to PSD-95 or SAP102 links the receptor to different intracellular trafficking pathways, which is essential for its trafficking from the ER/Golgi complex to the PSD.31–34 Changes in the expression of transcripts encoding NMDA receptor subunits in the prefrontal cortex have previously been described in post-mortem schizophrenic brains.35–37 However, relatively little is known about protein expression of these subunits in schizophrenia. Additionally, expression of the NMDA receptor-interacting PSD molecules in schizophrenia have to date only been analyzed on transcriptional level, and primarily in subcortical regions in post-mortem brain.38–41 Although the analysis of transcript expression plays an essential role in describing the pathophysiology of schizophrenia at anatomical and molecular levels, it does not account for any post-transcriptional changes that might affect protein translation, trafficking and protein stability. Schizophrenia is characterized by behavioral and cognitive disturbances, which has been mapped to brain structures including the prefrontal cortex (PFC).42,43 As the circuitry of corticocortical and thalamocortical projections in the PFC by and large relies on glutamate signaling, expression of glutamate receptors and their interacting PSD molecules in this area have been hypothesized to be altered in schizophrenia. In order to complement and expand on previous studies of abnormal expression of transcripts for glutamatergic proteins in schizophrenia, the present work contains an analysis of protein expression of NMDA receptor subunits and their interacting PSD molecules, PSD-95, SAP102, PSD-93 and NF-L in dorsolateral PFC (DLPFC) and anterior cingulate cortex (ACC). In addition, we have also analyzed

Molecular Psychiatry

transcript expression of these same PSD proteins in these two cortical areas.

Materials and methods Post-mortem brain tissue from 24 subjects with schizophrenia and 16 comparison subjects was obtained from the Mount Sinai Medical Center Brain Bank and used for in situ hybridization and Western blotting experiments (Table 1). Owing to limited availability of this tissue, overlap of subjects represented in both transcript and protein expression studies was 17% (seven out of 40). No significant differences in age, post-mortem interval (PMI) or pH between diagnostic groups were identified for subjects studied by either experimental approach (Table 1). The subjects studied were well-diagnosed patients with schizophrenia and matched comparison individuals, with no sign of neurodegenerative disorders or other discernable neuropathologies, including Alzheimer’s disease.44 Tissue preparation Dissected blocks of brain tissue containing DLPFC and ACC were kept at 801C until further processing. For in situ hybridization, tissue was thawed to 201C, cryosectioned at 15 mm in a Lipshaw cryostat/microtome onto Fisherbrand Superfrost/Plus positively charged microscope slides (Fisher Scientific at no: 12-550-15), dried and returned for storage at 801C until initiation of hybridization experiments. For protein determination studies, approximately 1 cm3 of tissue was homogenized (10% w/v) in 50 mM Tris-HCl (pH 7.0) for 30 s with a polytron homogenizer and stored at 801C in 0.5 ml aliquots. Protein concentration of tissue homogenates was determined by the Bradford method.45 Antibodies Most antibodies were commercially available: NR1C2 0 (#06-311), NR1C2 (#06-314), NR2A (#06-313), NR2B (#06-600), PSD-95 (#05-494), b-Tubulin (#05-661) (Upstate Biotechnology), NF-L (#13-0400), PSD-93 (#34-4700) (ZYMED Laboratories) and SAP102 (#124 202) (Synaptic Systems). The antibodies directed 0 against the NR1C2 and NR1C2 spliceforms recognize entirely different C-termini and do not crossreact.46,47 We prepared antisera specific for NR2C and NR2D by custom peptide synthesis followed by immunization of rabbits. Peptide synthesis, immunization and antisera titer determination by enzyme-linked immunosorbent assay (ELISA), were performed by commercial vendor (Research Genetics Inc., AL, USA). The following peptide sequences were used to generate these antisera: PALLLTSLLGAWARL corresponding to the N-terminal sequence of NR2C (Accession number NP_000826) and PPPCPYLDLEP, which corresponds to amino acids 1101–1110 in NR2D (Accession number NP_000827). Based on the distinctive sizes of the NR2C (140 kDa) and NR2D (150 kDa) proteins, which is considerably smaller


Table 1

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Summary of subject characteristics

Subject no.

