Rat Frontal Cortex: Identification by
Differential Display
Len V. Hua, Marty Green, Jerry J. Warsh, and Peter P. Li
Background: Substantial evidence indicates that lithium
may exert its therapeutic effects through progressive
adaptive changes at the level of gene expression; however,
the study of lithium-regulated genes has been primarily
undertaken with the “candidate gene” approach based on
a specific testable hypothesis. The aim of our study was to
identify lithium-regulated genes that would not be
pre-dicted a priori by the candidate gene approach.
Methods: Differential display polymerase chain reaction
was used to isolate and identify messenger RNAs (mRNAs)
that are differentially expressed in the frontal cortex of
rats given lithium for 5 weeks to achieve plasma lithium
concentrations of 0.6 to 0.9 mmol/L.
Results: A putative lithium-regulated complementary
DNA fragment (LRG1) was identified. Northern blot
analysis revealed that 5 weeks of lithium treatment, but not
1 week, significantly reduced LRG1 mRNA levels. LRG1
mRNA levels were similarly reduced by 5 weeks of
carbamazepine, but not valproate administration.
Se-quence analysis and search of the GenBank database
revealed that LRG1 is analogous to the sequence of the
gene for rat aldolase A.
Conclusions: These results demonstrate that chronic
ad-ministration of lithium, but not short-term adad-ministration,
downregulates the levels of aldolase A mRNA, suggesting
this effect may play a role in mediating the therapeutic
action
of
this
agent. Biol
Psychiatry
2000;48:
58 – 64 © 2000 Society of Biological Psychiatry
Key Words: mRNA, differential display, aldolase A,
lithium, carbamazepine, gene expression, bipolar disorder
Introduction
A
lthough lithium is the primary drug used in the acute
and prophylactic treatment of bipolar affective
disor-der, the neurobiological substrates underlying its
therapeu-tic efficacy are still poorly understood. Recent evidence
suggests that molecular and cellular changes in critical
neuronal circuitry thought to be dysregulated in bipolar
disorder may be involved in its mood-stabilizing effects
(reviewed in Jope 1999; Li et al 2000; Manji et al 1995).
These compensatory and homeostatic responses, which
include modulation of intracellular signaling cascades,
produce substantial and long-lasting changes in neuronal
function, effects that reset the postulated signaling
abnor-malities in bipolar affective disorder to their normal
functional range (Hudson et al 1993; Jope 1999; Li et al
2000; Manji et al 1995).
Since the clinical action of lithium is marked by a
latency of onset, regulation of gene expression has been
hypothesized to be one of the potential mechanisms by
which lithium exerts its mood-stabilizing effects (Hyman
and Nestler 1996; Jope 1999). Indeed, a growing body of
evidence supports the notion that lithium, at clinically
effective doses, is capable of regulating the expression of
a number of genes in rat brain and cultured-cell models.
For example, chronic lithium treatment reduces the mRNA
levels of G
a
s, G
a
i1, and G
a
i2(Li et al 1991), but increases
those of adenylyl cyclase types I and II in the rat cortex
(Colin et al 1991). Long-term treatment with lithium also
increases the mRNA levels of dynorphin A, prodynorphin,
preprotachykinin (Sivam et al 1988, 1989), and dopamine
D
2receptor (Dziedzicka-Wasylewska and Wedzony 1996)
in the rat striatum. Moreover, maintained exposure to
lithium increases the levels of 2
9
,3
9
-cyclic nucleotide
3
9
-phosphodiesterase mRNA in rat C6 glioma cells (Wang
and Young 1996) and the expression of nitric oxide
synthase type 2 in rat astrocytes (Feinstein 1998). The
transcriptional effects of lithium may be mediated by
alterations in stimulus-transcriptional coupling through
dampening of the intracellular second messenger systems
From the Section of Biochemical Psychiatry, Centre for Addiction and Mental Health, Clarke Site (LVH, MG, JJW, PPL), and the Departments of Pharma-cology (LVH, JJW, PPL) and Psychiatry (JJW, PPL) and Institute of Medical Science (JJW), University of Toronto, Toronto, Canada.
