Expression of the Rap1 guanine nucleotide exchange factor, MR-GEF, is altered in individuals with bipolar disorder.

Abstract

In the rodent forebrain GABAergic neurons are generated from progenitor cells that express the transcription factors Dlx1 and Dlx2. The Rap-1 guanine nucleotide exchange factor, MR-GEF, is turned on by many of these developing GABAergic neurons. Expression of both Dlx1/2 and MR-GEF is retained in both adult mouse and human forebrain where, in human, decreased Dlx1 expression has been associated with psychosis. Using in situ hybridization studies we show that MR-GEF expression is significantly down-regulated in the forebrain of Dlx1/2 double mutant mice suggesting that MR-GEF and Dlx1/2 form part of a common signalling pathway during GABAergic neuronal development. We therefore compared MR-GEF expression by in situ hybridization in individuals with major psychiatric disorders (schizophrenia, bipolar disorder, major depression) and control individuals. We observed a significant positive correlation between layers II and IV of the dorso-lateral prefrontal cortex (DLPFC) in the percentage of MR-GEF expressing neurons in individuals with bipolar disorder, but not in individuals with schizophrenia, major depressive disorder or in controls. Since MR-GEF encodes a Rap1 GEF able to activate G-protein signalling, we suggest that changes in MR-GEF expression could potentially influence neurotransmission.

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Expression of the Rap1 Guanine Nucleotide Exchange
Factor, MR-GEF, Is Altered in Individuals with Bipolar
Disorder
Angela Bithell1¤a, Tony Hsu2., Apsara Kandanearatchi1.¤b, Sabine Landau3, Ian P. Everall1,2¤c, Ming T.
Tsuang2, Gursharan Chana1,2¤c, Brenda P. Williams1*¤a
1Department of Psychological Medicine, Institute of Psychiatry, King’s College London, London, United Kingdom, 2Center for Behavioral Genomics, Department of
Psychiatry, University of California San Diego, San Diego, California, United States of America, 3Department of Biostatistics and Computing, Institute of Psychiatry, King’s
College London, London, United Kingdom
Abstract
In the rodent forebrain GABAergic neurons are generated from progenitor cells that express the transcription factors Dlx1
and Dlx2. The Rap-1 guanine nucleotide exchange factor, MR-GEF, is turned on by many of these developing GABAergic
neurons. Expression of both Dlx1/2 and MR-GEF is retained in both adult mouse and human forebrain where, in human,
decreased Dlx1 expression has been associated with psychosis. Using in situ hybridization studies we show that MR-GEF
expression is significantly down-regulated in the forebrain of Dlx1/2 double mutant mice suggesting that MR-GEF and Dlx1/
2 form part of a common signalling pathway during GABAergic neuronal development. We therefore compared MR-GEF
expression by in situ hybridization in individuals with major psychiatric disorders (schizophrenia, bipolar disorder, major
depression) and control individuals. We observed a significant positive correlation between layers II and IV of the dorso-
lateral prefrontal cortex (DLPFC) in the percentage of MR-GEF expressing neurons in individuals with bipolar disorder, but
not in individuals with schizophrenia, major depressive disorder or in controls. Since MR-GEF encodes a Rap1 GEF able to
activate G-protein signalling, we suggest that changes in MR-GEF expression could potentially influence neurotransmission.
Citation: Bithell A, Hsu T, Kandanearatchi A, Landau S, Everall IP, et al. (2010) Expression of the Rap1 Guanine Nucleotide Exchange Factor, MR-GEF, Is Altered in
Individuals with Bipolar Disorder. PLoS ONE 5(4): e10392. doi:10.1371/journal.pone.0010392
Editor: Katharina Domschke, University of Muenster, Germany
Received June 16, 2009; Accepted March 3, 2010; Published April 28, 2010
Copyright: ? 2010 Bithell et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Stanley Foundation (http://www.stanleyfoundation.org/), the Wellcome Trust (http://www.wellcome.ac.uk/) and the
Medical Research Council (http://www.mrc.ac.uk/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: brenda.p.williams@kcl.ac.uk
. These authors contributed equally to this work.
