Prefrontal GABAA receptor α-subunit expression in normal postnatal human development and schizophrenia

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Abstract

Cortical GABA deficits that are consistently reported in schizophrenia may reflect an etiology of failed normal postnatal neurotransmitter maturation. Previous studies have found prefrontal cortical GABAA receptor α subunit alterations in schizophrenia, yet their relationship to normal developmental expression profiles in the human cortex has not been determined. The aim of this study was to quantify GABAA receptor α-subunit mRNA expression patterns in human dorsolateral prefrontal cortex (DLPFC) during normal postnatal development and in schizophrenia cases compared to controls. Transcript levels of GABAA receptor α subunits were measured using microarray and qPCR analysis of 60 normal individuals aged 6 weeks to 49 years and in 37 patients with schizophrenia/schizoaffective disorder and 37 matched controls. We detected robust opposing changes in cortical GABAA receptor α1 and α5 subunits during the first few years of postnatal development, with a 60% decrease in α5 mRNA expression and a doubling of α1 mRNA expression with increasing age. In our Australian schizophrenia cohort we detected decreased GAD67 mRNA expression (p = 0.0012) and decreased α5 mRNA expression (p = 0.038) in the DLPFC with no significant change of other α subunits. Our findings confirm that GABA deficits (reduced GAD67) are a consistent feature of schizophrenia postmortem brain studies. Our study does not confirm alterations in cortical α1 or α2 mRNA levels in the schizophrenic DLPFC, as seen in previous studies, but instead we report a novel down-regulation of α5 subunit mRNA suggesting that post-synaptic alterations of inhibitory receptors are an important feature of schizophrenia but may vary between cohorts.

Introduction

The protracted maturation of cortical GABAergic circuitry into adolescence in primates coincides with the vulnerable period for schizophrenia onset, suggesting that the neuropathology of schizophrenia may result, in part, from a failure in normal postnatal development of the GABAergic system (Lewis et al., 2004, Reynolds and Beasley, 2001). Indeed, there is increasing evidence from postmortem studies that alterations in cortical GABA neurotransmission, via deficits in GABA synthesis, transport and receptor binding, contribute to the pathogenesis of schizophrenia (Coyle, 2004, Lewis et al., 2005). One of the most replicated findings in postmortem studies of schizophrenia is decreased glutamic acid decarboxylase, 67 kDa isoform (GAD67) mRNA and protein in the cerebral cortex, supporting a GABAergic deficit that is consistent and widespread in the disease (Akbarian et al., 1995b, Guidotti et al., 2000, Hashimoto et al., 2003, Impagnatiello et al., 1998, Thompson et al., 2009, Volk et al., 2000). GABAA receptor binding and density is also altered in the cortex of people with schizophrenia (Benes et al., 1996, Hanada et al., 1987), indicating that post-synaptic deficits may contribute to cortical dysfunction. The affinity of GABA for the major GABAA receptor is particularly dependent upon the composition of α subunits (Levitan et al., 1988, Sigel et al., 1990) and it is the temporal and spatial distribution of GABAA receptor α subunits that regulates the physiological response to GABA-modulating agents (Luddens and Wisden, 1991, Pritchett and Seeburg, 1990). GABAA receptor α subunits show pronounced developmental mRNA and protein regulation in the rat cortex and differential patterning in the adult rat cortex (Laurie et al., 1992, Wisden et al., 1992, Yu et al., 2006). Alterations in the expression of GABAA receptor α subunits have been detected in schizophrenia, including a decrease in α1 mRNA expression (Hashimoto et al., 2008), and increases in α2 protein expression (Volk et al., 2002) and in α5 mRNA expression (Impagnatiello et al., 1998) in the PFC. Yet, several studies have reported no change in cortical expression of α subunits (Akbarian et al., 1995a) or even an increase in α1 mRNA expression (Impagnatiello et al., 1998, Ohnuma et al., 1999), leaving a need in the field for further replication of these studies.