Address reprint requests to Peter P. Li, Ph.D., Centre for Addiction and Mental Health, Clarke Site, Section of Biochemical Psychiatry, 250 College Street, Toronto Ontario M5T 1R8, Canada.
Received October 12, 1999; revised February 2, 2000; accepted February 7, 2000.
(Jope 1999; Li et al 1993). Accordingly, it has been shown
that chronic lithium treatment decreases basal c-fos
mRNA levels and differentially modulates the
stimulus-induced c-fos expression in the rat brain (Miller and Mathe
1997). Long-term administration of lithium also leads to
increased transcription factor binding to activator
pro-tein-1 (AP-1) and cyclic AMP-responsive element (CRE)
in the rat brain (Ozaki and Chuang 1997). Similarly, in
vitro incubation of rat C6 glioma cells and human
SH-SY5Y cells with therapeutically relevant concentrations of
lithium altered AP-1 or CRE DNA binding activity
(As-ghari et al 1998; Wang et al 1999; Yuan et al 1998). Thus,
modulation by lithium of c-fos expression or transcription
factor activity may have the potential to regulate
down-stream gene expression, ultimately promoting the cellular
adaptation in critical neuronal circuits that lead to mood
stabilization.
The changes in gene expression induced by lithium that
have been tested to date have been primarily hypothesis
driven. With the candidate gene approach, transcriptional
changes in other genes may be overlooked unless they
occur in the neurobiological cascade(s) of those genes for
which expression changes have already been found.
Dif-ferential display polymerase chain reaction (dd-PCR) is an
emerging technique that permits systematic comparison of
the entire transcript repertoire in various cells or tissues
(Liang and Pardee 1992). As an initial step in uncovering
other known or novel genes that display transcriptional
changes following chronic lithium administration, we
applied the technique of dd-PCR to screen and identify
genes that are differentially regulated in the frontal cortex
of rats treated chronically with lithium. We report here the
downregulation of aldolase A expression after 5 weeks of
lithium treatment but not after 1 week, an effect that was
shared by chronic carbamazepine (CBZ) administration
but not by valproate (VPA) administration.
Methods and Materials
Chemicals
Lithium carbonate (Li2CO3), CBZ, and sodium VPA were
obtained from Sigma Chemical Co. (St. Louis). [a-32
P]Deoxy-cytidine triphosphate ([a-32
P]dCTP; ;3000 Ci/mmol) and [a-33
P]deoxyadenosine triphosphate ([a-33
P]dATP;;2000 Ci/ mmol) were purchased from New England Nuclear (Boston). Other reagents were acquired as molecular biological grade from commercial sources.
Animal Treatments
Male Wistar rats (300 –350 g; Charles River, St. Constant, Canada) were individually housed in a temperature-controlled room (21°C61°C) and maintained on a 12-hour light/dark cycle with free access to food and water for at least a week before
experiments. Animals were fed rat chow in pellet form contain-ing Li2CO3(2.2 g/kg diet; Bioserve, Frenchtown, NJ) for 1 or 5
weeks. Water and 2.6% saline were provided ad libitum to all animals. Rats receiving CBZ were fed with food pellets contain-ing 0.25% CBZ for the first 4 days followed by 0.5% CBZ (Bioserve) for the next 31 days. In the VPA comparison group, animals were maintained on chow pellets containing 0.4% VPA (Bioserve) for 5 weeks. Separate control groups of rats were fed the regular rat chow for the indicated period. At the beginning of the experiment, there was no difference in weights of control (3266 6 g, mean6 SEM, n56) and mood-stabilizing drug treatment groups (lithium, 33168 g; CBZ, 323611 g; VPA, 325 6 3 g; n 5 6 in each group). Animals receiving mood-stabilizing drugs had weight gains [lithium, 104616 g; CBZ, 110610 g; VPA, 12665 g; F(3,20)50.865, p5.4] similar to those of control subjects (1206 7 g) during the treatment periods. Animals used in this study were cared for in strict accordance with guidelines of the Canadian Council on Animal Care, and the study was approved by the local Animal Care Committee.
