JOURNAL OF NEUROCHEMISTRY| 2016 | 138 | 448–456,1

doi: 10.1111/jnc.13661,1,1*Department of Chemistry, Graduate School of Science, Chiba University, Inage-ku, Chiba, Japan†Animal Resource Development Unit and Genetic Engineering Team, Riken Center for Life Science
Technologies, Kobe, Japan

Abstract
The g isozyme of diacylglycerol kinase (DGK) is highly
expressed in the hippocampus and Purkinje cells in thecentral nervous system. Recently, several genome-wide
association studies have implicated DGKg in the etiology ofbipolar disorder (BPD). However, it is still unknown whether
DGKg is indeed related to BPD. In this study, we generated
DGKg-knockout (KO) mice and performed behavioral tests
such as the open ﬁeld test, the elevated plus maze test and tailsuspension test using the KO mice to investigate the effects of
DGKg deﬁcits on psychomotor behavior. Intriguingly, DGKgKO mice displayed an overall behavioral proﬁle that is similar
to human mania, including hyperactivity, less anxiety and lessdepression-like behavior. In addition, these phenotypes weresigniﬁcantly attenuated by the administration of a BPD (mania)

remedy, namely, lithium. Moreover, DGKg-KO mice showed
impairment in glycogen synthase kinase (GSK) 3b signaling,which is closely related to BPD. These ﬁndings clearly support
the linkage between BPD and DGKg that is implicated bygenome-wide association studies. Moreover, this study provides DGKg-KO mice as a previously unrecognized modelthat reﬂects several features of human BPD with manic
episodes and revealed an important role for DGKg in
regulating behavior and mood through, at least in part, GSK3b
signaling.
Keywords: bipolar disorder, depression, diacylglycerol,
diacylglycerol kinase, lithium, mania, phosphatidic acid,signal transduction.
J. Neurochem. (2016) 138, 448–456.

Bipolar disorder (BPD) is a mental disorder characterized
by unusual shifts in mood from the heights of mania to thedepths of depression (Martinowich et al. 2009). The
lifetime prevalence of BPD was thought to be approximately 1%, but current reports indicate that this ﬁgure may
be closer to 5% (Martinowich et al. 2009). Because of theelevated morbidity and mortality suffered by individuals
with the disorder, BPD has been increasingly recognized asa major health problem. Despite advances in its diagnosis
and recognition, the underlying neurobiology of BPD
remains largely unknown.
Recently, several genome-wide association studies(GWASs) conducted by Baum et al. (2008), Weber et al.
(2011) and Zeng et al. (2011) have successively implicated
DGKg (diacylglycerol kinase (DGK) g gene) in the
etiology of BPD. Moreover, Moya et al. (2010) also foundthat in patients with BPD, DGKg mRNA levels are
signiﬁcantly increased. Therefore, DGKg is attracting muchattention as a BPD-related candidate gene. However, it isstill unknown whether DGKg is indeed related to BPD
because DGKg gene-deﬁcient animals have not yet been
established.
DGK phosphorylates diacylglycerol to generate phosphatidic acid (Goto et al. 2006; Sakane et al. 2007; Merida
et al. 2008; Topham and Epand 2009). Ten mammalian
DGK isozymes (a–j), which commonly contain two or three448

Received March 7, 2016; revised manuscript received April 12, 2016;
accepted April 14, 2016.
Address correspondence and reprint requests to Fumio Sakane,
Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan. E-mail:sakane@faculty.chiba-u.jp
1
These authors contributed equally to this work.
Abbreviations used: ADHD, attention deﬁcit hyperactivity disorder;
BPD, bipolar disorder; DGK, diacylglycerol kinase; GSK, glycogensynthase kinase; GWAS, genome-wide association study; KO, knockout.

