mRNA and protein expression for novel GABAA receptors [theta] and [rho]2 are altered in schizophrenia and mood disorders; relevance to FMRP-mGluR5 signaling pathway

Abstract

Fragile X mental retardation protein (FMRP) is an RNA-binding protein that targets ∼5% of all mRNAs expressed in the brain. Previous work by our laboratory demonstrated significantly lower protein levels for FMRP in lateral cerebella of subjects with schizophrenia, bipolar disorder and major depression when compared with controls. Absence of FMRP expression in animal models of fragile X syndrome (FXS) has been shown to reduce expression of gamma-aminobutyric acid A (GABA _A ) receptor mRNAs. Previous work by our laboratory has found reduced expression of FMRP, as well as multiple GABA_A and GABA_B receptor subunits in subjects with autism. Less is known about levels for GABA_A subunit protein expression in brains of subjects with schizophrenia and mood disorders. In the current study, we have expanded our previous studies to examine the protein and mRNA expression of two novel GABA_A receptors, theta (GABRθ) and rho 2 (GABRρ2) as well as FMRP, and metabotropic glutamate receptor 5 (mGluR5) in lateral cerebella of subjects with schizophrenia, bipolar disorder, major depression and healthy controls, and in superior frontal cortex (Brodmann Area 9 (BA9)) of subjects with schizophrenia, bipolar disorder and healthy controls. We observed multiple statistically significant mRNA and protein changes in levels of GABRθ, GABRρ2, mGluR5 and FMRP molecules including concordant reductions in mRNA and proteins for GABRθ and mGluR5 in lateral cerebella of subjects with schizophrenia; for increased mRNA and protein for GABRρ2 in lateral cerebella of subjects with bipolar disorder; and for reduced mRNA and protein for mGluR5 in BA9 of subjects with bipolar disorder. There were no significant effects of confounds on any of the results.

Citation: Transl Psychiatry (2013) 3, e271; doi:10.1038/tp.2013.46

& 2013 Macmillan Publishers Limited All rights reserved 2158-3188/13 http://www.nature.com/tp

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SH Fatemi1,2,3, TD Folsom1, RJ Rooney4 and PD Thuras5

Fragile X mental retardation protein (FMRP) is an RNA-binding protein that targets B5% of all mRNAs expressed in the brain. Previous work by our laboratory demonstrated signicantly lower protein levels for FMRP in lateral cerebella of subjects with schizophrenia, bipolar disorder and major depression when compared with controls. Absence of FMRP expression in animal models of fragile X syndrome (FXS) has been shown to reduce expression of gamma-aminobutyric acid A (GABAA) receptor mRNAs. Previous work by our laboratory has found reduced expression of FMRP, as well as multiple GABAA and GABAB receptor subunits in subjects with autism. Less is known about levels for GABAA subunit protein expression in brains of subjects with schizophrenia and mood disorders. In the current study, we have expanded our previous studies to examine the protein and mRNA expression of two novel GABAA receptors, theta (GABRh) and rho 2 (GABRq2) as well as FMRP, and metabotropic glutamate receptor 5 (mGluR5) in lateral cerebella of subjects with schizophrenia, bipolar disorder, major depression and healthy controls, and in superior frontal cortex (Brodmann Area 9 (BA9)) of subjects with schizophrenia, bipolar disorder and healthy controls. We observed multiple statistically signicant mRNA and protein changes in levels of GABRh,

GABRq2, mGluR5 and FMRP molecules including concordant reductions in mRNA and proteins for GABRh and mGluR5 in lateral cerebella of subjects with schizophrenia; for increased mRNA and protein for GABRq2 in lateral cerebella of subjects with bipolar disorder; and for reduced mRNA and protein for mGluR5 in BA9 of subjects with bipolar disorder. There were no signicant effects of confounds on any of the results.

Translational Psychiatry (2013) 3, e271; doi:http://dx.doi.org/10.1038/tp.2013.46

Web End =10.1038/tp.2013.46 ; published online 18 June 2013

Introduction

Impairment of the gamma-aminobutyric acid (GABA) signaling system is believed to partially account for behavioral and cognitive decits associated with schizophrenia and mood disorders.1,2 Reduction of GABAA mediated signal transmission has also been associated with anxiety, panic, impaired learning and memory.35 GABAA receptors are responsible for mediating the fast inhibitory action of GABA,6 and are important sites for clinical action of a number of drugs including benzodiazepines, barbiturates and anesthetics.Recent work has suggested that proper GABAergic neuro-transmission is required for network oscillations that facilitate the processing of information both in and between various brain regions and that this may be required for normal cognition.1 Altered expression of GABAA receptor subunits could impair these oscillations and result in improper cognitive function. Little is currently known about GABAA receptor subunit expression in schizophrenia and mood disorders, although it is likely that changes in GABAA receptor expression would result in reduced GABAergic transmission.

Keywords: bipolar disorder; FMRP; GABRr2; GABRy; mGluR5; schizophrenia

mRNA and protein expression for novel GABAA receptors h and q2 are altered in schizophrenia and mood disorders; relevance to FMRP-mGluR5 signaling pathway

Recent evidence79 provides a linkage between GABA neurotransmission and fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that has been estimated to regulate translation of 842 transcripts in the brain.10 In animal models of fragile X syndrome (FXS), the absence of FMRP is accompanied by reduced mRNA expression of GABAA receptor subunits including alpha 1 (a1), a3, a4, beta 1 (b1), b2, delta (d), gamma 1 (g1) and g2 in frontal cortex, whereas there was no change in the cerebellum.79 A functional consequence of this reduced expression has been observed in fragile X mental retardation 1 (Fmr1)-knocked out mice that display impaired GABAergic signaling in striatal neurons, as measured by increased frequency of spontaneous and miniature inhibitory post-synaptic currents and reduced paired pulse ratio of inhibitory postsynaptic currents.11

FMRP normally represses metabotropic glutamate receptor 5 (mGluR5) signaling, whereas the absence of FMRP has been hypothesized to lead to unregulated mGluR5 signaling, and ultimately result in the various abnormal phenotypes

1Division of Neuroscience Research, Department of Psychiatry, University of Minnesota Medical School, Minneapolis, MN, USA; 2Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN, USA; 3Department of Neuroscience, University of Minnesota Medical School, Minneapolis, MN, USA;

4Genome Explorations, Memphis, TN, USA and 5VA Medical Center, Department of Psychiatry, Minneapolis, MN, USACorrespondence: Dr SH Fatemi, Division of Neuroscience Research, Department of Psychiatry, University of Minnesota Medical School, 420 Delaware St SE, MMC 392, Minneapolis, MN 55455, USA.