Analysis

Comparison subjects (N = 16) 1 2 In situ 3 In situ 4 In situ 5 6 In situ 7 In situ 8 In situ 9 In situ 10 In situ 11 12 13 14 In situ 15 In situ 16 In situ

Sex

Age (years)

PMI (h)

pH

Cause of death

F F F F F F F F F F M M M M M M

86 79 96 90 74 64 102 73 79 84 88 70 55 69 93 101

4.7 3.0 3.3 4.2 3.0 19.1 7.1 3.4 7.7 18.5 4.8 8.0 10.0 4.3 19.0 4.7

6.5 6.3 6.7 6.0 6.0 6.1 6.5 6.3 6.5 6.2 5.9 6.0 5.7 6.3 6.4 6.8

Myocardial infarction Cardiopulmonary failure Cardiorespiratory failure Cardiopulmonary failure Cardiorespiratory failure Pulmonary edema Acute myocardial infarction Acute myocardial infarction Acute myocardial infarction Myocardial infarction Cardiac Lower GI bleed Cancer Cancer of lung Congestive heart failure Coronary artery disease

F

86

6.9

5.8

WB WB

F F F

84 86 76

15.6 5.5 8.5

6.2 6.2 6.1

WB WB WB

F F F

65 69 79

5.8 13.7 20.4

5.9 6.2 7.1

WB WB WB WB

F F F M M M M

76 82 79 54 61 84 69

21.2 18.8 9.9 8.2 3.5 6.2 4.5

6.1 6.6 6.8 6.0 6.5 6.5 6.4

M M M M M M M M M M

72 63 87 68 85 73 66 97 66 68

20.6 6.2 11.2 5.6 5.3 7.9 12.1 9.3 8.4 17.3

6.6 5.9 6.5 6.8 6.3 6.5 6.5 6.5 6.7 6.6

Respiratory insufficiency, renal failure Unknown Cardiac, pneumonia Cardiopulmonary failure, breast cancer Cardiopulmonary failure Cardio respiratory failure Cardiopulmonary arrest, cancer of pancreas Cardiogenic shock Cardiopulmonary arrest Cardiac arrest Acute myelocytic leukemia Cardiopulmonary failure Cardiopulmonary failure Cardiac infarction, renal failure Cardiopulmonary failure Cardiopulmonary failure Cardiopulmonary failure Cardiopulmonary failure Cardiopulmonary arrest Cardiorespiratory failure Acute cardiac failure Cardiopulmonary arrest Cardiopulmonary arrest Cardiopulmonary arrest

WB WB WB WB WB

WB WB WB

Schizophrenia subjects (N = 24) 17 In situ 18 19 20

In situ

21 22 23

In situ In situ In situ

24 25 26 27 28 29 30

In situ In situ In situ

31 32 33 34 35 36 37 38 39 40

In situ In situ

In In In In In In In In

situ situ situ situ situ situ situ situ

WB WB WB WB

Summary of subject characteristics per experimental procedure Analysis N Sex Age (years) In situ (7s.d.) WB (7s.d.) ANOVA in situ WB

29 21

13 M/16 F 11 M/10 F

79.8 (711.2) 74.8 (711.8) P = 0.072 P = 0.134

PMI (h)

PH

Diagnosis

10.1 (76.0) 7.7 (75.1) P = 0.275 P = 0.072

6.4 (70.3) 6.3 (70.4) P = 0.537 P = 0.169

11 con/18 scz 8 con/13 scz

Abbreviations: ANOVA, analysis of variance; con, comparison group; F, female; M, male; PMI, post-mortem interval; scz, schizophrenia; s.d., standard deviation; WB, Western blot.