© 2016 International Society for Neurochemistry, J. Neurochem. (2016) 138, 448--456

Diacylglycerol kinase g and bipolar disordercharacteristic cysteine-rich, zinc ﬁnger-like C1 domains anda catalytic region, are subdivided into ﬁve groups according
to their structural features. DGKg belongs to type II DGK(Sakai and Sakane 2012). Type II DGKs commonly have apleckstrin homology domain at their N-termini and acatalytic domain that is divided into two subdomains. We
revealed that DGKg positively regulates the epidermal
growth factor receptor pathway through the activation of
C-Raf in HeLa cervical cancer cells (Yasuda et al. 2009).
Intriguingly, this DGK isozyme is most abundantly
expressed in the brain (Klauck et al. 1996; Usuki et al.
2015). The expression of DGKg increased between 1 and4 weeks, which is coincident with synapse formation of thebrain (Usuki et al. 2015). Moreover, a substantial amount of
DGKg was detected in layers II–VI of the cerebral cortex; inthe CA1, CA2, and dentate gyrus regions of the hippocampus; in the mitral cell and glomerular layer of the olfactorybulb; and in the Purkinje cells in the cerebellum of one- to
32-week-old mice (Usuki et al. 2015). However, the functionof DGKg in the brain remains unclear.
The purpose of our present work, based on the results of
GWASs (Baum et al. 2008; Weber et al. 2011; Zeng et al.
2011), was to investigate the involvement of DGKg in BPD togain insight into the neurogenic mechanisms of BPD. For thispurpose, we have generated DGKg-knockout (KO) miceand used these mice to perform behavioral and pharmacologicaltests.

Materials and methods
MiceThis study received approval from the Animal ExperimentCommittee of Chiba University (permission number: 2012–95,
2013–149, 2013–251, 2014–096, and 2014–370) and the Institutional Animal Care and Use Committee of the RIKEN Kobe
Branch (Permit Number: AH13-03-65). All procedures relating toanimal care and treatment conformed to the animal careguidelines of the committees. All efforts were made to minimize
both the suffering and the number of animals used. The animals(male, 10–14 weeks old) were housed at 24  2°C under a 12 h
light–dark cycle (lights on from 7:00 to 19:00) with ad libitum
access to food and water. DGKg-KO mice were originated from
C57BL/6 and CBA background (RIKEN, Kobe, Japan), and thenbackcrossed with C57BL/6 mice (Japan SLC, Hamamatsu, Japan)
for at least ﬁve generations to eliminate any background effects
on the observed phenotypes. In all experiments, we used wildtype (WT) littermates as a control group for DGKg-KO mice.
Behavioral experiments were performed between 10:00 and
18:00.
Anti-DGKg antibody
For preparation of anti-human DGKg antibody, an oligopeptide
corresponding to amino acids 600–620 of human DGKg
(IGKPSSQKAVKPREIMLRANS) was used as the antigen. Rabbits
were immunized by multiple subcutaneous injections of 100 lg of
the peptide emulsiﬁed with an equal volume of Freund’s complete

449

adjuvant (Sigma-Aldrich, St. Louis, MO, USA). Injections wererepeated at 4-week intervals, and the serum was obtained after theﬁfth injection. The IgG was puriﬁed by column chromatography on
the antigen peptide-conjugated Hi-Trap NHS-activated Sepharose
HP (GE Healthcare Bio-Sciences; Piscataway, NJ, USA).
Western blotting
Western blotting of the brain was performed using the anti-DGKg
antibody (see above), the anti-glycogen synthase kinase (GSK) 3b(H-76, Santa Cruz Biotechnology, Santa Cruz, CA) and the antiphospho-GSK3b (Ser9, Cell Signaling Technology, Danvers, MA,
USA) as previously described (Usuki et al. 2015).
PCR of the DGK gene and RT-PCR amplification of the DGK mRNA
As indicated in Supplemental Fig. 1, genomic DNA was subjected
to PCR using the following primers: A) 50 -TTCGTCGCTTTAAA
CAGTTGC-30 , B) 50 -CGAAGAGGTTCACTAGTTCTAGAGC-30 ,and C) 50 -GTCAATCACCAGCACCATTC-30 . The PCR conditions
were 94°C for 3 min; 32 cycles of 94°C for 30 s, 55°C for 30 s, and72°C for 60 s; and 72°C for 5 min.
RT-PCR was performed as previously described (Usuki et al.
2015). Brains were homogenized in QIAzol lysis reagent, and total
RNA was isolated with the RNeasy Lipid Tissue Midi Kit (Qiagen,
Venlo, Netherlands). cDNA synthesis was performed with theTranscriptor First-Strand cDNA Synthesis Kit (Roche Diagnostics),
using 0.5 lg of total RNA and random hexamer primers. PCR
ampliﬁcation was performed using Ex Taq DNA polymerase (Takara
Bio, Kusatsu, Japan). The DGKg primer sequences were as follows:forward primer D (nucleotide positions 65–88), 50 -GCAGAGAACCCTATGAGGTGGCCC-30 and reverse primer E (nucleotide
positions 473–496) 50 -GGCCAAGGGGACAGACGGGGATGG-30 .
The PCR conditions were 94°C for 5 min; 45 cycles of 94°C for 30 s,
65°C for 30 s, and 72°C for 60 s; and 72°C for 5 min.
Open field test
Each mouse (WT: n = 21, DGKg-KO: n = 28) was placed in the
periphery of the open ﬁeld apparatus (length 60 9 width 60 9height 40 cm). The time spent in the center area (length 30 9 width
30 cm), the number of entries into the center area and the number ofbehavioral switches were analyzed for 25 min for each test session
in a blind manner by a single observer. The total distance moved in
the arena was quantiﬁed using Image J software (NIH, Bethesda,
MD, USA).
Elevated plus maze test
The elevated plus maze test apparatus consisted of two open arms
(length 30 9 width 5 cm) and two closed arms of the same size,
along with a wall (height 15 cm) and central platform (length 5 9width 5 cm). These arms and central platform were elevated 50 cm
above the ﬂoor. Each mouse (WT: n = 15, DGKg-KO: n = 18) was
placed in the central platform facing one of the open arms. The
number of entries into the open arms and the time spent in the open
arms of the apparatus were scored.
Tail suspension test
For the tail suspension test, a mouse (WT: n = 18, DGKg-KO:
n = 20) was tail-suspended with an adhesive tape 50 cm above theﬂoor, and their behavior was recorded for 7 min.