E-mail: mailto:[email protected]

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Received 27 February 2013; revised 11 April 2013; accepted 22 April 2013

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Materials and methods

Brain procurement. The Institutional Review Board of the University of MinnesotaSchool of Medicine has approved this study. Post-mortem lateral cerebella were obtained from the Stanley Foundation Neuropathology Consortium under approved ethical guidelines. Post-mortem superior frontal cortex (BA9) was obtained from the McLean 74 Cohort, Harvard Brain and Tissue Resource Center. DSM-IV diagnoses were established prior to death by psychiatrists using information from all available medical records and family interviews. Details regarding the subject selection, demographics, diagnostic process and tissue processing were collected by the Stanley Medical Research Foundation, and the Harvard Brain and Tissue Resource Center. The Stanley collection consisted of 15 subjects with schizophrenia, 15 with bipolar disorder, 14 with major depression without psychotic features and 14 normal controls (Table 1). The McLean 74 Cohort consists of 20 subjects with schizophrenia, 19 with bipolar disorder and 29 normal controls (Table 2).All groups were matched for age, sex, race, post-mortem interval and hemispheric side.

Sodium dodecyl sulfate polyacrylamide gel electro phoresis and western blotting. Brain tissue was prepared as previously described.18,2328 For lateral cerebellum, 60 mg

of tissue was used, whereas for BA9, 30 mg of tissue was used. For mGluR5 and FMRP, we used 6% resolving gels, whereas for GABRy, GABRr2, neuronal-specic enolase (NSE) and b-actin we used 10% resolving gels. We minimized interblot variability by including samples from subjects of each group (control, schizophrenia, bipolar disorder and major depression) on each gel. Samples were run in duplicate. Samples were electrophoresed for 15 min at75 V, followed by 55 min at 150 V. Samples were then electroblotted onto nitrocellulose membranes for 2 h at 300 mAmp at 4 1C. Blots were blocked with 0.2% I-Block (Tropix, Bedford, MA, USA) in phosphate-buffered saline with 0.3% Tween 20 for 1 h at room temperature (RT), followed by an overnight incubation in primary antibodies at 4 1C. The primary antibodies used were anti-GABRy

Table 1 Demographic information for the four diagnostic groups from Stanley Medical Research Institute

Bipolar Control Schizophrenia Depression F or w2 P

Age 42.33 (11.72) 46.64 (9.46) 44.53 (13.11) 46.57 (9.66) 0.49a 0.69 Sex 6F, 9M 5F, 9M 6F, 9M 6F, 8M 0.15b 0.99 Race 14W, 1B 13W, 1B 12W, 3A 14W 14.12b 0.12 PMI 32.53 (16.12) 24.5 (9.85) 33.67 (14.62) 27.57 (11.13) 1.51a 0.22 pH 6.18 (0.23) 6.26 (0.25) 6.16 (0.26) 6.18 (0.23) 0.51a 0.68 Side of brain 7L, 8R 7L, 7R 9L, 6R 9L, 5R 1.20b 0.75 Brain weight 1441.2 (171.5) 1511 (165.4) 1471.7 (108.2) 1443.57 (127.56) 0.71a 0.55 Family hx 0.93 (0.8) 0.13 (0.52) 1.13 (0.83) 0.73 (0.46) 27.19b 0.0001 Suicidal death 9 (5 Violent) 0 6 (2 Violent) 9 (4 Violent) 14.04b 0.029 Drug/Alc hx 0.8 (0.77) 0.36 (0.74) 0.53 (0.74) 0.43 (0.64) 5.08b 0.45 Age of onset 21.47 (8.35) 23.2 (7.96) 33.36 (13.68) 3.63a 0.001 Duration of illness 20.13 (9.67) 21.67 (11.24) 12.29 (11.37) 2.47a 0.018 Severity of substance abuse 1.93 (1.98) 0.14 (0.54) 1.20 (1.86) 1.15 (2.04) 22.21b 0.10 Severity of alcohol abuse 2.27 (1.98) 1.14 (1.03) 1.47 (1.59) 1.93 (2.02) 9.52b 0.85 Fluphenazine (lifetime) 20 826.67 (24 015.96) 52 266.67 (62 061.57) 6.21a 0.019

aANOVA. bw2 test.

Bold values indicate signicant (Po0.05) values.

associated with FXS.12 Animal studies using antagonists of the mGluR5 receptor have rescued learning and behavioral decits associated with FXS, and reduced seizures in FMR1-knock out mice.1317

Recently, we reported on reduced protein expression of FMRP in lateral cerebellum from subjects with schizophrenia, bipolar disorder and major depression.18 These results are novel, as gene association studies have not identied fragile X mental retardation 1 (FMR1), the gene that codes FMRP, as a candidate gene for schizophrenia.1920 However, a recent study veried our earlier study, nding reduced FMRP expression in peripheral blood lymphocytes from subjects with schizophrenia.21 Kovcs et al.21 found that age of onset and IQ predicted FMRP levels, but chlorpromazine-equivalent antipsychotic dose did not. Importantly, none of the study subjects showed the CGG triplet expansion that normally causes silencing of the FMR1 gene in subjects with FXS.21

Combined with our ndings of reduced FMRP expression in the cerebellar vermis and prefrontal cortex of subjects with autism,2223 who were not comorbid for FXS, reduction of

FMRP expression may be a hallmark of multiple psychiatric disorders.

