GABAergic dysfunction in schizophrenia and mood disorders as reflected by decreased levels of glutamic acid decarboxylase 65 and 67 kDa and Reelin proteins in cerebellum

doi:10.1016/j.schres.2004.02.017

Schizophrenia Research

Volume 72, Issues 2–3, 1 January 2005, Pages 109-122

https://doi.org/10.1016/j.schres.2004.02.017 Get rights and content

Abstract

Background: Glutamic acid decarboxylase (GAD) is the rate limiting enzyme responsible for conversion of glutamate to gamma-aminobutyric acid (GABA) regulating levels of glutamate and GABA in the mammalian brain. Reelin is an extracellular matrix protein that helps in normal lamination of the embryonic brain and subserves synaptic plasticity in adult brain. Both GAD and Reelin are colocalized to the same GABAergic interneurons in several brain sites. We hypothesized that levels of GAD and Reelin would be altered in cerebellum of subjects with schizophrenia and mood disorders differentially vs. controls. Methods: We employed SDS-PAGE and Western blotting to measure levels of GAD isomers 65 and 67 kDa and Reelin isoforms 410-, 330- and 180-kDa proteins as well as β-actin in cerebellum of subjects with schizophrenia, bipolar disorder and major depression vs. controls (N=15 per group). Results: GAD 65- and 67-kDa levels were decreased significantly in bipolar, depressed and schizophrenic subjects (p<0.05) vs. controls. Reelin 410- and 180-kDa proteins decreased significantly (p<0.05) in bipolar subjects vs. controls. Reelin 180 kDa was decreased significantly (p<0.05) in schizophrenics vs. controls. β-Actin levels did not vary significantly between groups. There were no significant effects of confounding variables on levels of various proteins. Conclusion: This study demonstrates for the first time significant deficits in GABAergic markers Reelin and GAD 65 and 67 proteins in bipolar subjects and global deficits in the latter proteins in schizophrenia and mood disorders, accounting for the reported alterations in CSF/plasma levels of glutamate and GABA in these disorders.

Section snippets

Tissue preparation, SDS-PAGE and Western blotting

Approximately 200 μg of cerebellar tissue was homogenized in lysis buffer (20 mM Tris, pH 8.0, 0.2 mM EDTA, 150 mM NaCl, 3% Igepal (v/v), 1% sodium deoxycholate (w/w), 0.1% SDS (w/v), 50 mg/ml leupeptin, 0.2 mM PMSF, 1 mM sodium orthovanadate, 30 μl/ml Aprotinin) using a Kontes hand pestle. Homogenates had an additional 1 μl of PMSF added, were iced for 30 min, then centrifuged at 10,000×g for 20 min. The supernatants were collected and protein was assayed by BioRad Protein Assay with a BSA

Background and brain variables across groups

Table 1 displays demographic information on the brain specimens obtained from the Stanley Foundation Neuropathology Consortium. To test for differences across diagnostic groups on demographic and brain variables, chi-square tests were performed on categorical variables and Analyses of Variance (ANOVAs) were performed on continuous variables. Across the four groups, significant differences were found on Cause of Death, Family History of Psychiatric Disorders, Freezer Storage Time, and Severity

Protein levels across diagnostic groups

The overall multivariate test for the main effect of diagnostic group on the six protein levels was significant, F(18, 149)=1.93, p=0.017. Tests of between-subjects (univariate) effects revealed significant main effects for Reelin 180, F(3, 56)=4.04, p=0.011, GAD 65, F(3, 56)=4.75, p=0.005, and GAD 67, F(3, 56)=4.34, p=0.008. Because the assumption of equal covariance matrices across groups on the dependent variables was not met, F(63, 7348)=1.55, p=0.004, a non-parametric test (Kruskal–Wallis)

Reelin 180

Post hoc tests using Tukey's HSD revealed significant differences between bipolar and normal subjects, p=0.042, and between schizophrenic and normal subjects, p=0.018, on Reelin 180. Compared with normal subjects (M=34.16, S.D.=12.6), bipolar subjects had significantly lower levels of Reelin 180 (M=22.54, S.D.=11.00), as did schizophrenic subjects (M=21.10, S.D.=11.95).

GAD 65

Post hoc tests using Tukey's HSD revealed significant differences between all three disordered groups (bipolar, depression, and schizophrenia) and normal subjects, p=0.017, p=0.033, and p=0.009, respectively. Compared with normal subjects (M=3.7, S.D.=2.45), bipolar subjects (M=1.8, S.D.=1.8), depressed subjects (M=2.0, S.D.=1.2), and schizophrenic subjects (M=1.67, S.D.=0.92) had significantly lower levels of GAD 65.

GAD 67

Post hoc tests using Tukey's HSD revealed significant differences between bipolar and normal subjects, p=0.015, and depressed and normal subjects, p=0.019, on GAD 67. Compared with normal subjects (M=1.4, S.D.=0.96), bipolar subjects and depressed subjects had significantly lower levels of GAD 67 (M=0.63, S.D.=0.64; M=0.65, S.D.=0.41, respectively) (Fig. 1).