© 2016 International Society for Neurochemistry, J. Neurochem. (2016) 138, 448--456

(b)Frequency

∗∗∗

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40

(c) 20

∗∗∗

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75

Ratio (%)

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(Times/25 min)(a)Frequency

T. Isozaki et al.

(Times/10 min)

450

50
25

WT

WT

KO

10
5
0

0

0

∗∗∗

15

KO

WT

KO

Fig. 1 Open ﬁeld test of WT and diacylglycerol kinase (DGK)g-KO mice.
WT and DGKg-KO mice were placed in the open ﬁeld apparatus tomeasure their frequency of behavioral switching for 10 min (a), theirfrequency of entering the center area for 25 min (b) and the ratio of staying
time in the center area for 25 min (c). The values are presented as themean  SEM (WT: n = 21, KO: n = 28). ***p < 0.005 versus WT mice.

Barnes maze test
Barnes maze consisted of a round table (90 cm in height) with 20
holes evenly distributed around the edge. An escape box was placed
underneath one of the holes. Three Visual cues with different colors
and shapes were placed around the maze. The maze was rotateddaily, with the spatial location of the target unchanged with respect
to the distal visual cues, to prevent a bias based on an olfactory cueor proximal cues within the maze. As a mildly aversive stimulus,
two table lights located above the center of the maze top that
produced approximately brightness. On the ﬁrst test day, eachmouse (WT: n = 10, DGKg-KO: n = 8) was gently guided to theescape box, which was then covered for 3 min. After these 3 min,the mouse was kept in the start box for 10 s in the center of theplatform, and the following was recorded by a video camera. The
mouse was allowed to walk around the maze for 3 min to ﬁnd andenter the escape box. The escape box was placed underneath thetarget hole. If the mouse located the escape box, it was allowed 60 s
inside before being returned to its home cage. If the mouse failed toenter the escape box within 3 min, it was also gently guided to thebox and remained in the box for 60 s prior to being returned to its
home cage. For four successive days these trials were conducted atthree per day with 25 min intervals between trials. The number ofnose pokes into the holes (frequency) and time spent to ﬁnd thetarget hole (latency) were measured during the experiment as
outcome factors.

homologous recombination, we deleted part of the catalytic
domain encoded by exons 5 and 6 of the DGKg gene inmice (Fig. S1A). The knockout allele followed Mendelian
distribution of inheritance. The mice lacking DGKg
(dgkh–/–) (Accession No. CDB0606K: http://www2.clst.
riken.jp/arg/mutant%20mice%20list.html) had a normal
gender ratio, were grossly normal, and did not express
truncated or full-length DGKg mRNA (Fig. S1B). Moreover, we conﬁrmed that the DGKg protein was notdetected in DGKg-KO mice (Fig. S1C). We veriﬁed that
deleting DGKg did not signiﬁcantly affect protein expression of other DGK isozymes (a, b, c, d, j, e, f, ι, and h)in brain tissue (Fig. S2).