At the end of the experiments, animals were decapitated. The brain was rapidly removed and the frontal cortex was dissected over ice, frozen on dry ice, and stored at270°C until use. Blood samples were collected immediately after decapitation from the cervical trunk and put into heparinized test tubes. Plasma was separated by centrifugation (900 g, 20 min) for subsequent determination of drug levels. Plasma lithium levels were deter-mined using the Vitros Li Slides (Johnson & Johnson, Missi-sauga, Canada), a colorimetric assay for lithium– crown– ether dye complex, and ranged from 0.62 to 0.92 mmol/L in the lithium-treated rats. Mean (6SEM) plasma CBZ concentrations were 1664mmol/L, as determined by the Vitros CRBM Slides (Johnson & Johnson), an immunoassay using anti-CBZ antibody. Plasma VPA levels were quantitated by the TDxFLx valproic acid assay system using fluorescence polarization immunoassay (Abbott Laboratories, Abbott Park, IL) and were 1364mmol/L in the VPA-treated rats.
Extraction of RNA
Total RNA from the frontal cortex was isolated by the guani-dinium isothiocyanate– cesium chloride method as previously described (Li et al 1993). Residual chromosomal DNA was removed from total cellular RNA using the MessageClean Kit (GenHunter, Nashville) according to the manufacturer’s instruc-tions. Ribonucleic acid quantitation was determined by absor-bance at 260 nm, and the integrity evaluated by electrophoresis on 1% formaldehyde agarose gels and ethidium bromide staining.
mRNA Differential Display
microliters of the cDNA was subjected to PCR amplification in duplicate in 20mL of PCR buffer (10 mmol/L Tris-HCl [pH 8.3], 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.001% [wt/vol] gelatin)
with 2 mmol/L each of 29-deoxynucleoside 59-triphosphate (dNTP), 0.2mmol/L of various arbitrary primers, 0.2mmol/L of the respective anchor primer, 0.3 mL of [a-33
P]dATP (2000 Ci/mmol, New England Nuclear), and 1 U of AmpliTaq (Perkin-Elmer, Branchburg, NJ). The PCR was conducted using the following program on an MJ Research (Waltham, MA) thermal cycler (model PTC-200): an initial denaturation at 95°C for 2 min followed by 40 cycles at 94°C for 30 sec, 40°C for 2 min, and 72°C for 30 sec, then a final extension at 72°C for 5 min, and rapid cooling at 4°C. The radiolabeled amplified cDNA frag-ments were then separated on a 6% (wt/vol) denaturing poly-acrylamide sequencing gel. The gel was vacuum dried at 80°C for 2 hours and autoradiographed (24 to 48 hours). Autoradio-graphic bands that were visually different in intensity between control and lithium-treated animals were excised from the dried gel and rehydrated, and the DNA was reamplified by PCR using the appropriate set of primers under the PCR conditions de-scribed above, except that the dNTP concentration was 25
mmol/L and no radioisotope was included.
DNA Cloning
The reamplified DNA was electrophoresed on a 1.5% agarose gel and the band of interest was excised and purified using a QIAEX Gel Extraction Kit (Qiagen, Chatsworth, CA) according to the manufacturer’s instructions. The recovered DNA was ligated into the pGEM-T vector system (Promega, Madison, WI). Ampicil-lin-resistant colonies were selected from ampicillin X-Gal LB agar plates and plasmid minipreps were prepared using standard procedures. Plasmids harboring inserts were identified by either restriction enzyme digestion using sites on the vector flanking the cloning sites or PCR screening using primers across the cloning sites.
Complementary DNA fragments isolated from plasmids were labeled with [a-32
P]dCTP using a random-primed DNA-labeling kit (Boehringer Mannheim Canada, Laval) to a specific activity of 13109
cpm/mg and used as probes for Northern blot analysis.