¤a Current address: Centre for the Cellular Basis of behaviour, The James Black Centre, Institute of Psychiatry, King’s College London, London, United Kingdom
¤b Current address: Centre for Immunology, St Vincent’s Hospital/University of New South Wales, Sydney, New South Wales, Australia
¤c Current address: Department of Psychiatry, The University of Melbourne, Royal Melbourne Hospital, Parkville, Victoria, Australia
Introduction
Abnormalities in cortical GABAergic interneurons have been
associated with psychiatric illness [1,2]. In schizophrenia, both
synthesis and reuptake of GABA are disrupted [3–7] and
parvalbumin expressing chandelier neurons are preferentially
affected [8–10]. Expression of GABAA[11,12,13] and GABAB
[14,15] receptors is also altered; possibly reflecting compensation
by the brain for decreased GABA. Similar GABAergic neuronal
changes occur in bipolar disorder [5,15].
GABABreceptors signal via guanine nucleotide binding proteins
(G proteins) whose activity is regulated by guanine nucleotide
exchange factors (GEFs) that activate signalling, and GTPase-
activating proteins (GAPs) that inhibit signalling. We have shown
that expression of a GEF, called mr-gef, is turned on in developing
rodent GABAergic neurons [16]. The human homologue of this
gene, called M-Ras-regulated GEF (MR-GEF or RAPGEF5),
specifically activates the small GTPase Rap1 [17], known to
regulate neurite outgrowth and synaptic transmission [18,19].
Since many susceptibility genes for schizophrenia influence
neurotransmission, synaptogenesis or synaptic plasticity [20,21],
it is possible that some of the observed GABAergic neuronal
abnormalities in schizophrenia and bipolar disorder are associated
with alterations in G protein signalling. Indeed, microarray
analysis of gene expression differences associated with schizophre-
nia indicated that the most altered group of genes are those
involved in presynaptic secretory function and in particular the
regulator of G-protein signalling 4 gene (RGS4), encoding a
GTPase activating protein, the expression of which was dramat-
ically reduced in schizophrenia [22,23].
Rodent forebrain GABAergic neurons are generated from
progenitor cells that express the transcription factors Dlx1 and
Dlx2 [24]. mr-gef is expressed by many of these developing
GABAergic neurons and its expression is retained in adult rodent
[16] and human brain [17]. Dlx1 and Dlx2 are also expressed
widely throughout the adult mouse forebrain where they are
involved in the migration, proliferation and neuronal sub-type
specification of progenitor cells along the rostral migratory stream
[25–27], and also in the maturation and survival of interneurons
[28]. Little is known about Dlx expression in adult human brain;
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however, decreased Dlx1 expression in the thalamic mediodorsal
nucleus, the principal source of thalamic afferents to the cortex
that synapse on both pyramidal neurons and interneurons [29],
has been associated with psychosis [30].
Here, we report that mr-gef expression is significantly down-
regulated in mice that do not express Dlx1 and Dlx2, suggesting
that they form part of a common signalling pathway during
GABAergic neuronal development. Further, in the human
DLPFC we observed a significant positive correlation between
cortical layers II and IV in the percentage of MR-GEF expressing
neurons in individuals with bipolar disorder, but not those with
schizophrenia, major depressive disorder or controls. We further
observed a significant positive correlation in the two-dimensional
(2D) neuronal density between layers II and IV in individuals with
bipolar disorder and schizophrenia, possibly reflecting a common
pathology in these diseases.
Methods
Brain Tissue
Post-mortem brain sections (14 mm) from Brodmann area 9/46
(BA9/46) of the DLPFC were obtained from the Stanley
Foundation Brain Consortium, an established collection of brain
samples obtained with full consent of next of kin. The sample
consisted of 60 subjects: 15 controls, 15 with schizophrenia, 15
with bipolar disorder, and 15 with major depressive disorder.
None of these brains demonstrated evidence of neurodegenerative
changes or other pathologic lesions. This study was performed
blind to diagnosis on all 60 subjects, however 8 subjects were
excluded from analysis due either to severe tissue damage (n=1)
or due to severe difficulty in locating landmarks to compare Nissl
and MR-GEF slides because of poor tissue quality in the desired
region (n=7). A summary of demographic, clinical and histolog-
ical information of the samples used in this study is given in Table
S1. This study was carried out with the approval of the Institute of
Psychiatry/South London and Maudsley NHS Trust Ethical
Committee (ethical study number 153/01).
Dlx1/2 mutant mouse brains were the kind gift of John
Rubenstein and Stewart Anderson [24].