Drug treatment
LiCl was mixed into the drinking water at 600 mg/L andadministered for 10 days (Dehpour et al. 2002; Roybal et al.
2007; Kakefuda et al. 2010).
Statistical analysis
Data are presented as the means  SEM. Pairwise comparisonswere made by Student’s t-test (Figs 1–3 and 6). Other datasets wereanalyzed by one-way ANOVA followed by a Tukey’s post hoc test
(Figs 4 and 5). A p-value of 0.05 or less was considered statisticallysigniﬁcant.

Results
Generation of DGKg-deficient mice by homologous
recombination
Given the links between DGKg and BPD, we determined
whether mice carrying a mutation in the DGKg gene
exhibit mood-related behavioral abnormalities. By

DGKg-KO mice exhibit hyperactivity
BPD patients in the manic state often display hyperactivity,
low levels of anxiety and reduced depression. Therefore, we
determined whether the DGKg-KO mice displayed thesethree phenotypes. First, DGKg-KO and WT mice were
subjected to an open ﬁeld test, and their activity andstereotyped behavior were compared. The traveled distanceof the DGKg-KO mice was not signiﬁcantly different
compared to that of WT mice (data not shown). However,
we next took note of the frequency of behavioral switching,that is, transition to walking, standing, grooming, scratching,and jumping. Intriguingly, we found that the frequency of
behavioral switching in DGKg-KO mice was signiﬁcantly(approximately 1.4-fold, p = 0.000067) greater than that of
WT mice (Fig. 1a). The results show that the DGKg-KO
mice quickly changed their behavior, indicating that they
were in a hyperactive state.
DGKg-KO mice exhibit less anxiety
In the open ﬁeld test, we also assessed the anxiety level of
DGKg-KO mice by measuring their frequency of entering
into the center area of an open ﬁeld apparatus (anxietyprovoking area for mice). In this assessment, DGKg-KO
mice more frequently (approximately 1.4-fold, p = 0.0010)
entered into the center area of the apparatus than WT mice
(Fig. 1b). Moreover, the time spent in the center area for the
DGKg-KO mice was longer (approximately 1.3-fold,p = 0.00094) than that of the WT mice as well (Fig. 1c).

© 2016 International Society for Neurochemistry, J. Neurochem. (2016) 138, 448--456

Diacylglycerol kinase g and bipolar disorder

DGKg-KO mice decrease immobility in the tail suspension
test
The mouse model for mood disorders generally exhibitschanges in their depressive states, i.e., the mouse model ofmania exhibits lower depressive states (Roybal et al. 2007).
Thus, we evaluated the change in DGKg-KO mice using thetail suspension test. In this test, DGKg-KO mice showed areduced state of despair (approximately 0.7-fold,p = 0.0000038), as assessed by immobility time (Fig. 3).
DGKg-KO mice exhibited moderate impaired learning andmemory performance in the Barnes maze test
Cognitive impairment is implicated in mania episode in BPD(Daglas et al. 2015). We performed the Barnes maze test to
determine spatial learning and memory performance. As
shown in Fig. 4, DGKg-KO mice exhibited moderate
impaired learning and memory performance, with increased
latency to reach the target hole (Fig. 4b, p = 0.00029 (Day1)) and increased errors (Fig. 4a, p = 0.045 (Day 4)).
The manic-like behavior of DGKg-KO mice can be reversedwith LiCl treatment
Lithium is a commonly prescribed mood stabilizer that isparticularly effective in treating mania (Shastry 1997). To
determine whether the behavioral abnormalities of the
DGKg-KO mice can be reduced by a low-dose lithium
treatment, we administered LiCl in drinking water at
600 mg/liter for 10 days as described previously (Dehpouret al. 2002; Roybal et al. 2007; Kakefuda et al. 2010). This

100

∗∗∗
75

50

25

0
WT

(b) 40∗∗∗KO

Fig. 3 Tail suspension test of WT and diacylglycerol kinase (DGK)gKO mice. WT and DGKg-KO mice were tail-suspended with adhesive
tape 50 cm above the ﬂoor over a period of 7 min; the immobility time
was measured in only the last 6 min. The values are presented as themean  SEM (WT: n = 18, KO: n = 20). ***p < 0.005 versus WT
mice.

