Profile of changes in gene expression in cultured hippocampal neurones evoked by the GABAB receptor agonist baclofen

Mohamed T. Ghorbel1, Kevin G. Becker2 and Jeremy M. Henley1

1 Medical Research Council Centre for Synaptic Plasticity, Department of Anatomy, School of Medical Sciences, University of Bristol, Bristol, United Kingdom
2 DNA Array Unit, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Metabotropic {gamma}-aminobutyric acid receptors (GABABRs) play a critical role in inhibitory synaptic transmission in the hippocampus. However, little is known about a possible long-term effect requiring transcriptional changes. Here, using microarray technology and RT-PCR of RNA from cultured rat embryonic hippocampal neurones, we report the profile of genes that are up- or downregulated by activation of GABABRs by baclofen but are not changed by baclofen in the presence of the GABABR antagonist CGP-55845A. Our data show, for the first time, regulation of transcription of defined mRNAs after specific GABAB receptor activation. The identified genes can be grouped into those encoding signal transduction, endocytosis/trafficking, and structural classes of proteins. For example, butyrylcholinesterase, brain-derived neurotrophic factor, and COPS5 (Jab1) genes were upregulated, whereas Rab8 interacting protein and Rho GTPase-activating protein 4 were downregulated. These results provide important baseline genomic data for future studies aimed at investigating the long-term effects of GABABR activation in neurones such as their roles in neuronal growth, pathway formation and stabilization, and synaptic plasticity.

cDNA microarrays; G protein-coupled receptor; hippocampus; {gamma}-aminobutyric acid


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
{gamma}-AMINOBUTYRIC ACID (GABA), the predominant inhibitory neurotransmitter in the central nervous system (CNS), can act at two distinct types of receptors: the fast ligand-gated ionotropic GABAA and GABAc receptors and the slower G protein-linked, metabotropic GABAB receptors (GABABR) (20, 22, 25). GABABRs have both pre- and postsynaptic distributions in the mammalian brain. Presynaptic GABABRs suppress neurotransmitter release by inhibiting voltage-sensitive P-, N-, and L-type Ca 2+ channels (19, 25, 31, 35). Postsynaptic GABABR stimulation generally causes inhibition of adenylate cyclase (45) and activation of hyperpolarizing potassium channels (23, 38).

In addition to playing fundamental roles in regulating basic neurotransmission, GABABRs are also involved in synaptic plasticity and nociception (for reviews, see Refs. 6, 7, 10, and 29). GABABR activation can also initiate long-term effects on protein synthesis and has, for example, been reported to negatively regulate CREB-mediated transcription in the CNS (4, 39). GABABRs have been implicated in the development of some neuronal pathways. For example, GABAB1 and GABAB2 subunits are present in the rat neocortex from embryonic day 14 (E14) suggesting that functional GABABRs are present during prenatal development in vivo (27). It has been reported that during corticogenesis in the rat, CNS cortical plate cells release GABA, which acts as a chemoattractant for GABABR-containing ventricular zone neurones migrating from germinal regions (5). GABA and the selective GABABR agonist baclofen both stimulate Xenopus retinal ganglion cell neurite outgrowth in culture, and GABABR antagonists applied to the developing optic projection in vivo cause a dose-dependent shortening of the optic nerve (15). These studies suggest that GABABR activation can result in changes in protein synthesis, but little is known about the target genes or long-term effects involving alterations at the genomic level.

To gain insight into the pathways by which GABABR activation may influence long-term changes in synaptic plasticity and neuronal growth and morphology, we investigated changes in gene expression in cultured hippocampal neurones in the presence of TTX evoked by the GABABR agonist baclofen. We used cDNA microarray gene expression profiling (12, 14, 34) to identify candidate genes that are differentially expressed in cultured hippocampal neurones after GABABR activation. Overall, the microarray analysis suggested increased levels of transcription of 14 genes and decreased transcription of 6 genes. Importantly, none of these genes displayed any change in expression when baclofen was applied in the presence of the GABABR-specific antagonist CGP-55845A. Our results show that the expression of several different classes of genes alters after baclofen application.