Non-radioactive in situ hybridisation and Nissl staining
Sense and anti-sense probes were designed to specifically target
a region from within the open reading frame (ORF) of the rodent
(mr-gef) [16] or human (MR-GEF) transcript. The human MR-
GEF mRNA comprises a short 59 untranslated region (UTR), an
ORF of 1740 bp and a long 39 UTR of more than 3 kb that
generates a single transcript (see Figure S1) [17].
Additional cDNAs used for riboprobe synthesis were Lhx6
(Lhx6 cDNA was a gift from Vassilis Pachnis and Maria
Grigoriou, NIMR, London, UK) and Dlx1 (Dlx1 cDNA was a
gift from John Rubenstein, University College of San Francisco).
Sections were fixed for 10 minutes in cold 4% paraformalde-
hyde in 0.1 M phosphate buffer before being washed in Depc-PBS
(diethylpyrocarbonate treated phosphate buffered saline), incubat-
ed in acetylation solution for 10 minutes and permeabilised by
incubation in 1% Triton X-100 (Sigma) for 5 minutes (human
tissue) or 30 minutes (mouse tissue). After further washes in Depc-
PBS, slides were placed in a hybridisation chamber humidified
with 50% formamide and 5xSSC. Sections were pre-hybridised in
500 ml hybridisation buffer (50% formamide, 5xDepc-SSC, 5x
Denhardts, 0.25 mg/ml yeast RNA, 0.5 mg/ml herring sperm
DNA) for 2–6 hours at room temperature. DIG-labelled RNA
probes (100–200 ng/ml diluted in hybridisation buffer) were
denatured at 80uC for 5 minutes then cooled on ice. The probe/
hybridisation buffer mix was pipetted onto sections and the slides
covered with glass coverslips. Hybridisation was carried out at
65uC overnight in a sealed, humidified hybridisation chamber.
Sections then went through a series of washes (0.2xSSC at 65uC
for 1 hour; 0.2xSSC for 5 mins at room temperature; 0.1 M Tris
pH 7.5/150 mM NaCl for 5 mins at room temperature) before
being blocked in 10% heat inactivated sheep serum (Sigma,
diluted in 0.1 M Tris pH 7.5/150 mM NaCl) for 1–3 hours at
room temperature. To detect the DIG-labelled riboprobes, 500 ml
of anti-DIG Fab-AP antibody (Roche, diluted 1:5000) was added
to each slide and incubated overnight at 4uC in a humidified
chamber. Slides were then washed 365 minutes in 0.1 M Tris
pH 7.5/150 mM NaCl, followed by 5 minutes in 0.1 M Tris
pH 9.5, 100 mM NaCl, 50 mM MgCl2 with 0.24 mg/ml
levamisole (Sigma) to inhibit endogenous phosphatase activity.
Bound probes were visualised by incubation in the dark in colour
solution (50 ng/ml NBT [Roche] and 75 ng/ml BCIP [Merck]
with levamisole). Reactions were stopped in water or 1x TE (sense
and anti-sense reactions were stopped at the same time). In all
cases sense probes showed no specific labelling (see Figure S1).
Slides were mounted in Faramount aqueous mounting medium
(Dako) and then either photographed using a Nikon Eclipse
microscope and processed with Lucia G and Adobe Photoshop
imaging software, for mouse tissue, or analysed as described below
for human post-mortem tissue.
For human post-mortem analyses, Nissl staining on adjacent
sections to those processed for non-radioactive in situ hybridisation
was performed as described previously [31].
Image analysis and counting
Tissue sections were viewed using a 20x objective on a Leica
BMLB microscope equipped with a Hitatchi colour camera and a
Marzhauser x-, y-motorised stage. Similar regions in area BA46
were selected on each slide based on cytoarchitecture. Obvious
landmarks were chosen (blood vessels, contour of tissue) in Nissl
stained sections to allow the same region to be detected on
adjacent MR-GEF stained sections. Composite images from the
pial surface to the grey/white matter border were generated as
previously described [31,32]. The size of each composite image
was standard in the x-axis (600 mm) but varied in the y-axis
according to the cortical thickness. Layers were identified,
manually inserted onto the Nissl image and then overlaid onto
MR-GEF images to allow layer identification. The soma of stained
cells was outlined manually to ensure all cell outlines were accurate
and all non-neuronal material such as blood vessels were excluded.