∗∗

30

Ratio (%)

Frequency (Times)(a) 30paradigm produces a stable serum Li+ concentration of
approximately 0.4 mmol/L, which is at the low end of thetherapeutic range for human patients (Roybal et al. 2007).
To investigate the effect of lithium on the hyperactivity of
DGKg-KO mice, the frequency of behavioral switching in
the open ﬁeld test was measured. As shown in Fig. 5a, thefrequency of behavioral switching of the DGKg-KO mice
was reduced by a low-dose treatment of lithium comparedwith the vehicle-treated DGKg-KO mice (p = 0.00017) andreturned to basal levels (the values of WT mice). The
behavior of the WT mice was not signiﬁcantly affected bythis concentration of LiCl. The results indicate that thehyperactivity of the DGKg-KO mice is lithium-sensitive.
Next, the effect of low-dose LiCl on the frequency ofentering into the center area and the time spent in the centerarea of an open ﬁeld apparatus was determined. Both of thesemeasures were signiﬁcantly decreased in lithium-treated
DGKg-KO mice when compared with the vehicle-treated
DGKg-KO mice (p = 0.0000064 and p = 0.025,

Ratio (%)

To further assess the anxiety levels of the DGKg-KO
mice, we conducted another anxiety-measuring behavioral
test: an elevated plus maze test. In this test, the number of
entries into the open arms for the DGKg-KO mice wasmarkedly increased (approximately 1.7-fold, p = 0.000092)
compared with the entries for the WT mice (Fig. 2a). In
addition, the time spent in the open arms for the DGKg-KO
mice was also greater (approximately 3-fold, p = 0.0061)
than that in the WT mice (Fig. 2b). These results indicatethat the DGKg-KO mice showed less anxiety.

451

20

10

20
10
0

0
WT

KO

Fig. 2 Elevated plus maze test of WT and diacylglycerol kinase (DGK)
g-KO mice. WT and DGKg-KO mice were placed in the elevated plus
maze apparatus for 10 min to measure their frequency of entering the

WT

KOopen arms (a) and the ratio of staying time in the open arms (b). The
values are presented as the mean  SEM (WT: n = 15, KO: n = 18).
**p < 0.01, ***p < 0.005 versus WT mice.

© 2016 International Society for Neurochemistry, J. Neurochem. (2016) 138, 448--456

T. Isozaki et al.

Phosphorylation levels of GSK3b are decreased in the
cerebral cortex and the cerebellum of DGKg-KO mice
GSK3b is the downstream factor of diacylglycerol-dependent
protein kinase C (Baum et al. 2008; Kaidanovich-Beilin andWoodgett 2011) and of the MEK (mitogen-activated protein
kinase)/ERK (extracellular signal-regulated kinase) pathway(Saito et al. 1994; Kaidanovich-Beilin and Woodgett 2011),
which is activated by DGKg (Yasuda et al. 2009), and oneof target proteins of lithium (Jope 2003; Zhang et al. 2003;
Beaulieu et al. 2004). Using western blot analysis, weevaluated the phosphorylation level of GSK3b. As shown in
Fig. 6, the phosphorylation levels of GSK3b (Ser9) weresigniﬁcantly decreased in the cerebral cortex (p = 0.019) andthe cerebellum (p = 0.0036) of DGKg-KO mice, despite thenormal levels of total GSK3b proteins. These results indicatethat DGKg-KO mice showed impairment in GSK3b signaling, which is closely related to BPD pathogenesis (Kaidanovich-Beilin and Woodgett 2011).

respectively) and returned to basal levels (Fig. 5b and c). We
next analyzed the effects of LiCl on DGKg-KO mice in theelevated plus maze test. Lithium treatment attenuated thefrequency of entering the open arms (Fig. 6d, p = 0.00014)
and the time spent in open arms (Fig. 5e, p = 0.0011). The
behavior of the WT mice was not signiﬁcantly affected by
LiCl in the open ﬁeld and elevated plus maze tests. These
results indicate that the lowered anxiety of the DGKg-KO
mice is also lithium-susceptible.
Finally, we determined the effects of LiCl on DGKgKO mice in the tail suspension test. As shown in Fig. 5f,
lithium treatment reversed the reduction in the despair statein the tail suspension test (p = 0.0015). Lithium treatment
did not markedly affect the behavior of the WT mice.
Taken together, these results indicate that all of the manialike phenotypes of the DGKg-KO mice are sensitive tolithium.