    MATERIALS AND METHODS
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Cell Culture
Rat protocols were approved by the United Kingdom Home Office; rats were used in accordance with United Kingdom Home Office regulations. Hippocampal cultures were prepared as previously described (21). Briefly, hippocampi from E18 Wistar rats were dissected, and the neurones were dissociated by enzymatic digestion with trypsin for 15 min and mechanical dissociation. Cells were then plated at a density of 500,000 cells/60-mm-diameter poly-L-lysine (1 mg/ml, Sigma)-coated dish. The culture medium was composed of neurobasal medium (GIBCO) supplemented with horse serum (10%), B27 (GIBCO), and 2 mM glutamine. On the second day, the media was changed for neurobasal medium supplemented with B27, and neurones were then fed each week with this glutamine-free medium until use. Cultures were maintained at 37°C in a 5% CO2 humidified incubator and used for experiments at day in vitro 20. Control and experimental group cultures were incubated with 10 µM TTX for 15 min, followed by the addition of 100 µM baclofen, 100 µM baclofen plus 10 µM CGP-55845A, or vehicle alone. TTX was used to ensure that the effects observed were directly attributable GABABR activation rather than downstream events caused by depression of synaptic transmission. This could occur if experiments were done in the absence of TTX because baclofen activation of inhibitory GABABRs would reduced intrinsic neuronal activity compared with control conditions (vehicle or baclofen + CGP-55845A).

Total RNA Preparation
Each experimental group was compared as replicates of three. For each replicate, different batches of cultured hippocampal neurones were harvested after 2 h of incubation with or without baclofen or baclofen + CGP-55845A, and total RNA was isolated using the RNeasy Mini kit (Qiagen) according to the manufacturer's protocol. All RNA samples were treated with DNase to remove any contaminating genomic DNA using a DNA-free kit (Ambion) according to the manufacturer's protocol.

Microarray Target Labeling and Hybridization
Targets for cDNA microarrays were generated using 5 µg total RNA from control and baclofen-treated cultured hippocampal neurones in a standard RT reaction. RNA was annealed in 16 µl water with 1 µg of 24-mer poly(dT) primer (Invitrogen; Paisley, UK) by heating at 65°C for 10 min and cooling on ice for 2 min. The RT reaction was performed by adding 8 µl of 5x first-strand RT buffer (Life Technologies; Rockville, MD), 4 µl of 20 mM dNTPs minus dCTP (Pharmacia), 4 µl of 0.1 M DTT, 40 units of RNAse OUT (Life Technologies), and 6 µl of 3,000 Ci/mmol [33P]dCTP (ICN Biomedicals) to the RNA-primer mixture to a final volume of 40 µl. Two microliters (400 units) of Superscript II reverse transcriptase (Life Technologies) was then added, and the sample was incubated for 30 min at 42°C, followed by an additional 2 µl of Superscript II reverse transcriptase and another 30-min incubation. The reaction was stopped by the addition of 5 µl of 0.5 M EDTA. The samples were incubated at 65°C for 30 min after the addition of 10 µl of 0.1M NaOH to hydrolyze and remove RNA. The samples were pH neutralized by the addition of 45 µl of 0.5 M Tris (pH 8.0) and purified using Bio-Rad 6 purification columns. The NIA NeuroArray consists of 1,152 cDNAs printed on a nylon membrane in duplicate (41). The arrays were hybridized with [{alpha}-33P]dCTP-labeled cDNA probes overnight at 50°C in 4 ml of hybridization solution. Hybridized arrays were rinsed in 50 ml of 2x SSC and 1% SDS twice at 55°C, followed by two washes in 2x SSC and 1% SDS at 55°C for 15 min each. The microarrays were exposed to phosphorimager screens for 1–3 days. The screens were then scanned with a Molecular Dynamics STORM PhosphorImager (Sunnyvale, CA) at 25-µm resolution.

Microarray Data Analysis: Z Normalization
ImageQuant software (Molecular Dynamics; Sunnyvale, CA) was used to convert the hybridization signals on the image into raw intensity values, and the data thus generated were transferred into Microsoft Excel spreadsheets, predesigned to associate the ImageQuant data format to the correct gene identities. Raw intensity data for each experiment were normalized by z-transformation. The intensity data were log10 transformed and used for the calculation of z-scores. z-Scores were calculated by subtracting the average gene intensity from the raw intensity data for each gene and dividing that result by the standard deviation of all the measured intensities. Gene expression differences between any two experiments were calculated by taking the difference between the observed gene z-scores. The significance of calculated z-differences can be directly inferred from measurements of the standard deviation of the overall z-difference distribution. Assuming a normal distribution profile, z-differences are assigned significance according to their relation to the calculated standard deviation of all the z-differences in any one comparison. To facilitate comparison of z-differences between several different experiments, z-differences were divided by the appropriate standard deviation to give the z-ratios (41). All microarry data have been submitted to the GEO database maintained by the National Center for Biotechnology (13) (http://www.ncbi.nlm.nih.gov/geo/) and can be accessed using the following accession numbers: GSM28446, GSM28534, GSM28535, GSM28536, GSM28537, and GSM28538.