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than NR2A and NR2B (B175 kDa), we were able to identify specific immunolabeling by these antisera. In addition, we undertook additional characterization when we had these antisera prepared. We obtained full-length clones of each NMDA subunit in an expression vector from Dr Stephen Heinemann at the Salk Institute. Each clone was transcribed in the ‘sense’ direction to make mRNA, and translated in a cell-free translation system to generate protein. We performed Western blot analysis on blots containing each translated protein, and our NR2C and NR2D antibodies identified the proteins against which they were raised, at their predicted molecular weights, and did not react with any other NMDA receptor subunit. In situ hybridization [35S]-labeled nucleotide probes specific for human PSD-95, NF-L, PSD-93 and SAP102 were generated from linearized plasmids containing B0.5 kb polymerase chain reaction (PCR)-amplified cDNA clones identical to coding regions within these molecules, as described previously.48,49 For each probe, two slides per subject were fixed in 4% (w/v) formaldehyde for 1 h and processed according to our previously published protocol for in situ hybridization.38,50 Slides from each in situ experiment were apposed to autoradiography film (Kodak Biomax MR #871-5187) for the following time periods: PSD-95, 8 days; PSD93, 14 days; SAP102, 18 days and NF-L, 35 days. Capturing of images was done using a northern light illuminator (Imaging Research Inc., model B-95) with a XC-77 CCD video camera module (Sony Corporation, model XC-77) connected to an image-acquisition and analysis software package (Scion Corporation, version Beta 4.0.2). For each subject, tissue background values from adjacent white matter were subtracted from gray matter gray scale values (GSVs) and converted to optical density units (OD) and values averaged. The amount of radioactivity per gram of tissue (nCi/g) was calculated using a [14C] micro-scale standard (Amersham Biosciences) which was exposed on the same film as the slides in each study.51 Bound radioactivity was converted to concentration of transcript per gram of tissue (fmol/g) by taking into account the number of radioactively labeled uridine nucleotides in each probe. Western blotting For preparation of samples for sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), tissue homogenates (20 mg/lane) were mixed with 6 sample buffer (4.5% SDS, 170 mM Tris-HCl (pH 6.8), 36% glycerol, 15% b-mercaptoethanol, 0.018% bromophenol blue) and heated at 951C for 4 min. Samples were loaded in triplicate onto 7.5% 15-well precast Tris-HCl gels (Bio-Rad Laboratories Inc.) in a mini-protean 3 electrophoresis module (Bio-Rad Laboratories Inc. #161-1118) and separated for 75 min at 120 V in electrophoresis buffer (25 mM Tris-base, 192 mM glycine, 0.1% SDS). Transblotting to nitrocellulose membranes was carried out in an SD


semi-dry transfer cell (Bio-Rad Laboratories Inc.). Protein containing membranes were washed briefly in dH2O, blocked for 30 min in phosphate-buffered saline (PBS) or Tris-buffered saline (TBS) containing 2–5% dry milk powder and incubated overnight with primary antibody (41C on a rocking platform). For each antibody, optimized protocols for washing buffer (PBS vs TBS), dry milk and antibody concentrations were identified before actual experiments. Following several washes in PBS or TBS, membranes were incubated with horseradish peroxidase-coupled secondary antibody for 2 h (goat anti-mouse or goat antirabbit from Upstate Biotechnology) washed as above and developed using enhanced chemiluminescence (ECL; Amersham Biosciences). Film exposures of immunopositive bands were captured for each subject, using the image-acquisition package described above, film background was subtracted from specific GSVs and averaged for each subject. In order to avoid differences owing to loading, blots were additionally probed for b-Tubulin. Measures of GSVs for PSD proteins and b-Tubulin were averaged for each subject and experimental values expressed as the ratio of PSD proteins to b-Tubulin expression. Statistical analysis Data analysis for in situ hybridization and protein quantification experiments was performed using the Statistica software package 7 (Statsoft Inc.). Analysis of variance (ANOVA) was used for all experiments except in cases when significant association with independent measures were found, in which case analysis of covariance was utilized. Independent measures included age, PMI and pH. For in situ hybridization experiments, factorial two-way ANOVA was performed with isodense cortical bands and diagnosis as categorical independent variables. For Western blot analysis, data were analyzed by univariate ANOVA. For all tests a = 0.05.