Reverse Northern Blot Analysis
Plasmids containing the cDNAs of interest or cyclophilin were denatured by boiling in 200mL of 0.4 mol/L NaOH and 10 mmol/L EDTA for 10 min, and neutralized with an equal volume of 2 mol/L ammonium acetate, pH 7.0. Each plasmid was spotted onto two replicas of nylon membrane (GeneScreen Plus, New England Nuclear) at three concentrations ranging from 0.2 to 1.8
mg using the Bio-Dot microfiltration apparatus (Bio-Rad Labo-ratories, Richmond, CA). The duplicate blots were UV-crosslinked in a UV-Stratalinker 2400 (Stratagene, La Jolla, CA), and prehybridized for 4 hours at 42°C in 53 SSPE, 50% formamide, 53Denhardt’s solution, 1% sodium dodecyl sulfate, 10% dextran sulfate, and 200 mg/mL denatured salmon sperm DNA. Hybridization was performed in the same buffer overnight at 42°C in the presence of equal amounts (13106
cpm/mL) of the cDNA probe from control or lithium-treated animals. The
cDNA probes were prepared to a specific activity of 13 107
cpm/mg from 30mg of each of the two RNA samples by reverse transcription in a reaction mixture containing 50 mmol/L Tris-HCl, pH 8.3; 75 mmol/L KCl; 3 mmol/L MgCl2; 4mmol/L oligo
(dT18); 100mCi [a 32
P]dCTP; 300mmol/L each of dATP, dTTP, and dGTP; 4mmol/L DTT; and 1000 U SuperScript II reverse transcriptase (GIBCO-BRL), and incubation at 42°C for 1 hour. The hybridization signals between duplicate blots were quanti-tated using a phosphoimaging detection system and the Image-Quant program (STORM, Molecular Dynamics, Sunnyvale, CA) and normalized against cyclophilin. As a negative control sub-stance, plasmid without the insert was included on each blot.
Northern Blot Analysis
Ten micrograms of heat-denatured total RNA from individual control and experimental animals were separated by electro-phoresis on 1% agarose gels containing 0.66 mol/L formalde-hyde. The RNA was transferred by capillary action onto Gene-Screen Plus membranes with 103 SSC, following by UV crosslinking and baking at 80°C for 2 hours. The blots were prehybridized at 42°C for 2– 4 hours in 53SSPE, 50% form-amide, 53Denhardt’s solution, 1% sodium docecyl sulfate, 10% dextran sulfate, and 200mg/mL denatured salmon sperm DNA, and subsequently hybridized with a32
P-labeled, random-primed cDNA probe (1 3106
cpm/mL) prepared from a gel-purified insert. After stringency washing, hybridization signals were obtained by autoradiography using phosphoscreens. The blots were stripped and rehybridized with 32
P-labeled rat cyclophilin cDNA to normalize for RNA loading and transfer.
DNA Sequence Analysis
The pGEM-T clone inserts were manually sequenced from both directions with T7 or M13-Reverse primer using the Thermo-Sequenase sequencing kit (Amersham Pharmacia Biotech, Baie d’Urfe´, Canada). Homology search was performed using the GenBank DNA database and the BLAST algorithms.
Statistical Analysis
Statistical analysis of the data was performed using either one-way analysis of variance, followed by post hoc Tukey’s test to assess differences between cell means or two-tailed unpaired Student’s t test. Values of p,.05 were considered statistically significant. Data were expressed as mean6SEM.
Results
mRNA Differential Display
lithium-treated rats. A subset of 10 clones was found to be
upregulated by chronic lithium treatment, whereas a subset
of four clones was downregulated. No apparent
differ-ences were observed for the remaining six clones.
One of these clones, representing a cDNA fragment of
;
550 base pairs (bp) that was amplified to a significantly
lesser extent from the RNA of the lithium-treated rats
(LRG1), was selected for further analysis (Figure 1A).
LRG1 hybridized with high stringency to an mRNA of 1.4
kilobases (kb) in the rat frontal cortex (Figure 1B). The
frontocortical levels of LRG1 mRNA were significantly
decreased [t(16)
5 2
2.33, p
5
.03] following chronic
lithium treatment (Figure 2). In contrast, short-term (1
week) lithium treatment was without effect [t(10)
5
0.38,
p
5
.71] on the abundance of LRG1 mRNA (Figure 2).