Multiplex RT-PCR
A two-step semiquantitative RT-PCR was employed for validation as follows. First-strand cDNA was synthesized using the RETROscript kit (Ambion) in a final volume of 20 µl containing 1x RT buffer, 1.5 µg total RNA, 5 µM random decamers primer, 200 µM dNTPs, and 100 units Moloney murine leukemia virus (MMLV) reverse transcriptase. For the PCR, gene-specific primers were generated based on the rat gene sequence. Primers specific for 18S were added as an internal control following the manufacturer's protocol (Ambion). One microliter of first-strand cDNA was added to a PCR mixture and amplified for 20–24 cycles by incubation at 95°C for 1 min, 63°C for 40 s, and 72°C for 1 min with a final incubation at 72°C for 5 min. The PCR cycle number for each gene was choosen from the determined linear range (20–24 cycles). Aliqots of the resultant products (15–20 µl) were subjected to 2% agarose gel electrophoresis. Gels were stained using Syber gold (Molecular Probes), and images were captured using a gel documentation system (GDS-8000 System, UVP BioImaging Systems; Cambridge, UK). PCR bands were quantified using NIH ImageJ software (Bethesda, MD). All of the RT-PCR experiments were conducted in triplicate using three separate RNA samples extracted from different culture preparations.

Statistics
RT-PCR data were analyzed by an unpaired two-tailed Student's t-test. Results were considered significant when P values were <0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Microarray findings
The cDNA microarray used in the present study is the National Institute on Aging Neuroarray. This array contains 1,152 genes relevant to neurobiology. NIA Neuroarray probes were hybridized in triplicate with targets derived from control and baclofen-treated cultured hippocampal neurones. Data analysis by Z-normalization of the hybridization signals identified 20 candidate regulated genes. Fourteen of these genes were upregulated by specific GABAB receptor activation, and six of these genes were downregulated (Table 1).


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Table 1. Genes showing up- or downregulation in hippocampal neurones after GABABR activation

 
The identified genes can be grouped into the following general protein categories.

Cell signaling proteins.
The group of genes involved in cell signaling represented the most hits and can be divided in to several subgroups: 1) growth and cell cycle factors: brain-derived neurotrophic factor (BDNF) and CDC28 protein kinase subunit 2 (CKS2); the genes in this group were upregulated on baclofen stimulation; 2) G protein-coupled receptors (GPCRs): one upregulated gene, ß2-adrenergic receptor (ADRB2), and one downregulated gene, corticotropin-releasing hormone receptor 1 (CRHR1), were detected on the chip array assays; and 3) signaling enzymes: butylcholinesterase (BchE), mitogen-activated protein kinase kinase 4 (MAP2K4), and connector enhancer of KSR-like kinase (CNK1) were all upregulated.

Cytoskeleton organization proteins.
Rho GTPase-activating protein 4 (ARHGAP4) was downregulated by baclofen treatment.

Endocytosis/trafficking proteins.
The Rab 18 small GTPase was upregulated, whereas Rab8-interacting protein (Rab8IP) and mitogen-activated protein kinase kinase 6 (MAP2K6) were downregulated.

Proteins involved in transcription and translation regulation.
Subunit 5 of the COP9 signalosome (COPS5, also known as Jab1) was upregulated, and the BRF1 homolog, a subunit of RNA polymerase III transcription initiation factor IIIB, was downregulated.

Intracellular transport proteins.
Two upregulated genes, the mitochondrial import receptor Tom22 (protein transporter) and cytochrome c oxidase subunit VIIc (electron transporter), were detected. In addition, there was downregulation of the nucleotide transporter ATP-binding cassette subfamily A member 4 transporter (ABCA4).

RT-PCR Validation
To confirm the array data, we selected six of the most potentially interesting genes and semiquantitatively (3) assessed their differential expression in the hippocampal mRNA of cultured neurones using multiplex RT-PCR (Figs. 1 and 2). Three separate mRNA samples, extracted from different culture preparations, for each experimental group were used in the RT-PCR. Of the six genes, the mRNAs for five were significantly altered after baclofen treatment in the RT-PCR assays. Consistent with the microarray data, BchE, BDNF, and COPS5/Jab1 were upregulated, whereas ARHGAP4 and Rab8IP were downregulated. Although ADRB2 showed a trend to increase in mRNA levels in the PCR assays, this increase was not significant (data not shown). It is important to note that the fold change obtained by the PCR assays was smaller than z-ratios in the array data. This is because the z-ratios (41) are z-differences divided by standard deviation and not fold change ratios.