Results NMDA receptor subunit protein expression in schizophrenia Using subunit-specific antibodies, the expression of NMDA receptor subunits was quantified by Western blotting in two brain regions in schizophrenic patients and a comparison group. As alternative splicing of the C-terminal C2 exon of the NR1 subunit 0 generates two distinct C-termini (C2 and C2 ) associated with different membrane kinetics of the NMDA receptor, it was of interest to distinguish between these subunits in our analysis. We detected immunopositive bands corresponding to the two alternatively 0 spliced NR1 isoforms, NR1C2 and NR1C2 (B118 kDa), as well as NR2A (B175 kDa), NR2B (B175 kDa), NR2C (B140 kDa) and NR2D (B150 kDa) in DLPFC and ACC in all subjects. Additionally, we probed all blots for b-Tubulin, which was used as internal lane loading control. b-Tubulin has previously been used as internal control52,53 and several studies have found


expression of b-Tubulin to be unaffected in schizophrenic brain.54,55 However, in order to further minimize the potential effect of dendritic loss on bTubulin expression, we used an antibody that recognizes not only the neuron-specific isoform III, but all b-Tubulin isoforms (I, II, III, IVa and IVb), representing both neuronal and non-neuronal tubulin expression. Examples of representative immunoblots are shown in Figure 1a. Before statistical analysis of diagnosis effects, linear regression, including age, pH and PMI, was performed on all protein data, as previous studies have shown that these variables might associate, in a protein-specific manner, with protein stability, although predominantly at more extended PMIs of > 18 h (in this study, mean PMI was 7.7 h) (Table 1).56–59 Correlation analysis showed no association between any of the NMDA receptor subunits and age with the exception of NR2C in DLPFC (r = 0.46, P = 0.03). In the ACC, only NR2B (r = 0.46, P = 0.04) was significantly associated with PMI. We found no significant association between any of the NMDA receptor subunits and age in ACC or with PMI in DLPFC. There were no significant correlation between pH and any NMDA receptor subunit in either region. Main effect of diagnosis was identified only for NR1C2 protein expression. Expression of the NR1C2 0

0

isoform was selectively increased in ACC in schizophrenia (F = 5.19, df = 1, 18, P = 0.035) with no change of its expression in DLPFC (Figure 1b). We did not find any effect of diagnosis on the expression of the NR1C2 isoform, NR2A, NR2B, NR2C or NR2D subunits in ACC or DLPFC.

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Transcript expression of NMDA receptor-interacting PSD molecules in schizophrenia We detected expression of transcripts for PSD-93, NFL, SAP102 and PSD-95 in distinct isodense bands in both ACC and DLPFC (Figure 2). The distribution of each transcript into isodense bands was similar between ACC and DLPFC, and expression levels in corresponding isodense bands were not significantly different between the two areas for any of the molecules studied. Linear regression analysis included age, PMI and pH as independent variables as all three factors might affect mRNA stability in post-mortem tissue.57,60,61 Correlation analysis showed a significant association between PSD-93 and age in ACC (r = 0.38, Pvalue = 0.000009). Expression of PSD-93, SAP102 and NF-L were significantly associated with PMI in DLPFC (r = 0.32, P = 0.0008; r = 0.45, P = 0.000001; r = 0.38, P = 0.00008) and NF-L in ACC (r = 0.30; P = 0.0003). Finally, expression of PSD-93 in DLPFC

Figure 1 Expression of NMDA receptor subunits in0 ACC and DLPFC. (a) Representative immunoblots from ACC showing expression of NMDA receptor subunits NR1C2, NR1C2 , NR2A, NR2B, NR2C and NR2D and b-Tubulin, with indication of size in kilodaltons (kDa). Shown are triplicate lanes from control and schizophrenic individuals. Inset shows an immunoblot for NR1C2 and b-Tubulin in the same membrane with indication of molecular weight markers. (b) Quantification of NMDA receptor subunit expression in ACC and DLPFC. Quantification of protein expression in ACC and DLPFC in schizophrenia expressed as the percent of comparison subjects. Bars represent specific NMDA receptor subunits as indicated below each diagram. Values are means7s.e.m. (*P < 0.05). Molecular Psychiatry


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Figure 2 Quantification of transcript expression for PSD-95, NF-L, PSD-93 and SAP102 in DLPFC and ACC. For each probe, pairs of bars indicate transcript expression levels in either five or six discrete isodense bands. Values are means7s.e.m. Representative cortical sections with indication of isodense bands used for quantification are shown for each probe. For NF-L in DLPFC, and PSD-95 and PSD-93 in ACC, significantly increased expression in schizophrenia when compared to comparison subjects. *P < 0.05. Scale bar = 10 mm.