WT
KO

15

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(b) 200

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DAY3

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∗∗∗

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DAY4

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+

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+

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(f)

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25

LiCl

Ratio (%)

(Times/10 min)Frequency

DAY4

0

−
WT

LiCl

DAY3

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∗∗∗

100

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LiCl

DAY2

Fig. 4 Barnes maze test of WT anddiacylglycerol kinase (DGK)g-KO mice.
The frequencies of errors (a) and latency
to target (b) were measured. The values arepresented as the mean  SEM (WT:n = 10,KO:n = 8).
*p < 0.05,***p < 0.005 versus WT mice.

Ratio (%)

(Times/10 min)Frequency

DAY2

120

(d)

KO

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5

(a)

WT

∗∗∗

(Times/25 min)Frequency(Times/3 min)20

Latency(a)

Ratio (%)

452

∗∗
∗∗∗

75

+
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∗∗

50
25
0

−

KO

+
WT

Fig. 5 Assessment of the lithium effect on diacylglycerol kinase (DGK)
g-KO mice in the open ﬁeld test, the elevated-plus maze test and thetail suspension test. Before (–) and after (+) 10-days of LiCl treatment,
WT and DGKg-KO mice were subjected to the open ﬁeld test (a–c), theelevated plus maze test (d and e) and the tail suspension test (f). In the
open ﬁeld test, the frequency of behavioral switching (a), the frequency

−

+
KO

LiCl

−

+
WT

−

+
KO

of entering the center area (b) and the ratio of staying time in the centerarea (c) were measured. In the elevated-plus maze test, the frequency
of entering open arms (d) and the ratio of staying time in open arms (e)were assessed. The values are presented as the mean  SEM (WT:n = 8, KO: n = 10). *p < 0.05, **p < 0.01, ***p < 0.005: KO mice with
LiCl treatment versus KO mice without LiCl treatment.

© 2016 International Society for Neurochemistry, J. Neurochem. (2016) 138, 448--456

Diacylglycerol kinase g and bipolar disorder453

Western blotting(a)P-GSK3β

GSK3β
WT

KO

WT

Cerebralcortex

Cerebralcortex∗

1.5

Cerebellum

Cerebellum∗∗

1.5

P-GSK3β/GSK3β(b)

P-GSK3β/GSK3β

Fig. 6 Western
blot
analysisofphosphorylated GSK3b. (a) Western
blotting of lysates from WT or KO mouse
cerebral cortexes and cerebellums was
performed to detect expression of total andphosphorylated
GSK3b(Ser9).
(b)
Quantitative analysis of western blotting.
The values of WT mice were set to 1. The
values are presented as the mean  SEM(WT: n = 3, KO: n = 3). *p < 0.05,**p < 0.01 versus WT mice.