Based on the evidence from animal models that decrease in FMRP expression results in reduced expression of GABAA receptor subunit mRNA and our nding of signicantly reduced FMRP in cerebella of subjects with schizophrenia and mood disorders,18 we hypothesized that we would observe reduced expression of the GABAA receptor subunits in lateral cerebella from the same diagnostic groups. An initial screen of several GABA receptor subunits in lateral cerebella of subjects with schizophrenia, bipolar disorder and major depression found reductions in GABAB receptor subunits one and two (GABBR1 and GABBR2).24 Here, we report novel ndings regarding alterations in levels of mRNA and protein for GABAA receptor theta (GABRy) and GABAA receptor rho 2 (GABRr2), as well as mGluR5 and FMRP levels in the lateral cerebellum and Brodmann Area 9 (BA9) of subjects with schizophrenia and mood disorders. These results demonstrate the disruption of the GABAergic and FMRP-mGluR5 signaling systems in subjects with schizophrenia and mood disorders.

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Table 2 Demographic information for the three diagnostic groups from the McLean 74 Cohort

Bipolar Control Schizophrenia F, t or w2 P

Age 61.75 (19.20) 58.59 (15.12) 60.71 (12.07) 0.24a 0.79 Sex 4M:15F 17M:12F 14M:6F 10.38b 0.006 PMI 22.25 (5.39) 21.76 (3.85) 23.86 (7.20) 0.92a 0.41 pH 6.45 (0.76) 6.45 (0.17) 6.49 (0.30) 0.07a 0.93 Side of brain 10L:9R 14L:15R 10L:10R 0.087b 0.96 Suicidal death 4 (1 violent) 0 0 10.96b 0.027 Drug/Alc hx 0.42 (0.51) 0.69 (0.66) 0.30 (0.47) 6.84b 0.15 Severity of substance abuse 0.58 (1.39) 0.41 (0.95) 0.70 (1.45) 0.33b 0.72 Severity of alcohol abuse 0.95 (1.43) 0.86 (1.73) 0.60 (1.23) 0.39b 0.75 Age of onset 23.53 (7.67) 20.52 (3.52) 1.45c 0.16 Duration of illness 39.69 (18.27) 40.18 (12.09) 0.091c 0.93 Use of MS 9 1 9.17b 0.002

Abbreviation: MS, mood stabilizer.

aANOVA. bw2 test. ct-test for bipolar versus schizophrenia. Bold values indicate signicant (Po0.05) values.

(ab49188, Abcam (Cambridge, MA, USA) 1:1000), anti-GABRr2 (ab83223, Abcam, 1:500), anti-FMRP (MAB2160,

Millipore (Temecula, CA, USA), 1:500), anti-mGluR5 (ab53090, Abcam, 1:300), anti-NSE (1:2000; Abcam) and anti-b actin (A5441, Sigma Aldrich (St Louis, MO, USA), 1:5 000). Blots were washed for 30 min in phosphate-buffered saline supplemented with 0.3% Tween 20 (PBST) for 30 min at RT, and were subsequently incubated in the proper secondary antibodies. Secondary antibodies were goat anti-mouse IgG (A9044, Sigma Aldrich, 1:80 000) and goat anti-rabbit IgG (A9169, Sigma Aldrich, 1:80 000). Blots were washed twice in PBST for 15 min each. Following the second wash, bands were visualized using the ECL-plus detection system (GE Healthcare, Buckinghamshire, UK) and exposed to CL-Xposure lm (Thermo Scientic, Rockford, IL, USA). The molecular weights of B224 (dimer) and 112 kDa (monomer) for mGluR5; 73 kDa (FMRP); 70 kDa (GABRy); 54 kDa (GABRr2); 46 kDa (NSE) and 42 kDa (b-actin) immunoreactive bands were quantied with background subtraction using a Bio-Rad GS-800 Calibrated

Densitometer (Bio-Rad, Hercules, CA, USA) and Quantity One 1-D Analysis software (Bio-Rad). Sample densities were analyzed blind to nature of diagnosis. Results obtained are based on at least two independent experiments.

Quantitative real-time polymerase chain reaction (qRTPCR). We performed qRT-PCR as previously described.28

Raw data were analyzed as previously described28 using the Sequence Detection Software RQ Manager (ABI, Foster City, CA, USA), whereas relative quantitation using the comparative threshold cycle (CT method) was performed in

Bioconductor using the ABqPCR package in Microsoft Excel (ABI Technote no. 2: Relative Gene Expression Quantitation). Calculations were done assuming that 1 delta CT equals a two-fold difference in expression.

Signicance values were determined using unpaired t-tests. The probe IDs used were: (1) GABAA receptor theta (GABRQ): Hs00610921_m1; (2) GABAA receptor rho 2 (GABRR2): Hs00266703_m1; (3) fragile X mental retardation 1 (FMR1): Hs00924547_m1; (4) metabotropic glutamate 5 (GRM5): Hs00168275_m1; (5) beta actin: Hs99999903_m1 and (6) glyceraldehyde 3-phosphate dehydrogenase (GAPDH): Hs99999905_m1.

Statistical analysis. All protein measurements for each group were normalized against b-actin and NSE, and expressed as ratios of GABRy/b-actin, GABRr2/b-actin,

FMRP/b-actin, mGluR5/b-actin, GABRy/NSE, GABRr2/NSE, FMRP/NSE and mGluR5/NSE. Statistical analysis was performed as previously described,18,23,25 with Po0.05

considered signicant. Group comparisons were conducted using analysis of variance (ANOVA). Follow-up independent t-tests were then conducted if the results were signicant. Group differences on possible confounding factors were explored using w2 tests for categorical variables, and ANOVA for continuous variables. Where group differences were found, analysis of covariance was used to explore these effects on group differences for continuous variables, and factorial ANOVA with interaction terms for categorical variables. All analyses were conducted using SPSS v.17 (SPSS, Chicago, IL, USA).