(r = 0.19, P = 0.047) and NF-L in ACC (r = 0.29, P = 0.0004) were significantly associated with pH. PSD-95 transcript expression did not associate with any of these variables. Using factorial statistical analysis, we found main effects for diagnosis on transcript expression for PSD95, PSD-93 and NF-L. Expression of PSD-95 and PSD93 transcripts in ACC were significantly increased in schizophrenia across all cortical isodense layers (F = 16.3, df = 1, 102, P = 0.0001 (PSD-95); F = 21.3, df = 1, 119, P = 0.00001 (PSD-93)) (Figure 2). Increased expression of these molecules was specific to the ACC as their expression in DLPFC was not altered (Figure Molecular Psychiatry

2). NF-L transcript expression was significantly increased across all cortical isodense layers in DLPFC in schizophrenia (F = 17.3, df = 1, 89, P = 0.00007) with no change in ACC. These significant effects were across the collapsed layers, and none showed isolated significant changes in a specific isodense band. There were no effects of diagnosis on SAP102 transcript expression in ACC and DLPFC. Protein expression of NMDA receptor-interacting PSD molecules in schizophrenia Increased expression of transcripts for the NMDA receptor-interacting PSD molecules suggests abnor-


false-positive finding, thus not included in subsequent analyses. For all other PSD-associated proteins, no significant associations with independent variables were present. (Figure 3) We detected main effects for diagnosis for PSD-93, PSD-95 and NF-L, with significantly decreased expression of PSD-93 (F = 5.29, df = 1. 19, P = 0.03) and PSD-95 (F = 4.95, df = 1. 19, P = 0.04) in ACC, and of NF-L (F = 7.73, df = 1. 18, P = 0.01) in DLPFC in schizophrenia (Figure 3A). No changes in the expression of SAP102 were detected in any of the cortical brain areas examined.

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Discussion In the present work, cortical expression of NMDA receptor subunits and its interacting proteins of the PSD have been analyzed. Using antibodies directed against the two alternatively spliced C2 -exon isoforms of the NR1 subunit, we found increased expression of the NR1C2 isoform in ACC in schizophrenia with no significant alterations of the NR1C2 isoform in either DLPFC or ACC. Additionally, we found abnormal transcript and protein expression of NF-L in DLPFC and PSD-95 and PSD-93 in ACC. 0

Figure 3 Protein expression for PSD-95, PSD-93, SAP102 and NF-L in DLPFC and ACC. (a) Quantification of protein expression in ACC and DLPFC in schizophrenia expressed as the percent of comparison subjects. Bars represent specific PSD proteins as indicated below each diagram. Values are means7s.e.m. (*P < 0.05). (b) Representative Immunoblots from ACC showing expression of PSD proteins and b-Tubulin with indication of size in kilodaltons (kDa). Shown are triplicate lanes from control and schizophrenic individuals.

mal processing of these proteins in schizophrenia. We therefore next analyzed protein expression of NF-L, PSD-93, PSD-95 and SAP102 in ACC and DLPFC. By Western blot analysis, we detected immunopositive bands corresponding to all four NMDA-related PSD molecules in both ACC and DLPFC. Figure 3b shows representative immunoblots for NF-L (B68 kDa), PSD-93 (B103–110 kDa), PSD-95 (B95 kDa) and SAP102 (B102 kDa), as well as for b-Tubulin (B55 kDa), which was used as internal control. The double-band detected for PSD-93, in accordance with earlier reports, likely represent different splice variants of this protein.62,63 Correlation analysis, using age, pH and PMI as independent measures, identified significant associations between PMI and NF-L expression in DLPFC (r = 0.50, P = 0.02) and PSD-93 and PMI in ACC (r = 0.45, P = 0.04). In addition, we found PSD-95 expression in ACC negatively associated with pH (r = 0.44, 0.048) (i.e., lower pH = increased PSD-95 concentration). As a negative association with pH is contrary to what one would predict (higher, more physiological pH = increased protein stability), we consider this association a likely