Effects of Chronic Treatment of Other Mood
Stabilizers on LRG1 mRNA Levels
To assess whether the regulation of LRG1 mRNA level
was unique to lithium or common to other
mood-stabiliz-ing agents, the effects of chronic lithium, VPA, and CBZ
treatment on the levels of LRG1 mRNA were studied
(Figure 3). The analysis of variance showed a significant
main effect of drug on LRG1 mRNA levels [F(3,16)
5
5.70, p
5
.008]. Post hoc analysis revealed that the levels
of LRG1 mRNA were significantly reduced in the rat
frontal cortex following either chronic CBZ [t(8)
5
2
4.92, p
5
.006] or lithium [t(8)
5 2
3.75, p
5
.04]
treatment. There was a tendency for a decrease in LRG1
mRNA level (84% of control group) following chronic
VPA administration; however, these changes were very
small and not statistically significant [t(8)
5 2
2.15, p
5
.36].
Sequence Analysis and Homology
Results from the DNA sequencing revealed that LGR1
contained 540 bp and had the expected primers at its 5
9
and 3
9
ends. LRG1 contained a putative polyadenylation
signal that was located 21 bp upstream from the poly A
1tail, further supporting the identity of this cDNA fragment
as the 3
9
-untranslated end of the corresponding transcript.
Using the BLAST algorithms to search the GenBank
database, the sequence of LRG1 displayed a near perfect
homology (
.
99%) to the nucleotide sequence (921–1441)
for rat aldolase A mRNA (Figure 4).
Discussion
In our study the technique of dd-PCR was used to identify
genes that show transcriptional changes in response to
chronic lithium administration. In particular, the
expres-sion of a cDNA fragment (LRG1) was downregulated by
lithium in the rat frontal cortex following a 5-week
treatment period. Northern blot analysis confirmed the
decreased expression of this transcript by chronic but not
short-term lithium treatment.
Comparison of the sequence for LRG1 against those in
the GenBank database revealed that this fragment
corre-sponds to aldolase A, a key enzyme in glycolysis
produc-ing energy. This is further supported by the findproduc-ing that the
LRG1 probe hybridized to a single transcript of 1.4 kb, as
previously reported for aldolase A (Joh et al 1985).
The biochemical role of aldolase A was originally
described in relation to the glycolysis pathway catalyzing
the aldolol cleavage of fructose 1,6-diphosphate. Previous
studies have shown that lithium in vitro inhibits the
activity of regulatory metabolic enzymes such as fructose
1,6-bisphosphatase (Marcus and Hosey 1980) and
glyco-gen synthase kinase-3
b
(GSK-3
b
; Klein and Melton
1996). Thus, the reduction in aldolase A mRNA levels
could conceivably be related to the effect of lithium on
carbohydrate metabolism; however, the associations
be-tween the temporal effect of lithium on aldolase A
expression and the apparent delay in the therapeutic
response to lithium in bipolar affective disorder patients
suggest that the changes in aldolase A expression may be
therapeutically relevant. Indeed, aldolase A has been
reported to modulate several other cellular functions that
on closer perusal suggest effects that could be important to
the therapeutic action (or side effects) of lithium. For
example, aldolase A has been shown to interact physically
Figure 1. (A) Differential display of messenger RNA (mRNA) comparing gene expression in the frontal cortex from represen-tative control and lithium-treated rats. The autoradiogram of [a-33
P]deoxyadenosine triphosphate–labeled differentially dis-played products, using T12MC as 39 primer and AP3
with tubulin and actin filaments, an action that may
contribute to the regulation of cytoskeletal structures and
cell mobility (Kao et al 1999; Kusakabe et al 1997; Volker
and Knull 1997). Second, the presence of aldolase A in the
nucleus and its ability to interact with certain DNA
sequences suggest a potential role of aldolase A in
modulating transcriptional activity (Ronai et al 1992).