Results

Western blotting results for GABRh, GABRq2, FMRP and mGluR5 in the lateral cerebellum. All protein measurements were normalized against b-actin or NSE. In lateral cerebella, ANOVA identied group differences for GABRy/b-actin (F(3,48) 5.49, Po0.003), GABRy/NSE

(F(3,48) 5.61, Po0.002), GABRr2/b-actin (F(3,40) 2.90,

Po0.047), GABRr2/NSE (F(3,40) 3.05, Po0.039),

mGluR5 monomer/b-actin (F(3,46) 4.15, Po0.011) and

mGluR5 monomer/NSE (F(3,46) 5.18, Po0.004)

(Figure 1; Table 3). There was a group difference for FMRP/NSE (F(3,50) 4.93, Po0.004) (Figure 1; Table 3).

Follow-up t-tests found signicant reductions in protein for GABRy/b-actin (Po0.001), GABRy/NSE (Po0.001), mGluR5 monomer/b-actin (Po0.050), mGluR5 monomer/

NSE (Po0.030) and FMRP/NSE (Po0.001) in subjects with schizophrenia (Table 3; Figures 2 and 3). In lateral cerebella from subjects with bipolar disorder, there were signicant reductions in GABRy/b-actin (Po0.012), GABRy/NSE (Po0.005), mGluR5 monomer/b-actin (Po0.001), mGluR5 monomer/NSE (Po0.004) and FMRP/NSE (Po0.003), and signicant increased expression of GABRr2/b-actin (Po0.0044) and GABRr2/NSE (Po0.009) (Table 3;

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Figure 1 Representative bands for mGluR5, FMRP, GABRy, GABRr2, NSE and b-actin in lateral cerebellum (a) and BA9 (b) of subjects with schizophrenia and mood disorders. B, bipolar disorder; C, control; D, major depression; S, schizophrenia. FMRP and b-actin images for lateral cerebellum reprinted from Fatemi et al.,18 with permission from Elsevier.

Table 3 Western blotting results for FMRP, GABRy, mGluR5 and GABRr2 values expressed as ratios to b-actin and neuronal-specic enolase (NSE) in lateral cerebellum

ANOVA Control Schizophrenia Bipolar disorder Major depression

F P Protein P Protein P Protein P Protein P

GABRy/b-actin 5.49 0.003 0.4250.105 RG 0.2310.136 0.001 0.2970.090 0.012 0.3010.138 0.014 GABRr2/b-actin 2.90 0.047 0.0230.013 RG 0.0480.042 NC 0.0470.014 0.0044 0.0580.038 0.0085 mGluR5 dimer/b-actin 2.14 NC 0.1760.111 RG 0.1250.10 NC 0.1270.101 NC 0.2110.064 NC mGluR5 monomer/b-actin 4.15 0.011 0.0380.030 RG 0.0150.023 0.05 0.00860.0098 0.001 0.0190.020 NC

FMRP/b-actina 6.22 0.001 0.0700.052 RG 0.0170.029 0.039 0.0290.033 0.014 0.0230.021 0.005 b-actin 0.83 NC 25.82.07 RG 25.22.07 NC 26.92.91 NC 26.14.38 NC

GABRy/NSE 5.61 0.002 0.870.18 RG 0.480.30 0.001 0.580.20 0.005 0.620.24 0.012 GABRr2/NSE 3.05 0.039 0.0450.025 RG 0.10.094 NC 0.0960.052 0.009 0.130.085 0.006 mGluR5 dimer/NSE 1.99 NC 0.320.20 RG 0.260.23 NC 0.260.22 NC 0.440.12 NC mGluR5 monomer/NSE 5.18 0.004 0.080.48 RG 0.0320.049 0.03 0.0170.021 0.004 0.0410.041 0.047 FMRP/NSE 4.93 0.004 0.0920.07 RG 0.0230.04 0.001 0.0420.05 0.003 0.0330.033 0.001 NSE 0.93 NC 20.23.34 RG 20.11.67 NC 20.23.24 NC 21.62.87 NC

Abbreviations: ANOVA, analysis of variance; NC, no change; RG, reference group.

aFMRP data reprinted from Schizophrenia Research, 124(13):246-247, Fatemi, S.H., Kneeland, R.E., Liesch, S.B., Folsom, T.D., Fragile X mental retardation protein levels are decreased in major psychiatric disorders, page 246, Copyright (2010), with permission from Elsevier.

Bold values indicate signicant (Po0.05) values.

Figures 2 and 3). In subjects with major depression, follow-up t-tests found signicant reductions in GABRy/b-actin (Po0.014), GABRy/NSE (Po0.012), mGluR5 monomer/

NSE (Po0.047) and FMRP/NSE (Po0.001), and signicantly increased expression of GABRr2/b-actin (Po0.0085) and

GABRr2/NSE (Po0.006) (Table 3; Figures 2 and 3). There were no signicant changes in protein levels of mGluR5 dimer in lateral cerebella.