Protein levels between bipolar and schizophrenic subtypes

A follow-up multivariate analysis of variance conducted between bipolar subjects with and without psychosis on protein levels revealed no significant main effect for presence of psychosis, F(6, 8)=0.840, p=0.573. Tests of between-subjects effects confirmed that the two groups did not differ significantly from each other on any of the proteins. However, given the small number of subjects in each group (n=11 with psychosis; n=4 without psychosis), the power to detect differences between groups at

Exploratory pairwise comparisons

Exploratory pairwise comparisons between diagnostic groups on protein levels were conducted using multiple t-tests. Table 2 displays the results of both the overall univariate test results with post hoc comparisons based on Tukey's HSD and the pairwise comparisons that do not correct for inflated Type I error. The less restrictive pairwise comparisons revealed the same significant findings with the addition of a significant difference between bipolar and normal subjects on Reelin 410, t

Relationship between protein levels and demographic and brain variables

Table 3, Table 4 display relationships between protein levels and demographic and brain variables. Of all the demographic and brain variables, significant relationships were found between protein levels and PMI, brain pH, and Severity of Substance Abuse. There was a significant relationship between PMI and β-actin, r(60)=0.266, p=0.04. In addition, there was a significant relationship between brain pH and all protein levels except β-actin, r(60)=0.462, .473, .433, .456, and .340, for Reelins

Demographic and brain variables as possible confounds

Each demographic and brain variable listed in Table 3, Table 4 was entered as a covariate in the MANOVA model in order to determine whether it accounted for the significant main effects between diagnostic group and protein levels that were found. The only covariate with a main effect on protein level was brain pH, F(6, 50)=3.545, p=0.005. However, this covariate did not alter the significant main effect of group on protein levels for Reelin 180, F(3, 55)=3.533, p=0.021; GAD 65, F(3, 55)=3.975, p

Acknowledgements

Postmortem brains were donated by The Stanley Medical Research Institute’s Brain Collection courtesy of Drs. Michael B. Knable, E. Fuller Torrey, Maree J. Webster, Serge Weis, and Robert H. Yolken. SHF is supported by the Stanley Medical Research Institute. We are grateful to Dr. A. Goffinet for his generous gift of anti-Reelin antibody. We appreciate the secretarial help provided by Ms. Janet Holland and Ms. Laurie Iverson.

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GABAergic dysfunction in schizophrenia and mood disorders as reflected by decreased levels of glutamic acid decarboxylase 65 and 67 kDa and Reelin proteins in cerebellum

Abstract

Section snippets

Tissue preparation, SDS-PAGE and Western blotting

Background and brain variables across groups

Protein levels across diagnostic groups

Reelin 180

GAD 65

GAD 67

Protein levels between bipolar and schizophrenic subtypes

Exploratory pairwise comparisons

Relationship between protein levels and demographic and brain variables

Demographic and brain variables as possible confounds

Acknowledgements

Curr. Opin. Pharmacol

Neuron

Biol. Psychiatry

Brain Res

Biol. Psychiatry

J. Psychiatr. Res

Brain Res. Bull

Schizophr. Res

Biol. Psychiatry

Brain Res., Dev. Brain Res

Neuron

Biol. Psychiatry

Neuron

Neuroscience

J. Affect. Disord

Pharmacol. Biochem. Behav

J. Biol. Chem

Neurosci. Lett

Trends Pharmacol. Sci

Schizophr. Res

J. Biol. Chem

Gene expression for glutamic acid decarboxylase is reduced without loss of neurons in prefrontal cortex of schizophrenics

Arch. Gen. Psychiatry

Systemic administration of dizocilpine maleate (MK-801) or l-dopa reverses the increases in GAD65 and GAD67 mRNA expression in the globus pallidus in a rat hemiparkinsonian model

Synapse

Time of origin or corresponding cell classes in the cerebral cortex of normal and reeler mutant mice: an autoradiographic analysis

J. Comp. Neurol

Disrupted synaptic development in the hypoxic newborn brain

Proc. Natl. Acad. Sci. U. S. A

The human reelin gene: isolation, sequencing, and mapping on chromosome 7

Genome Res

The axonal chemorepellant semaphorin 3A is increased in the cerebellum in schizophrenia and may contribute to its synaptic pathology

Mol. Psychiatry

Comparative localization of two forms of glutamic acid decarboxylase and their mRNAs in rat brain supports the concept of functional differences between the forms

J. Neurosci

Two new mutants, Trembler and ‘Reeler’, with neurological actions in the house mouse

J. Genet

Reelin mutations in mouse and man: from reeler mouse to schizophrenia, mood disorders, autism and lissencephaly

Mol. Psychiatry

The role of Reelin in pathology of autism

Mol Psychiatry