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Fig. 1. Multiplex RT-PCR validation of upregulated genes. Total RNA was isolated from control (Ct) and 100 µM baclofen (Bac)-stimulated cultured hippocampal neurones at day in vitro 21. Left: multiplex RT-PCR amplification was performed using gene-specific and 18S RNA primers. Right: histograms represent the signal of the specific gene normalized to the 18S internal control. RT-PCRs were carried out in triplicate for each experimental group. BChE, butyrylcholinesterase; BDNF, brain-derived neurotrophic factor. *P < 0.05.

 


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Fig. 2. Multiplex RT-PCR validation of downregulated genes. Total RNA was isolated from control and 100 µM baclofen-stimulated cultured hippocampal neurones at day in vitro 21. Left: multiplex RT-PCR amplification was performed using gene-specific and 18S RNA primers. Right: histograms represent the signal of specific gene normalized to the 18S internal control. RT-PCRs were carried out in triplicate for each experimental group. ARHGAP4, Rho GTPase-activating protein 4; Rab8IP, Rab8-interacting protein. *P < 0.05; **P < 0.01.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Microarray analysis provides the means to perform parallel analysis of multiple genes in a single assay (26, 47) resulting in a semiquantitative assessment of changes in gene expression. They represent a powerful tool to investigate alterations in mRNA levels that accompany, and may regulate, physiological change. Our cDNA microarray analysis revealed 20 genes as being differentially expressed in the hippocampal neuronal as a consequence of specific GABABR activation (Table 1). The 2-h exposure to baclofen was employed to ensure maximum effect. GABABRs are extremely stably expressed at the surface of neurons with a half-life in the order of days (10). The physiological importance of this is unknown, but it is noteworthy that baclofen is administered by a regular intrathecal injection to treat intractable spasticity. Interestingly, in contrast to, for example, opiates, the same fixed dose of baclofen can be used for years without any diminution in therapeutic effects. The stability and lack of downregulation of GABABRs may underlie this phenomenon. It is not possible for us to distinguish if these changes are due to pre- or postsynaptic GABABRs but of particular potential interest are our observations of changes in the following examples.

BChE
Acetylcholinesterase (AChE) and BuChE are coregulators of the duration of action of acetylcholine in cholinergic neurotransmission and are also implicated in neuronal growth and development. BuChE activity in thalamic neurones plays an important role in neurotransmission in the human nervous system (11). Functional BChE activity is present in all human hippocampal and temporal neocortical areas known to receive cholinergic input with a substantial presence in neuroglia and their processes (30). There have been no previous reports that BChE can be regulated by GABABRs, but, consistent with our findings, AChE expression is increased by baclofen in mixed neuronal-glial primary cultures from the fetal rat medial septum (24). These results suggest that increased levels of cholinesterases evoked by GABABR activation may play a role in neuronal maturation and stabilization.

BDNF
BDNF is a neurotrophin that can regulate neuronal survival via high-affinity membrane tyrosine kinase receptors (17). It is widely distributed in the CNS, and, in addition to its survival-promoting actions on a variety of CNS neurones, the interplay between BDNF and signal transduction modulators has been suggested to play a key role in certain types of synaptic plasticity (42). BDNF is a molecular target of CREB and, in turn, can regulate CREB transcription as well as synapsin I, a protein that is involved in synaptic transmission.

We show that BDNF expression is increased by ~200% by baclofen, a GABABR agonist. It has been reported previously that a single, physiologically active and nonconvulsive dose of GABABR receptor antagonist in vivo can increase BDNF mRNA levels by 200–400% in the rat neocortex, hippocampus, and spinal cord (18). These results demonstrate that BDNF gene expression is regulated by GABABR signaling and, consistent with the regulation of cholinesterases, suggest that GABABRs are likely to play an important role in neuronal development, maturation, and stability.

Baclofen reduces the transcriptional stimulation evoked by both forskolin and KCl (4). Specifically, in cerebellar granule neurones, the specific agonist baclofen inhibits forskolin-initiated CREB-transcriptional programs by lowering cytosolic cAMP or Ca2+ levels (4). Although we did not detect a baclofen-evoked alteration in the expression of the transcription factor ATF4 (CREB2), we and others have shown that CREB2 binds directly to the GABABR1 subunit via the coiled-coil domains present in both proteins (32, 43, 44). We found that activation of GABABRs in hippocampal neurones caused a dramatic translocation of ATF4 out of the nucleus and into the cytoplasm (but see Ref. 44), suggesting that this interaction could represent a novel neuronal signaling pathway. A possible role for the GABABR-ATF4 interaction is in gene regulation (48). ATF4 is a member of a family of cAMP response element-binding proteins that has been shown to negatively regulate CREB (1, 2, 46). CREB itself has been widely implicated in memory formation, and, interestingly, CREB is activated during long-term potentiation and other forms of synaptic plasticity (1, 37). Therefore, docking of ATF4 to somatodendritic GABABR could prevent its nuclear function and thereby effect the transcriptional regulation of proteins. In this way, GABABRs may influence the expression of proteins, including those important for synaptic plasticity, by both modulating the levels of cAMP (4) and by a direct interaction between GABABR and ATF4.