Western blotting results for GABRh, GABRq2, FMRP and mGluR5 in BA9. In BA9, ANOVA identied group differences for GABRy/b-actin (F(2,64) 4.04, Po0.022),

GABRy/NSE (F(2,62) 4.54, Po0.014), mGluR5 monomer/

b-actin (F(2,63) 10.72, Po0.001), mGluR5 monomer/NSE

(F(2,63) 8.14, Po0.001), FMRP/b-actin (F(2,59) 3.85,

Po0.027) and FMRP/NSE (F(2,60) 4.26, Po0.019)

(Figure 1; Table 4). Follow-up t-tests found signicant reductions of GABRy/b-actin, mGluR5 monomer/b-actin and FMRP/b-actin (Po0.017, Po0.001 and Po0.018, respectively), and GABRy/NSE, mGluR5 monomer/NSE and FMRP/NSE (Po0.019, Po0.003 and Po0.029, respectively) in BA9 of subjects with schizophrenia (Table 4,

Figures 4 and 5). In subjects with bipolar disorder, follow-up t-tests found signicant reductions in GABRy/b-actin,

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Figure 2 Expression of GABRy/b-actin (a), GABRr2/b-actin (b), mGluR5 dimer/b-actin (c), mGluR5 monomer/b-actin (d), FMRP/b-actin (e), and b-actin (f) in lateral cerebella of healthy controls versus subjects with bipolar disorder, major depressive disorder and schizophrenia. Histogram bars shown as means.e., *Po0.05. FMRP and b-actin data reprinted from Fatemi et al.,18 with permission from Elsevier.

mGluR5 monomer/b-actin and FMRP/b-actin (Po0.024, Po0.001 and Po0.030, respectively), and GABRy/NSE, mGluR5 monomer/NSE and FMRP/NSE (Po0.011,

Po0.001 and Po0.011, respectively) (Table 4; Figures 4 and 5). There were no signicant changes in protein levels for GABRr2 or mGluR5 dimer in BA9.

Analysis of confounds for protein data in lateral cerebellum and BA9. In the analysis of protein data from lateral cerebella, no signicant differences were found between groups on hemisphere side, ethnicity, gender, history of substance abuse, severity of alcohol abuse or substance abuse, post-mortem interval, age, pH or brain weight (Table 1). We also compared the groups on family history and suicide, and found signicant differences (Po0.0001 and Po0.029, respectively), but further analysis revealed that these factors had no signicant impact on any of the results. We did nd that subjects with schizophrenia and bipolar disorder had signicantly longer duration of illness than did those with depression

(t(47) 2.47, Po0.018). Age of onset was signicantly later

for subjects with major depression compared to subjects with schizophrenia or bipolar disorder (t(41) 3.63, Po0.001).

ANOVA controlling for age of onset and duration of illness did nd that subjects with depression displayed signicantly higher mGluR5 dimer/NSE (F(2,31) 4.31, Po0.02) and

mGluR5 dimer/b-actin (F(2,31) 4.31, Po0.02) than sub

jects with bipolar disorder or schizophrenia while controlling for duration. However, as mGluR5 dimer values did not change signicantly between the groups, this nding is not meaningful.

For protein data from BA9, no signicant differences were found between diagnostic groups on hemisphere side, history of substance abuse, severity of alcohol abuse or substance abuse, post-mortem interval, age or pH (Table 2). Nor did we nd signicant differences on use of barbiturates, opiates, amphetamines, cocaine or propoxyphene. We also compared subjects with schizophrenia versus subjects with bipolar disorder on disease duration, age of onset, use of anti-psychotic, antidepressant and anticonvulsant medications,

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Figure 3 Expression of GABRy/NSE (a), GABRr2/NSE (b), mGluR5 dimer/NSE (c), mGluR5 monomer/NSE (d), FMRP/NSE (e), and NSE (f) in lateral cerebella of healthy controls versus subjects with bipolar disorder, major depressive disorder and schizophrenia. Histogram bars shown as means.e., *Po0.05.

Table 4 Western blotting results for FMRP, GABRy, mGluR5 and GABRr2 values expressed as ratios to b-actin and neuronal-specic enolase (NSE) in BA9

ANOVA Control Schizophrenia Bipolar disorder

F P Protein P Protein P Protein P

GABRy/b-actin 4.04 0.022 1.340.40 RG 1.060.32 0.017 1.070.44 0.024 GABRr2/b-actin 0.38 NC 0.230.17 RG 0.210.14 NC 0.260.23 NC mGluR5 dimer/b-actin 1.39 NC 0.780.38 RG 0.570.42 NC 0.700.49 NC mGluR5 monomer/b-actin 10.72 0.001 0.190.13 RG 0.0890.058 0.001 0.0710.042 0.001

FMRP/b-actin 3.85 0.027 0.810.40 RG 0.540.40 0.018 0.560.29 0.030 b-actin 0.19 NC 7.061.68 RG 7.311.23 NC 7.291.82 NC GABRy/NSE 4.54 0.014 1.510.40 RG 1.230.40 0.019 1.200.36 0.011

GABRr2/NSE 0.54 NC 0.260.20 RG 0.230.14 NC 0.300.27 NC mGluR5 dimer/NSE 0.66 NC 0.900.59 RG 0.720.67 NC 0.740.50 NC mGluR5 monomer/NSE 8.14 0.001 0.220.17 RG 0.110.07 0.003 0.0860.052 0.001 FMRP/NSE 4.26 0.019 1.050.55 RG 0.710.50 0.029 0.640.36 0.011 NSE 0.01 NC 6.481.79 RG 6.542.27 NC 6.442.61 NC

Abbreviations: ANOVA, analysis of variance; NC, no change; RG, reference group.

Bold values indicate signicant (Po0.05) values.

and found no signicant differences. We did nd that 21.1% of bipolar patients died by suicide versus none for the other diagnostic groups (w2(2) 10.96, Po0.027). We also found

that there were signicantly more female subjects (w2(2) 10.38, Po0.006) in the bipolar group (78.9%) than

in either normal controls (41.4%) or subjects with

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Figure 4 Expression of GABRy/b-actin (a), GABRr2/b-actin (b), mGluR5 dimer/b-actin (c), mGluR5 monomer/b-actin (d), FMRP/b-actin (e) and b-actin (f) in BA9 of healthy controls versus subjects with bipolar disorder and schizophrenia. Histogram bars shown as means.e., *Po0.05.

schizophrenia (30%). We also found higher rates of mood stabilizer use in patients with bipolar disorder (47.4%) than in patients with schizophrenia (5%) (w2(1) 9.17,