NMDA receptor subunit expression in schizophrenia Cortical expression of transcripts encoding NMDA receptor subunits has previously been studied in schizophrenia by in situ hybridization and quantitative real-time PCR.35,37 These studies found no changes in transcript expression for NR2A, NR2B and NR2D, a small but significant decrease for NR2C in the frontal cortex in schizophrenia, and identified increased transcript expression for NR2A and PSD-95 in occipital cortex.35 In addition, these studies reported either increased or unchanged expression of the NR1 transcript, which might be attributed to a difference in the prefrontal cortical area studied (Brodmann area 10 vs 46). For NR2A-D, our present protein data are consistent with previous reports of unaltered expression of these subunits at the transcriptional level. NR2C expression in PFC is generally quite low64 and a small decrease of this subunit mRNA, as reported previously, might not be detected 0 on the protein level. The observed increase in NR1C2 expression in ACC in the present work likely was detected as a result of differentiating between the two NR1 C2-terminal variants. With a specific increase of 0 approximately 32% of the NR1C2 subunit in a total pool consisting of two NR1 splice variants, it may be 0 less likely that the observed increase in NR1C2 would substantially alter the total pool of NR1 subunit in schizophrenia. Increased protein expression of this differentially spliced NR1 isoform in ACC may therefore be consistent with previous studies of unchanged NR1 transcript expression. A different ratio between 0 NR1C2 and NR1C2 splice variants in ACC suggests altered cell processing of the NMDA receptor in schizophrenia. An increase in the proportional avail0 ability in the ER of the NR1C2 variant, which is Molecular Psychiatry


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associated with increased NMDA receptor cell surface expression and different synaptic targeting,15,65 likely modulates cellular processing of the NMDA receptor including ER export and trafficking. An increase in the NR1C2 subunit in the ACC with its different, more rapid, ER-to-membrane trafficking, may be a cellular response to compromised function of glutamate receptor complexes at the cell membrane brought about by disturbed cellular receptor trafficking and interaction with associated PSD mediator proteins, partially associated with decreased NMDA receptor signaling. These possibilities would lend support to a hypoglutamatergic hypothesis of schizophrenia.66 However, from the present data it cannot be determined if the increased ratio of NR1C2 /NR1C2 in schizophrenia causes changes in ER assembly of the NMDA receptor, or whether this increased amount of NR1C2 in the complete NR1 pool, which is already expressed in surplus in the ER, is degraded through the endogenous ERAD mechanism.4,5 Future studies on how NR1 splice variants are recruited for receptor assembly or degradation in the ER are needed to further understand the implications of our finding. 0

0

0

Expression of NMDA-interacting PSD molecules in schizophrenia NMDA receptor subunit expression in schizophrenia has been analyzed in both cortical and subcortical structures, typically identifying only modest changes.38,67,68 It has therefore been suggested that rather than the receptor itself, expression of interacting PSD molecules may be abnormal in schizophrenia.1 We found increased expression of transcripts for NF-L in DLPFC, and PSD-95 and PSD-93 in ACC in schizophrenia. Interestingly, we found opposite, but matching changes in protein expression of these molecules with decreased expression of NF-L in DLPFC and PSD-95 and PSD-93 in ACC. SAP-102 was neither altered at transcriptional nor translational levels. NF-L, PSD-95 and PSD-93 are molecules that are involved in trafficking and stabilization of the NMDA receptor in the cell membrane, and that additionally connect the receptor to downstream signaling molecules in the postsynaptic cell.12,19,23,69,70 Abnormal expression of these proteins could affect NMDA receptor function. Interestingly, previous studies have reported transcript expression for these PSD molecules of opposite polarity, possibly depending on the neuroanatomical structure and subject characteristics of the cohort studied. Using the same cohort of elderly schizophrenic patients as used for the present study, increases in transcripts for NF-L, PSD-95 and SAP102 were found in the thalamus, associated with diminished NMDA receptor expression in this region.48 However, in a younger group of schizophrenic patients, decreased transcripts were measured for NF-L, PSD-95 and SAP102 in the thalamus, as well as for PSD-95 and SAP102 in the striatum.38,41 We have suggested that those contradictory findings represent Molecular Psychiatry