COPS5/Jab1
COPS5 is one of the eight subunits of the COP9 signalosome, a highly conserved protein complex that functions as an important regulator in multiple signaling pathways. The COP9 signalosome can act as a positive regulator of E3 ubiquitin ligases. It can also act as a coactivator that increases the specificity of Jun/activator protein (AP)-1 transcription factors; specifically, COPS5 selectively potentiates transactivation by c-Jun or JunD, stabilizes their complexes with AP-1 sites, and increases the specificity of target gene activation by AP-1 protein (9). COPS5 binds to and induces specific downregulation of the cyclin-dependent kinase inhibitor p27, a central mediator in the imposition and maintenance of quiescence in the cell cycle (8, 40). The physiological implications of our finding that activation of GABABRs increases COPS5 expression remain unclear. However, it is intriguing that this protein is involved in the regulation of ubiquitination, a process that tags membrane proteins for internalization and/or degradation and also cell quiescence. Modulation of these pathways, for example, by removal of excitatory channels or receptors, would be consistent with a model in which GABABR activity could dampen down cell activity in the long term.

ARHGAP4
Rho GTPases are molecular switches that control many cellular functions via the regulation of the actin cytoskeleton (28). In neurones, they are involved in neuronal migration, growth cone guidance, and synaptic formation (28). Rho GTPase-activating proteins are modulators of Rho GTPase activity in neurones (16). ARHGAP4 can stimulate the GTPase activity of three members of Rho GTPases: Rac1, Cdc42, and RhoA (16). ARHGAP4 mRNA is expressed at high levels throughout the developing and adult CNS, but protein levels are most abundant in specific regions including the hippocampus (16). In resting neurones, ARHGAP4 associates with the Golgi complex and is also present in the tips of differentiating neurites of PC12 cells (16). The fact that baclofen evoked a decrease in ARHGAP4 mRNA levels suggests that GABABR activation may reduce one or more of the cellular events mediated by this Rho GTPase-activating protein. For example, it could be envisaged that stimulation of GABABRs may oppose the induction of late-phase long-term potentiation by reducing ARHGAP4 levels and, in turn, inhibiting neurite outgrowth and synapse formation/remodeling.

Rab8IP
Rab8 is a small GTP-binding protein belonging to the RAS oncogene family. It plays a role in vesicular transport from the trans-Golgi network to the dendritic surface in hippocampal neurones. Rab8IP is a member of the serine/threonine protein kinase family similar to MAP4K2. Rab8IP undergoes autophosphorylation and is able to phosphorylate classical serine/threonine protein kinase substrates such as myelin basic protein and casein. GTP-dependent association of Rab8 with Rab8IP may be an important step in vesicle targeting or fusion (33). Rab GTPases regulate the movement of GPCRs through intracellular membrane compartments, and the activity of Rab GTPases may also influence GPCR function. Interestingly, GPCR activation may directly influence Rab GTPase activity. Thus, consistent with our finding that baclofen evokes a decrease in Rab8IP gene expression, GPCR activity could play a role in their own targeting between intracellular compartments (36).

In conclusion, we show that baclofen application to neuronal cultures in the presence of TTX alters the transcription of 20 of the 1,152 genes interrogated. Expression of these genes was unchanged when the GABABR-specific antagonist CGP-55845A was also included. While this represents only a fraction of the total number of genes of the rat genome, a number of interesting and potentially important changes have been identified. These novel data on downstream, late-phase consequences of GABABR signaling provide a basis for future studies to investigate these changes in gene expression at the protein, cell biology, and functional levels. Furthermore, the changes in gene expression identified will shed light on the multiple roles of GABABRs.


    GRANTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was supported by the Wellcome Trust, the Medical Research Council and European Commission Framework V (to J. M. Henley), the Wellcome Trust Value in People Award (to M. T. Ghorbel), and the National Institute on Aging Intramural Program (to K. G. Becker).


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: J. M. Henley, Dept. of Anatomy, School of Medical Science, Univ. of Bristol, Bristol BS8 1TD, UK (E-mail: j.m.henley{at}bris.ac.uk).


    REFERENCES
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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