KO

1

0.5

0

1

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WT

Discussion
Recently, GWASs implicated the DGKg gene in the etiology
of BPD (Baum et al. 2008; Weber et al. 2011; Zeng et al.
2011). Moreover, DGKg mRNA levels are increased inpatients with BPD (Moya et al. 2010). To test the linkages
between DGKg and BPD, DGKg-KO mice are required.
However, the generation of DGKg-KO mice has not beenaccomplished until now. In this study, we succeeded ingenerating DGKg-KO mice (Fig. S1) and performed acomprehensive behavioral analysis of the mice (Figs 1–5) toinvestigate the role of DGKg in higher brain functions, andthe relationship between DGKg and BPD.
DGKg-KO mice exhibited increased open ﬁeld activity(Fig. 1a), increased open ﬁeld center time/frequency (Fig. 1b
and c), increased open arm time/frequency in elevated plus
maze (Fig. 2), increased antidepressant-like behavior(Fig. 3). Moreover, these phenotypes were sensitive to a
BPD remedy, namely, lithium (Fig. 5). Furthermore, moderate cognitive impairment, which is implicated in mania
episodes in BPD (Daglas et al. 2015), was detected in
DGKg-KO mice (Fig. 4). The behavioral proﬁle (hyperactivity, lower anxiety, lower depressive states, and cognitive
impairment) of DGKg-KO mice is similar in several
behavioral dimensions to BPD patients in the manic state(Martinowich et al. 2009) (Figs 1–4), including the disappearance of their phenotypes by treatment with lithium(Fig. 5). These lithium sensitive-phenotypes were commonly
observed in representative BPD model mice such as Neurocan-KO (Miro et al. 2012), Clock-KO (Roybal et al. 2007),
glutamate receptor 6-KO (Shaltiel et al. 2008), DGKb-KO(Kakefuda et al. 2010; Shirai et al. 2010), and GSK3btransgenic (Spittaels et al. 2000; Prickaerts et al. 2006) mice.

KO

WT

KO

Therefore, these ﬁndings strongly suggest that DGKg is oneof the key enzymes involved in BPD pathogenesis andsupport the GWAS results. The lack of availability ofsuitable animal models of mania has been one of the greatest
impediments in the ﬁeld. Our results indicate that the DGKgKO mice would represent a bona ﬁde model of human BPD
with mania. Therefore, it is likely that these mice areparticularly useful for studying the pathophysiology ofmania.
DGKg has also been found to be associated with attention
deﬁcit hyperactivity disorder (ADHD) by GWAS (Weberet al. 2011). Moreover, mania-like behavior is similar to
ADHD symptoms. Therefore, DGKg-KO mice could also
represent a model for ADHD. Although the focus of thisstudy is on the role of DGKg in BPD, there may be apossible link between DGKg and ADHD in addition to BPD.
In addition to the common tests among the ﬁve representative BPD model mice described above, Neurocan-KO mice
showed increased preference for saccharine, increased marble burying and increased acoustic startle (Miro et al. 2012).
Clock-KO mice exhibited increased preference for sucroseand decreased hyponeophagia (Roybal et al. 2007). Glutamate receptor 6-KO mice showed increased aggression(Shaltiel et al. 2008). GSK3b-transgenic mice exhibited
increased acoustic startle (Spittaels et al. 2000; Prickaerts
et al. 2006). We performed marble burying, sucrose preference and cotton bud biting (aggression) tests. However, wehave not detected signiﬁcant increases in these tests so far(data not shown). Nevertheless, it is reasonable that thephenotypes of DGKg-KO mice may be moderately different
from other mania model mice, because BPD and mania are
multigenic disorders that result from a combination ofdifferent genetic proﬁles.

© 2016 International Society for Neurochemistry, J. Neurochem. (2016) 138, 448--456

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T. Isozaki et al.