Po0.002). Further analyses controlling for gender, mood stabilizer use and suicide found the initial differences on outcome measures as a function of diagnostic groups to be unchanged.

qRT-PCR results for GABRh, GABRq2, FMRP and mGluR5 in lateral cerebellum and BA9. For qRT-PCR experiments, all values were normalized against both b-actin and GAPDH, and these values were averaged. In the lateral cerebella, ANOVA identied group differences for GABRQ (GABRy; Po0.046), GABRR2 (GABRr2; Po0.017) and

GRM5 (mGluR5; Po0.034) (Table 5). There were signicantly reduced mRNA values for GABRQ (Po0.016) and

GRM5 (Po0.039) in the lateral cerebella of subjects with schizophrenia (Table 5), similar to protein changes in the same region. GABRR2 mRNA was signicantly increased

(Po0.019) in the lateral cerebella in subjects with bipolar disorder, mirroring similar changes in protein levels, and

GRM5 mRNA expression was signicantly reduced (Po0.009) in subjects with major depression (Table 5). In

BA9, ANOVA identied group differences for GABRR2 (Po0.003) and GRM5 (Po0.048) (Table 5). In BA9 of subjects with schizophrenia, there was signicantly increased mRNA for GABRQ (Po0.03). In BA9 of subjects with bipolar disorder, there was signicantly increased mRNA for GABRR2 (Po0.0001) and signicantly reduced mRNA for GRM5 (Po0.04), similar to changes in mGluR5 protein levels in the same region (Table 5). FMR1 mRNA values did not show signicant changes in either of the brain areas (Table 5).

Discussion

The current studies demonstrate abnormal processing of mRNA and protein expression for two novel GABAA

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Figure 5 Expression of GABRy/NSE (a), GABRr2/NSE (b), mGluR5 dimer/NSE (c), mGluR5 monomer/NSE (d), FMRP/NSE (e) and NSE (f) in BA9 of healthy controls versus subjects with bipolar disorder and schizophrenia. Histogram bars shown as means.e., *Po0.05.

receptors, y and r2, as well as FMRP and mGluR5 in lateral cerebella and BA9 of subjects with schizophrenia and mood disorders. The most salient results included: (1) FMRP protein levels were signicantly decreased in all the brain sites in schizophrenia, bipolar disorder and major depression; (2) mGluR5 protein levels were signicantly reduced in all the brain sites in schizophrenia and bipolar disorder; (3) mRNA levels for mGluR5 were signicantly reduced in lateral cerebellum of subjects with schizophrenia and major depression, and BA9 of subjects with bipolar disorder; (4) Protein levels for GABRy were reduced signicantly in all the brain sites in schizophrenia, bipolar disorder and major depression;

(5) mRNA levels for GABRy were elevated signicantly in BA9 of subjects with schizophrenia, in contrast mRNA for the same receptor was decreased signicantly in lateral cerebellum of subjects with schizophrenia; (6) Protein levels for GABRr2 were increased signicantly in lateral cerebellum of subjects with bipolar disorder and major depression; simultaneously,

mRNA for the same receptor was also increased signicantly in all the brain sites in subjects with bipolar disorder.

The GABRy gene (GABRQ) is clustered with GABAA receptor epsilon (GABRE) and GABAA receptor alpha 3 (GABRA3) at Xq28.29 In rat, GABRy mRNA has been shown embryonically (E17/E19) to localize to the hypothalamus, tegmentum, pontine nuclei and medulla, suggesting a possible role in midbrain development.30 GABRy mRNA is expressed in multiple brain regions in human including amygdala, dorsal raphe, hippocampus, hypothalamus, locus coeruleus and substantia nigra.31 The locus coeruleus is relevant to psychiatric disorders, as it is the largest nora-drenergic nucleus and has important roles in the regulation of anxiety states, vigilance, attention and memory functions.32

GABRy forms a functional receptor when coexpressed with alpha, beta and gamma subunits.31 The functional properties of GABAA receptors that include the y subunit have not been well characterized.30

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Table 5 qRT-PCR results for GABRQ, GABRR2, GRM5 and FMR1 in lateral cerebella, and BA9 of subjects with schizophrenia and mood disorders

Lateral cerebellum Schizophrenia Bipolar disorder Major depression

Gene ANOVA Fold change P Fold change P Fold change P

GABRQ 0.046 0.64 0.016 1.25 0.47 0.730 0.11 GABRR2 0.017 1.33 0.11 1.58 0.019 1.019 0.91 GRM5 0.034 0.49 0.039 0.80 0.39 0.530 0.009 FMR1 0.099 0.65 0.16 1.06 0.85 0.606 0.052

BA9 Schizophrenia Bipolar disorder Major depression

Gene ANOVA Fold change P Fold change P

GABRQ 0.075 1.43 0.03 1.03 0.78 NTA GABRR2 0.003 1.21 0.32 2.10 0.0001GRM5 0.048 1.05 0.68 0.77 0.04FMR1 0.705 0.88 0.31 0.96 0.68

Abbreviations: ANOVA, analysis of variance; FMR1, fragile X mental retardation 1; NTA, no tissue available; qRT-PCR, quantitative real-time polymerase chain reaction.

Note: for lateral cerebella, ANOVA is based on six comparisons: C versus S, C versus B, C versus D, S versus B, S versus D and B versus D.

For BA9, ANOVA based on three comparisons: C versus S, C versus B and S versus B.

Bold values indicate signicant (Po0.05) values.

Recent studies have investigated possible associations of the gene that codes for GABRy (GABRQ) with multiple disorders.3336 However, single-nucleotide polymorphisms of

GABRQ were not associated with susceptibility to bipolar disorder,3334 migraine35 or essential tremor.36 However, the GABRQ-478F allele showed an association with the improvement of tremor with ethanol use among men.36

The levels of GABRy receptor protein are reduced signicantly in both BA9 and lateral cerebellum of the subjects with schizophrenia (Figure 6). In contrast, mRNA for GABRy receptor is downregulated in the lateral cerebella, whereas in

BA9 its mRNA is upregulated (Figure 6). As both mRNA and protein are concordantly downregulated in the lateral cerebellum, a severe chronic receptor decit may be responsible for our observed results; while in BA9, increased mRNA expression may be a compensatory response to chronic receptor downregulation, suggesting that different mechanisms may be at work (Figure 6).