Furthermore, the finding that aldolase A binds inositol
1,4,5-trisphosphate (InsP
3) suggests a potential role for
this enzyme in regulating the free intracellular
concentra-tions of this second messenger, and subsequently
intracel-lular calcium dynamics (Baron et al 1999; Koppitz et al
1986). This is of particular pathophysiologic importance
given the well-documented observations of altered
cal-cium homeostasis in bipolar affective disorder (Dubovsky
et al 1992; Emamghoreishi et al 1997). Although it may
seem difficult at first to ascribe any direct significance of
these diverse cellular functions affected by aldolase A to
the therapeutic action of lithium, the observations that
chronic lithium treatment exerts modulatory effects on
neuronal cytoskeletal architectures, transcription factors
activity, InsP
3signaling, and intracellular calcium
dynam-ics (Jope 1999) shed a different light on the role of
aldolase A in these cellular changes regulated by lithium.
Moreover, it has recently been suggested that lithium
regulation of cytoskeletal networks and AP-1
transcrip-tional factor activity may be mediated via inhibition of
GSK-3
b
, another multifunctional metabolic enzyme (for a
review see Jope 1999).
With the above considerations, it is not unreasonable to
assume that some of these diverse cellular effects of
lithium may also involve other metabolic enzymes such as
aldolase A. The recent suggestion that GSK-3
b
may
represent a potential target of lithium in the treatment of
bipolar affective disorder (Agam and Levine 1998; Klein
and Melton 1996) underscores the importance of lithium
regulation of “multifunctional” metabolic enzyme(s) in its
psychotherapeutic mechanisms. Of additional interest in
this regard, increased serum aldolase A activity has been
reported in a much earlier study of a small group of manic
bipolar disorder patients (Meltzer 1968). Although the
significance of the increased aldolase A activity was not
clear at that time, it was suggested that an increase in cell
permeability or decreased clearance of the enzyme from
serum might be the peripheral manifestation of a
patho-logic process occurring in the central nervous system
(Meltzer 1968). Although this has not yet been proven, our
findings of reduced expression of aldolase A following
chronic lithium treatment offer a more interesting
possi-bility that dysregulation of aldolase A might occur in the
brain of bipolar disorder patients.
Although an array of neurochemical, cellular, and
mo-Figure 2. Effect of short-term and chronic lithium administration on the expression of LRG1 messenger RNA (mRNA) in the rat frontal cortex. Rats were fed with 0.22% Li2CO3 for 1 or 5
weeks, and levels of LRG1 mRNA were determined by Northern blot analysis. Representative autoradiograms are shown in the top panels of each bar graph. The results are expressed as the mean6SEM for six to nine animals. *p5.03 compared with control animals (Student’s t test).
lecular actions of lithium has been described (Jope 1999;
Li et al 2000; Manji et al 1995), it is often difficult to
identify which of these targets underlie the
mood-stabiliz-ing effect of lithium and which reflect secondary or side
effects of the drug. We have approached this question by
determining whether alteration in aldolase A expression is
shared by other mood-stabilizing agents. We found that
chronic CBZ administration, but not VPA administration,
also reduced the expression of aldolase A. The similar
changes in aldolase A mRNA produced by chronic lithium
and CBZ treatment may represent one of the molecular
correlates of the therapeutic overlap (or side effects) of
these mood stabilizers in the treatment of bipolar
disor-ders. On the other hand, the lack of effect of chronic VPA
administration on aldolase A expression may represent
dissimilar molecular mechanisms of action for lithium and
VPA, a notion supported by clinical evidence suggesting
that VPA is more efficacious than lithium in the treatment
of rapid-cycling bipolar affective disorder and mixed
manic states (Calabrese et al 1994).
In conclusion, in this study we have provided the first
evidence that chronic lithium administration regulates the
expression of aldolase A, a multifunctional enzyme, in the
brain. These findings raise the possibility that
downregu-lation of aldolase A expression may play a role in
mediating the therapeutic action of lithium.
This study was supported by a Canadian Psychiatric Research Founda-tion grant and an Ontario Mental Health FoundaFounda-tion grant to Dr. Li.
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