age-related differences in transcript expression, possibly owing to cellular adaptation to sustained abnormal protein processing. Analysis of protein expression might therefore be critical, in addition to transcript studies, to better understand the pathophysiology of schizophrenia. The divergence between transcript and protein expression in the present study suggests that increased cellular transcription might occur as a compensatory response to the primary loss of protein product and not as a compensatory result of a general diminished NMDA receptor expression. Decreased protein expression can result from several interacting causes such as failure of proper translation and protein folding in the ER–Golgi and/or increased protein turnover, possibly owing to abnormal posttranslational modifications (PTMs) of these proteins.71,72 Although decreased protein synthesis and compromised PTMs might generally be associated with older age,73 matching of subjects and correction of age effects in this study indicate that this is an effect related to schizophrenia rather than age per se. Thus, the present findings support the hypothesis of diminished excitatory glutamate signaling in schizophrenia74 brought about by abnormally expressed PSD proteins. A caveat of the present study is the relatively small overlap of subjects used for in situ and Western blot analyses (Table 1). Although both sample sets belong to the same cohort of elderly subjects, it is possible that our findings of increased transcript and decreased protein are a consequence of unrelated differences between the two groups. In an attempt to control for such within-group differences, we have compared independent variables (age, PMI and pH) between the two groups and found no significant difference (Table 1). Thus, despite the suboptimal comparison of transcripts and protein from different subgroups within the same patient cohort, comparison of age, PMI and pH support our interpretation that differences in transcript and protein alterations are primarily related to diagnosis. Functional implications of NMDA-related alterations The DLPFC and ACC are critical components of the brain circuitry underlying executive control and both are differentially affected in schizophrenia.75,76 Whereas the DLPFC is hypothesized to be primarily involved in performance initiation, ACC is mainly involved in cognitive control.77 Although altered transcript and protein expression have been reported for both areas, the changes reported here are primarily associated with ACC and could indicate that aspects of performance monitoring that involve NMDA receptor signaling are selectively impaired in ACC. In agreement with our finding of increased 0 NR1C2 expression, selective increase in NMDA receptor binding in the ACC has been reported in schizophrenia, which was hypothesized to be a cellular response to impaired glutamatergic neurotransmission.78


Implications for glutamatergic circuitry of the frontal cortex in schizophrenia Glutamatergic signaling in the frontal cortex is exerted principally by pyramidal neurons.43 Despite the fact that different cell types in all cortical layers express NMDA receptors,79 pyramidal cells account for approximately 70% of NMDA receptor expression in the frontal cortex.64,80 The primary afferent input to the frontal cortex is from thalamocortical projections, with synaptic endings in cortical layer IV.81 These projections to cortical layer IV likely interact primarily with non-NMDA receptor targets, as NMDA receptor expression is less abundant in layer IV than in layers II–III and V–VI.64 Owing to the layer-related differences in the expression of NMDA receptors in the frontal cortex, our observation of abnormal NMDA-related protein expression in schizophrenia therefore might suggest that the abnormalities of cortical NMDA-related circuitry primarily is of corticocortical nature. Through the complex connectivity of pyramidal neurons in the frontal cortex, consisting of layer II–III neurons projecting between cortical regions, the larger pyramidal neurons of layer V projecting to the thalamus, and layer VI neurons projecting to the hippocampus and striatum among other subcortical structures, the present findings of NMDA-related abnormalities in schizophrenia either directly or indirectly link with other areas affected in this psychiatric illness.43,82 In summary, this study found increased expression in ACC of an NR1 isoform associated with fast cell membrane kinetics. Additionally, we have found increased transcript and decreased protein expression of NF-L in DLPFC and PSD-95 and PSD-93 in ACC, suggesting abnormal cellular processing of the receptor, potentially involving receptor translation, transport and degradation. Altered cortical expression of the NMDA receptor and its interacting PSD molecules may affect corticocortical excitatory signaling, thus compromising cortical functioning in schizophrenia.

Acknowledgments This work was supported by MH53327 (Dr MeadorWoodruff) and MH45212 and MH064673 (Dr Haroutunian).

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