Two sections were analysed for each case and from each
composite image two fields were chosen for counting. Within
each field the total number of neurons within our two-dimensional
plane were counted using the Nissl stained sections. MR-GEF
labelled/Nissl stained cells were counted from an identical region
on the adjacent section and their percentage of the total Nissl
stained population calculated. Somal sizes were measured by a
computer algorithm within Image Pro Plus 4.0, converting pixels
covered by cell outlines to square microns by using a calibration
factor for the x20 objective used.
Statistical analysis
Identification of Adjustment Variables.
demographic variables were included as co-variates where they
were found to correlate with outcome at p,0.05. This included
assessment of medications via lifetime fluphenazine equivalents,
substance abuse and use of ethanol as supplied to us by the Stanley
Foundation. In addition, in order to control for potential
differential shrinkage of layers 2 and 4 for individual cases we
Post-mortem and
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calculated the percentage of the cortical width that each case
represented and analysed differences between groups at p,0.05.
We found no significant differences in the proportions of layer II
and IV to the whole cortical width between any of the psychiatric
groups versus controls.
Group Comparisons.
Statistical analysis was carried out
using a one-way analysis of variance (ANOVA) to test for
differences in estimates between psychiatric and control groups
using SPSS 18.0 (SPSS, Inc.) at a bonferroni adjusted significance
level of p,0.05/2=0.025. We determined that this analysis was
appropriate after showing that our data was normally distributed
for neuronal somal size; both mean and median neuronal somal
size was analysed and no significant difference were observed
between groups. The percentage of neurons that expressed MR-
GEF and the 2D areal density of neurons in layers II and IV was
analysed in sections from patient versus control groups. We also
carried out a pearson correlation analysis between layers II and IV
for the percentage of MR-GEF expressing neurons as well as total
neurons (expressing and non-expressing) within groups. The main
objective of the statistical analysis was to compare the outcome in
each of the patient groups compared with the control group and to
identify any relationships that may be specific to the major
psychiatric disorder under investigation.
Results
mr-gef expression is perturbed in the Dlx1/2 mutant
forebrain
During development, mr-gef expression showed a striking
overlap with the LIM-homeobox gene, Lhx6, but a reciprocal
pattern of expression with the transcription factor Dlx1
(Figure 1A–C). Lhx6 is expressed by, and required for, the
migration of GABAergic neurons during development [33],
while Dlx1, together with its family member Dlx2, is necessary
Figure 1. Association between mr-gef and Dlx1 expression. Regional expression of mr-gef, Lhx6 and Dlx1 in the E14.5 mouse ventral
telencephalon (A–C). mr-gef and Lhx6 are expressed in the SVZ and mantle of the MGE (A,B respectively). mr-gef is expressed in regions adjacent to
those in the VZ and SVZ expressing Dlx1 (A and C respectively) and in a band of cells reaching up into the Ctx (arrow A). A similar mr-gef expression
pattern is observed in the ventral telencephalon in the Dlx1/2 heterozygote (+/2, D), with highest levels in the striatum (white arrow) and septum.
Higher power magnification of striatum and cortex is shown in F and G respectively. In the Dlx1/2 double mutant telencephalon, mr-gef expression is
greatly reduced, particularly in the striatum (black arrow, D’). Expression in the mutant striatum and cortex is shown at higher power in F’ and G’
respectively. For comparison, Lhx6 expression is shown in adjacent sections from the same heterozygote and mutant telencephalon (E and E’
respectively), with a loss of Lhx6 expression in the cortex of the mutant (E’). Abbreviations: VZ, ventricular zone; SVZ, subventricular zone; MGE,
medial ganglionic eminence; Str, striatum; Ctx, cortex; Sep, septum. Scale bars: 250 mm.
doi:10.1371/journal.pone.0010392.g001
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for the correct development of many GABAergic neurons [24].
Both Lhx6 and mr-gef were expressed in the subventricular zone
(SVZ) of the striatal anlage (termed the lateral and medial
ganglionic eminences, LGE and MGE, respectively, in rodent);
in the mantle zone (MZ) composed of young neurons; and in a
band of cells reaching up into the cortex (Figure 1 arrows in A, B
) composed of migrating GABAergic neurons [33]. In contrast,
Dlx1 expression was restricted to the ventricular zone (VZ) and
the SVZ. Thus, mr-gef was expressed in cells lying directly
adjacent to Dlx1 expressing cells (Figure 1, compare A with C),
suggesting that mr-gef expression was turned on in young
neurons as they differentiated from Dlx1 (and Dlx2) expressing
progenitor cells.