In subjects with bipolar disorder, GABRy receptor protein is reduced in the lateral cerebellum while its mRNA is upregulated (Figure 7). By the same token, protein for this receptor is downregulated in BA9, with its mRNA level unchanged (Figure 7). Here, the mechanisms for these alterations may again be different in the two brain sites. In lateral cerebellum, chronic GABRy protein downregulation could lead to upregulation of its mRNA in a feedback loop. In

BA9, normal mRNA levels with decreased protein levels indicate a defective step either in processing of protein in rough endoplasmic reticulum or subsequent cell compartments (such as Golgi or secretory granules), leading to reduced protein production (Figure 7).

In subjects with major depression, although protein levels for GABRy are reduced signicantly in lateral cerebellum, its mRNA levels remain normal (Figure 8). This scenario again indicates that the decit lies at rough endoplasmic reticulum or a subsequent cellular compartment causing the chronic receptor protein downregulation (Figure 8). In the absence of available BA9 tissue, the fate of GABRy in major depression will await future determinations.

The gene that codes for GABRr2 (GABRR2) is localized to 6q15.37 GABRr2 mRNA is widely distributed in the brain, including prefrontal cortex, hippocampus and cerebellum.38,39

In adult rat cerebellum, GABRr2 has been localized to

Figure 6 Summary of mRNA and protein expression for GABRy, GABRr2, mGluR5 and FMRP in lateral cerebella and BA9 of subjects with schizophrenia.

Concordant results for mRNA and protein were obtained for GABRy and mGluR5 in lateral cerebellum. Decreased expression of GABRy protein in BA9 may lead to a positive feedback loop, increasing mRNA expression. Protein levels for mGluR5 and FMRP were reduced signicantly in both brain sets. m, increased expression; k, reduced expression, , no change.

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Purkinje cells and basket-like cells only.40 GABRr2 has been shown to associate with a1 and g2 receptor subunits.41

In cerebellum, GABAA receptors that include the r2 subunit help mediate a component of phasic inhibitory GABAergic transmission at interneuronPurkinje cell synapses.42

A recent study has demonstrated an association between an single-nucleotide polymorphism of GABRR2 (GABRr2)

(rs1570932) and a component of the bipolar phenotype, namely bipolar patients with psychotic symptoms, similar to those experienced by subjects with schizophrenia.33 A

second study found an single-nucleotide polymorphism (rs12201676) associated with bipolar disorder that is anked by GABRR1 (15 kb away) and GABRR2 (17 kb away) genes.43 GABRR2 has also been associated with alcoholism.44

Levels of GABRr2 mRNA and protein did not change in lateral cerebellum or BA9 of subjects with schizophrenia (Figure 6). However, in subjects with bipolar disorder, a concordant and signicant increase was observed in mRNA and protein levels of GABRr2 in lateral cerebellum, indicating chronic upregulation in gene and protein product in this disorder (Figure 7). Interestingly, in BA9 of bipolar subjects, GABRr2 mRNA levels were also elevated signicantly, but no protein change was observed (Figure 7). In major depression,

GABRr2 protein levels were also elevated but without any change in mRNA, indicating abnormalities in processing

GABRr2 protein in rough endoplasmic reticulum compartment or subsequent cellular stations (Figure 8). Thus, GABRr2 changes were conned to brains of subjects with mood disorders and were not seen in schizophrenia and, at least in the case of bipolar disorder, reect upregulation of GABRr2 mRNA/protein.

The FMR1 gene is located at Xq27.3. FMRP has been shown to localize in multiple regions of neurons, including the soma, dendrites, synaptic spines and the axon.11,45,46 FMRP

controls multiple post-transcriptional events, including splicing, nuclear export and translation.46,47 FMRP protein is

signicantly downregulated in schizophrenia, bipolar disorder and major depression in lateral cerebellum,18 and in BA9 for subjects with schizophrenia and bipolar disorder in the absence of any mRNA abnormalities (Figures 68). This

Figure 7 Summary of mRNA and protein expression for GABRy, GABRr2, mGluR5 and FMRP in lateral cerebella and BA9 of subjects with bipolar disorder.

Concordant results for mRNA and protein were obtained for GABRr2 in lateral cerebellum. Decreased expression of GABRy protein in lateral cerebellum may lead to a positive feedback loop, increasing mRNA expression. Protein levels for GABRy, mGluR5 and FMRP were decreased signicantly in both brain sites. m, increased expression; k, reduced expression, , no change.

Figure 8 Summary of mRNA and protein expression for GABRy, GABRr2, mGluR and FMRP in lateral cerebella of subjects with major depression. There were no concordant results in subjects with major depression. However, protein levels for GABRy and FMRP were reduced signicantly, whereas it increased for GABRr2 in major depression. m, increased expression; k, reduced expression, , no change.

Figure 9 Summary of relationships between GABRy, GABRr2, mGluR5 and FMRP in three major psychiatric disorders: schizophrenia, bipolar disorder and major depression. Although there are clear biochemical connections between FMRP, mGluR5 and GABRr2, no direct relationship can be established between

GABRy and FMRP. m, increased expression; k, reduced expression, , no change.