Although it is widely recognized that properties of cortical GABAergic neurons may differ substantially between rodents and primates (Ascoli et al., 2008) there are few studies of GABAA receptor α-subunit expression in the developing non-human primate cortex (Brooks-Kayal and Pritchett, 1993, Cruz et al., 2003, Hornung and Fritschy, 1996, Maldonado-Aviles et al., 2009). These indicate that the developmental shift in α-subunit expression is more protracted in primates compared to rodents, occurring through adolescence – a time of electrophysiological shift in GABAergic neurotransmission (Hashimoto et al., 2009). In order to determine the developmental significance of pre- and post-synaptic GABA alterations in schizophrenia, the expression of GABAA receptor α subunits must be characterized over the time course of normal human postnatal life, which has not been systematically explored previously. Furthermore, given the contradictory findings in postmortem tissue, further studies are required to support or refute the generality of α subunit dysregulation in the DLPFC of patients with schizophrenia.

In this study, we analyzed the molecular expression of the functionally critical GABAA receptor α subunits in the human dorsolateral prefrontal cortex (DLPFC) during postnatal development and in schizophrenia. The mRNA expression levels of α1–α5 subunits were assessed using microarray and qPCR analyses. α6 mRNA is expressed in the cerebellum only in mammals, and thus was not examined in this present study (Laurie et al., 1992). Gene expression analysis of tissue from 60 individuals aged from six weeks to 49 years indicates that α subunits have distinct and dynamic expression patterns that are protracted compared to rodents, similar to findings in non-human primates. The present study also confirms and extends the identification of GABA deficiencies in the DLPFC of patients with schizophrenia in a cohort of 37 patients and 37 matched controls, with decreased mRNA expression of both GAD67 and the GABAA receptor α5 subunit.

Section snippets

Human postmortem brain samples and tissue processing

For the developmental postmortem brain cohort, human DLPFC tissues were obtained from the National Institutes for Child Health and Human Development Brain and Tissue Bank for Developmental Disorders (UMBB; NICHHD contract #NO1-HD8–3283) from 68 individuals ranging in age from six weeks to 49 years and grouped into seven developmental periods (Supplementary Table 1) as defined in many previous publications (Choi et al., 2009, Romanczyk et al., 2002, Tunbridge et al., 2007, Weickert et al., 2009,

GABAA receptor α-subunit mRNA expression in the postnatal human prefrontal cortex

GABAA receptor α1–α5 subunit mRNAs were analyzed in the human DLPFC using both microarray hybridization and qPCR analysis (Fig. 1). We detected a significant 4-fold increase in GABAA receptor α1-subunit mRNA expression from birth to adulthood (r = 0.763; p = 1.03E-09), with a strong effect of developmental age group on α1 mRNA expression (F(6,38) = 24.74; p = 1.03E-11) (Fig. 1A). Expression differences between age groups were validated by qPCR analysis (F(6,49) = 3.827; p = 0.003) with a highly significant

Discussion

In this study, our objective was to analyze two postmortem brain cohorts: a schizophrenia case-control cohort and a postnatal developmental cohort, to assess how any changes in GABAA receptor α-subunit expression in schizophrenia may relate to normal human development. We confirmed the presence of a cortical GABAergic deficit in a newly assembled Australian schizophrenia cohort, yet we were unable to detect changes in α1 or α2 mRNA, despite their developmental regulation in the human prefrontal

Contributors

C. Duncan contributed to the study design, experimental work, data analyses and intepretation and was responsible for writing of the manuscript. M. Webster contributed to the study design and manuscript preparation. D. Rothmond contributed to data analysis and preparation of the manuscript. S. Bahn contributed her expertise in preparing RNA samples for microarray, microarray analysis support and editing of manuscript. M. Elashoff was responsible for statistical analysis of microarray data and

Role of funding sources

Funding for this study was provided by Schizophrenia Research Institute, utilizing funding from NSW Health and the Macquarie Group Foundation. These sources of funding 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.

Conflicts of interest

None declared.

Acknowledgements

We acknowledge the assistance of Dr. H. Ronald Zielke and Robert Vigorito of the University of Maryland Brain and Tissue Bank for Developmental Disorders. Tissues were also received from the Australian Brain Donor Programs NSW Tissue Resource Centre, which is supported by The University of Sydney, National Health and Medical Research Council of Australia, Schizophrenia Research Institute, National Institute of Alcohol Abuse and Alcoholism and NSW Department of Health. We would like to thank

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