To determine whether Dlx1 and 2 were required for mr-gef
expression, we compared mr-gef expression in the forebrain of mice
where both Dlx1 and Dlx2 have been knocked out (Dlx1/2 2/2
mice) with expression in heterozygote littermates as controls
(Figure 1 D, D’ – G, G’). In the E16.5 Dlx1/2 heterozygote
forebrain intense mr-gef expression was observed in the striatum
(str),corticalplate(ctx),septum(sep)andpallidum(Figure1Dand at
higher magnification in F and G). In sharp contrast, mr-gef
expression in the Dlx1/2 2/2 forebrain was dramatically reduced
within the striatum (Figure 1, compare D, D9 and F, F9) and cortical
plate (Figure 1, compare D, D9 and G, G9). This latter observation
could be explained by a failure of some ventrally-derived mr-gef
expressing cells to migrate into the cortex, since GABAergic
neuronal development is disrupted in these mice and migration
of Lhx6-expressing GABAergic neurons into the cortex is
inhibited [24,25]. This migration failure can be observed by
comparing Lhx6 expression in the heterozygote and mutant
forebrain (Figure 1 E and E9 respectively). We assume that
the remaining mr-gef expression in the cortex is regulated by
other transcription factors [16]. Thus, these studies suggest that mr-
gef expression in the majority of striatal neurons, and in
a subpopulation of cortical plate neurons, requires Dlx1 and/
or Dlx2.
MR-GEF & NISSL expression in human DLPFC layer II and
layer IV
Our observed reduction in striatal and cortical mr-gef
expression in Dlx1/2 2/2 mice, together with the previously
reported observation that Dlx1 expression is decreased in the
mediodorsal thalamic nucleus (MDTN) in patients with psychosis
[30], prompted us to determine whether MR-GEF expression was
perturbed in individuals with psychiatric disorders. The MDTN is
the major source of excitatory thalamic input to the DLPFC and a
decrease in the neuronal number and volume of this nucleus has
been reported in schizophrenia [34,35]. Although the pyramidal
neurons in cortical layers III and V are the primary targets of these
thalamic afferents, these pyramidal neurons also require GABAer-
gic input in order to function properly. Indeed, GABAergic
neurons may also receive a direct input from thalamic afferents
[29]. This intimate relationship between GABAergic interneurons,
pyramidal neurons and neuronal input from the MDTN, suggests
that the functional impairment of any one of these cell types could
cause dysregulation of excitatory input and output from this brain
region.
When comparing Nissl stained and MR-GEF labelled serial
sections it was apparent that MR-GEF expression in the human
DLPFC was not restricted to GABAergic neurons (Figure 2).
However, the developmental relationship observed between Dlx1,
mr-gef and GABAergic neurons prompted us to focus our initial
MR-GEF expression study on layers II and IV of the DLPFC since
these layers are rich in a variety of GABAergic neuronal subtypes
[10,36,37]. Because these layers also include small pyramidal
neurons, it is possible that the types of neurons expressing MR-
GEF could vary between patient groups and/or between patient
and control groups. We therefore compared the somal sizes of
neurons expressing MR-GEF in layers II and IV from each group
(Figure 3). We reasoned that a shift toward larger somal sizes
would indicate that more pyramidal neurons were expressing MR-
GEF while a shift toward smaller somal sizes would indicate that
more GABAergic neurons expressed this gene. The range of sizes
observed was very similar across all groups (see Figure 3).
However, in both layer II and IV there appeared to be fewer
neurons of all sizes in individuals with bipolar disease, and in layer
II there appeared to be more neurons of all sizes in individuals
with schizophrenia (Figure 3). This difference was not reflected in
Figure 2. MR-GEF labelled and Nissl stained sections. Compar-
ison of Nissl stained and MR-GEF labelled serial sections showing that
MR-GEF is expressed by cells throughout the cortex and is not confined
to GABAergic neurons.
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our statistical analysis looking at both mean and median somal
sizes, where no significant difference was observed between
groups.
When the percentage of neurons expressing MR-GEF was
calculated and compared across all groups and layers no
significant difference was observed. Neither was there any
Figure 3. Distribution of MR-GEF-expressing neuronal sizes by group for cortical layers II and IV. Comparison of the range of somal sizes
of MR-GEF expressing neurons in layer II (A) and layer IV (B) of the DLPFC. Abbreviations: BPD, bipolar disorder; MDD, major depressive disorder; SCZ,
schizophrenia.
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