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picture is similar to what we have described in idiopathic cases of autism without evidence of any effects in the gene for FMRP, and thus replicative of the post-transcriptional abnormalities affecting protein synthesis (Figure 9). Changes in mRNA expression do not always correlate with similar changes in protein expression, including expression of FMRP. A recent study found that in subjects with the FMR1 premutation (expanded 50 CGG repeat, but without full symptoms of FXS), there were both signicantly increased FMR1 mRNA and signicantly reduced levels of FMRP.48

Similarly, the recent ndings of Kovcs et al.21 demonstrated the downregulation of FMRP protein levels in the absence of any change in FMR1 mRNA or expansion of the 50 CGG triplet repeat in peripheral blood lymphocytes of subjects with schizophrenia. As reduced FMRP expression has been identied in four major psychiatric disorders, identifying the post-transcriptional abnormalities that may be responsible for this reduction would have a major impact on the etiology and treatment of these disorders. Additionally, verication of reduced FMRP protein levels in peripheral blood lymphocytes of subjects with schizophrenia conrm our data at least in schizophrenia, and validate our additional new ndings of reduced FMRP in BA9 of subjects with schizophrenia.

The gene for mGluR5 is located at 11q14.2-q14.3. mGluR5, like other metabotropic glutamate receptors contains seven membrane-spanning domains and a large extracellular N-terminus,49 and are G-protein coupled. Metabotropic glutamate receptors are found throughout the CNS, with high concentrations in cerebral cortex, hippocampus, striatum, hypothalamus, midbrain, cerebellum, medulla and pons.50

There were concordant and signicant reductions in levels of mRNA and protein for mGluR5 in lateral cerebellum of subjects with schizophrenia (Figure 6). In BA9, despite signicant reductions in protein levels, mRNA levels were normal (Figure 6). Thus, protein levels for mGluR5 were downregulated in both brain sites in schizophrenia. In a similar vein, mGluR5 protein levels were reduced signicantly in both lateral cerebellum and BA9 in subjects with bipolar disorder despite normal mRNA levels, indicating post-transcriptional abnormalities in the pathway for mGluR5 protein synthesis (Figure 7). Interestingly, in lateral cerebellum of subjects with major depression, despite downregulated mRNA for mGluR5, the protein levels were normal (Figure 8). It is possible that unknown mechanisms affecting transit for protein rescue the product for release, despite low turnover for its mRNA, in major depression.

Recently, Matosin et al.51 showed no signicant alteration in mGluR5-binding density or mGluR5 protein levels in dorsolateral prefrontal cortex of subjects with schizophrenia. However, close inspection of their western blotting data showed highly oversaturated bands for the monomeric mGluR5 protein levels for both control and subjects with schizophrenia, potentially masking any differences between the two groups. Although several other reports did not show any change in mGluR5 protein52,53 or mRNA5456 in prefrontal

cortex of subjects with schizophrenia, these results could be due to the use of different analytic techniques or brain regional effects. However, other reports have reported the decreases in mGluR5 mRNA in prefrontal cortex of subjects with schizoaffective disorder56 and in those with major

depression,57 supporting our current data showing signicant decreases in mGluR5 protein levels in lateral cerebellum and BA9 of subjects with schizophrenia and bipolar disorder, and decreases in mRNA levels in lateral cerebella of subjects with schizophrenia and major depression, and BA9 of subjects with bipolar disorder. Additionally, we have previously observed increased expression of mGluR5 protein in BA9 and cerebellar vermis of children with autism (Figure 9).22,23

However, although there is a great deal of overlap in the symptomologies of autism and FXS, there is less overlap between FXS and schizophrenia, and mood disorders.

Interactions between the aforementioned four proteins may alter GABAergic transmission. The cytoplasmic domains of GABRr1 and GABRr2 interact with MAP1B (Figure 9).58

Disruption of r-MAP1B interactions leads to a doubling of the inward current of GABAC receptors from bipolar cells in retinal slices in the presence of low levels of GABA.58 MAP1B mRNA is targeted by FMRP10 (Figure 9), and in FMR1-knock out mice there is an abnormal upregulation of MAP1B.59 With

reduced expression of FMRP, one might speculate that there would be increased expression of MAP1B in subjects with schizophrenia and mood disorders. However, a preliminary study involving anterior cingulate cortex found reduced expression of MAP1B protein in subjects with bipolar disorder, with no change in subjects with schizophrenia or major depression.60 Further experiments involving multiple brain sites would be required to see if this is a regional difference or if there is a global reduction. Altered expression of MAP1B could in turn cause changes in GABAergic neurotransmission through GABA receptors that contain r subunits. Currently, we do not know of a link between FMRP and GABRy (Figure 9). No changes in GABRy mRNA were identied among the GABAA receptor subunits that show reduced expression in animal models of FXS.79

In conclusion, FMRP is signicantly downregulated in the lateral cerebella18 and BA9 from subjects with schizophrenia, bipolar disorder and major depression, potentially causing GABA receptor changes and altered expression of mGluR5 in the three disorders in the absence of any FMR1 chromosomal abnormalities. Additionally, we have identied selective abnormalities in mRNA and protein levels of two novel GABAA receptors, namely GABRy and GABRr2, in subjects with schizophrenia and mood disorders. These changes could potentially explain changes in GABAergic transmission and consequent decits associated with these disorders including anxiety, panic, and impaired learning and memory. Our results also identify potential novel targets for future pharmacologic intervention. Lastly, despite signicance and novelty of these results, the study should be considered exploratory, requiring further future conrmation in other brain sites.

Conict of interest

The authors declare no conicts of interest. Dr Fatemi has patents related to Reelin as a marker of psychiatric disorders. He derives no income as yet from these patents.

Acknowledgements. Grant support by the National Institutes of Mental Health (Grant no. 1R01MH086000-01A2) to SHF is gratefully acknowledged. SHF is also supported by the Bernstein Endowed Chair in Adult Psychiatry. Tissue samples

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from the Stanley Medical Research Institute and assistance with demographic information from Dr Edwin Fuller-Torrey and Dr Maree J Webster to SHF is gratefully acknowledged. Tissue samples from the Harvard Brain Tissue Resource Center, which is supported in part by PHS grant number R24 MH068855 is gratefully acknowledged.

Disclaimer. NIMH had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report and in the decision to submit the paper for publication.

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