Elsevier

Neuropharmacology

Volume 59, Issue 3, September 2010, Pages 167-171
Neuropharmacology

Early developmental alterations in GABAergic protein expression in fragile X knockout mice

https://doi.org/10.1016/j.neuropharm.2010.05.002Get rights and content

Abstract

Fragile X syndrome is the most common heritable form of mental retardation. It is caused by silencing of the Fmr1 gene and the absence of the encoded protein. The purpose of this study was to examine global protein expression levels of GABAA and GABAB receptors, and GABAergic enzymes and trafficking proteins in fragile X knockout mice during brain maturation. Quantitative western blotting of homogenates of forebrain revealed that the levels of GABAA β1 and β3, GABAB-R1, NKCC1, KCC2, gephyrin and ubiquilin were not significantly different from wild-type mice at any of the postnatal time points examined.

In contrast, the GABAA receptor α1, β2, and δ subunits, and the GABA enzymes GABA transaminase and succinic semialdehyde dehydrogenase were down-regulated during postnatal development, while GAD65 was up-regulated in the adult knockout mouse brain. The GABAA receptor α1 and β2 subunits displayed a divergent pattern of developmental expression whereby α1 was reduced in the immature brain but regained a level of expression similar to wild-type mice by adulthood, while the expression of β2 was similar to wild-types at postnatal day 5 but reduced at day 12 and in the adult brain. The GABAA receptor δ subunit and the GABA catabolic enzymes GABA transaminase and succinic semialdehyde dehydrogenase were simultaneously but transiently decreased only at postnatal day 12. Our results demonstrate that GABAA receptor subunits and GABA enzymes display complex patterns of changes during brain development suggesting that dynamic interactions may occur between GABA transmitter levels and GABA receptors in fragile X syndrome.

Introduction

Fragile X syndrome (FXS) is caused by a dynamic CGG trinucleotide repeat expansion in the 5′ untranslated region of the Fmr1 gene leading to transcriptional silencing of the gene and the absence of the encoded protein, fragile X mental retardation protein (FMRP, Bassell and Warren, 2008, Verkerk et al., 1991). Persons with FXS display hyperactivity, social anxiety, autistic characteristics, and an increased incidence of epileptic seizures in childhood (Hagerman et al., 2009). Abnormal dendritic spine morphology manifested as an increase in immature spines is also a hallmark of the syndrome (de Vrij et al., 2008, Grossman et al., 2006, Weiler and Greenough, 1999).

FMRP is an RNA-binding protein that is expressed most abundantly in the brain. The absence of FMRP results in the dysregulation of cargo mRNAs which contributes to the abnormalities in brain maturation and function observed in FXS. Fmr1 knockout mice (Kooy et al., 1996) are widely used as the mammalian animal model of the disorder. The study of these mice has revealed abnormalities in neuronal synaptic plasticity manifested as enhanced long-term depression and possibly impaired long-term potentiation in several brains regions such as the hippocampus and cerebral cortex (Dolen and Bear, 2008, Huber et al., 2002, Zhang et al., 2009).

Increasing evidence has implicated the GABAergic system in FXS (D’Hulst and Kooy, 2009). GABAA receptors are involved in many processes that are perturbed in FXS patients such as neuronal excitability and seizures and the modulation of anxiety, cognition, and memory and learning (Martin et al., 2009, Sieghart et al., 1999). Several studies have demonstrated that the expression of mRNAs coding for GABAA receptor subunits and GABAergic enzymes, and GABAA receptor clustering proteins are decreased in adult Fmr1 knockout mice (D’Hulst et al., 2009, El Idrissi et al., 2005, Gantois et al., 2006). Additionally, experiments conducted on brain slices revealed abnormal GABA-mediated transmission in knockout mice (Centonze et al., 2008, Curia et al., 2008). Moreover, treatment with GABA in Fmr1 mutant flies was shown to reverse several phenotypes associated with FXS including mushroom body defects and abnormal courtship behaviour (Chang et al., 2008).

Prior studies examining the GABA system in Fmr1 knockout mice (D’Hulst et al., 2009, Gantois et al., 2006) have focused primarily on expression at the mRNA level, whereas validation with protein expression studies have been limited. Moreover, previous studies also focused on analysis of adult tissues and did not examine expression during the critical period of development when synaptogenesis and neuronal maturation occurs. In this study, we compared the temporal expression of GABAergic proteins in Fmr1 knockout mice and wild-type C57/BL6 mice. We demonstrate that the α1 and β2 GABAA receptor subunits show a divergent pattern of developmental expression in Fmr1 mice, and that the GABAA receptor δ subunit, GABA transaminase, and succinic semialdehyde dehydrogenase are transiently and simultaneously under-expressed at postnatal day 12 but not at day 5 or in adult animals. These findings suggest a potential link between the expression of some GABA receptor subunits and GABA metabolic enzymes.

Section snippets

Animal models

All animal experiments were carried out in accordance with the guidelines set out by the Canadian Council on Animal Care and were approved by the University Animal Care Committee of the University of Toronto. All wild-type and Fmr1 knockout mice (referred to herein as Fmr1 mice; kindly provided by Dr. William Greenough, University of Illinois) used were derived on the C57/Black 6 strain. Mice were assessed at three different developmental age points – postnatal days 5, 12, and adults (2–3

GABAA receptor subunit profile in the developing and adult brain of Fmr1 knockout mice

There are six GABAA receptor α subunits expressed in the CNS. Previous studies have shown that the mRNAs of the α1, α3, α4 subunits are down-regulated (D’Hulst et al., 2006), while α5 protein is reduced in the subiculum of adult Fmr1 knockout mice (Curia et al., 2008). We focused our efforts here on the α1 subunit because complexes containing α1 subunits are the most abundant and widely expressed GABAA receptors in the CNS, comprising roughly half of all GABAA receptors (Mohler, 2007, Olsen and

Discussion

In this study, we sought to quantitate global changes in GABAergic protein expression in the forebrain of the Fmr1 knockout mouse. The alterations that were observed in mouse forebrain likely reflect gross changes in GABAergic protein expression in the absence of FMRP, and additional alterations may be present in brain subregions that may not have been detected in the analysis of homogenates of whole forebrain. Nevertheless, our results demonstrate that changes in the expression of several GABAA

Acknowledgments

We thank Dr. William Greenough for kindly providing the Fmr1 mice, Dr. D.M. Broussard for helpful comments on the manuscript, Dr. Michael K. Gibson for the SSADH antibody and Dr. Soo Young Choi for the GABA-T antibody. The GAD65 antibody was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. The monoclonal antibodies against GABAA receptor β3, δ and GABAB-R2 were

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