DGKg mRNA is expressed much more in the dentate
gyrus than in the CA region of the hippocampus (Usuki et al.
2015). Dysgeneration of the dentate gyrus is known to be acause of BPD (Walton et al. 2012; Hagihara et al. 2013).
Therefore, it is possible that a high expression of DGKg in
the dentate gyrus is involved in the pathogenesis of BPD.
DGKg is also highly expressed in the Purkinje cells of thecerebellum (Usuki et al. 2015). Purkinje neurons are also
associated with BPD (Maloku et al. 2010). Therefore,
DGKg expression in Purkinje cells may play a role in thepathogenesis of BPD as well. Dysregulation of G-proteincoupled receptor activity is involved in the pathology ofmany psychiatric disorders, including BPD (Catapano andManji 2007). Rittiner et al. (2014) reported that DGKg
enhances G-protein-coupled receptor signaling by reducing
protein kinase C activation. Therefore, DGKg may regulate
pathogenesis of BPD through G-protein-coupled receptor.
Although GWAS has not implicated DGKb in the etiology
of BPD so far, DGKb-KO mice also exhibited mania-likephenotypes (Kakefuda et al. 2010; Shirai et al. 2010). DGKb
is widely distributed in the brain, particularly in the olfactorybulb, cerebral cortex, striatum, and hippocampus (Goto andKondo 1993). Therefore, the expression pattern of DGKb isdifferent from that of DGKg (Usuki et al. 2015) as describedabove. DGKb is stably distributed at the plasma membrane incells (Goto and Kondo 1993; Shirai et al. 2010), whereas
DGKg is translocated to the non-ionic detergent-resistantmembrane in punctate vesicles from the cytoplasm in responseto stress stimuli (Murakami et al. 2003; Matsutomo et al.
2013). Although further studies are required, these differencessuggest that DGKg and DGKb regulate BPD (mania)
pathogenesis through distinct mechanisms.
Phosphatidylinositol turnover, which generates DG, hasbeen hypothesized to play an important role in the mechanism of action of LiCl (Martinowich et al. 2009). DGK is
one of the components of the phosphatidylinositol turnover(Goto et al. 2006; Sakane et al. 2007; Merida et al. 2008;
Topham and Epand 2009). Moreover, we recently found thatthe pleckstrin domain of DGKg is strongly bound tophosphatidylinositol 4,5-bisphosphate, a product of thephosphatidylinositol turnover (Kume et al. 2016). We also
revealed that DGKg is a unique enzyme with high afﬁnity
for DG (Komenoi et al. 2015). In addition, DGKg is apositive regulator of the epidermal growth factor receptor
pathway (Yasuda et al. 2009), which drives phosphatidylinositol turnover and is implicated in BPD (Sklar et al. 2008).
It will be interesting to determine what role DGKg plays in
the phosphatidylinositol turnover-related, lithium-sensitive
molecular mechanisms of BPD pathogenesis.
Thus far, there have been no indications that DGKg-KO
mice cycle between mania and depression. However, futurestudies are needed to determine whether depression-like
symptoms occur in these mice after periods of stress.
Interestingly, in patients with BPD, DGKg mRNA levels

were increased (Moya et al. 2010). Because the depression/
normal state is considerably longer than the mania state in
BPD patients, many of these patients were in the depression
state. If this is the case, in consideration of the manic-likephenotypes of the DGKg-deﬁcient mice (Figs 1–5), theseresults suggest that abnormally low and high expression
levels of DGKg cause manic and depressive states of BPD,
respectively. Intriguingly, Klauck et al. (1996) and we(Murakami et al. 2003) reported that a glucocorticoid
treatment markedly increased the expression levels of
DGKg. In addition, excess glucocorticoid is associated withhuman depression and anxiety disorders (Holsboer 2000;
Korte 2001). It will be interesting to perform experiments
using the DGKg-transgenic mice and mice with highglucocorticoid levels to verify this hypothesis.
All of the SNPs in DGKg (Baum et al. 2008; Weber et al.
2011; Zeng et al. 2011) that are implicated in the etiology of
BPD by GWASs are located in introns. For example, the
SNP rs1170191, which is identiﬁed in three independent
reports (Baum et al. 2008; Weber et al. 2011; Zeng et al.
2011), is located in the ﬁrst intron of DGKg. Therefore, it islikely that the SNPs lead to dysregulation of the expressionof DGKg, which probably causes BPD as described above.
GSK3b lies downstream of diacylglycerol-dependent protein kinase C (Baum et al. 2008; Kaidanovich-Beilin andWoodgett 2011) and of the MEK/ERK pathway (Saito et al.
1994; Kaidanovich-Beilin and Woodgett 2011), and one oftarget proteins of lithium (Jope 2003; Zhang et al. 2003;
Beaulieu et al. 2004). Transgenic mice over-expressing
GSK3b display mania-like behavior (Prickaerts et al.
2006). Moreover, the decreases in levels of phosphorylated
GSK3b were also observed in the brains of BPD (mania)
model mice such as dopamine transporter-KO (Beaulieu
et al. 2004) and DGKb-KO mice (Kakefuda et al. 2010).
Therefore, to analyze the mechanisms of mania-like behavior
generation in DGKg-KO mice, we assessed the phosphorylation (inactivation) level of GSK3b in the cerebral cortex
and the cerebellum. We conﬁrmed that the phosphorylationlevels of GSK3b (Ser9) were signiﬁcantly decreased in the
cerebral cortex and the cerebellum of DGKg-KO mice
(Fig. 6). The results strongly suggest that DGKg regulates
behavior and mood through, at least in part, GSK3b
signaling and further support the linkage between DGKg
and BPD. GSK3b was reported to be phosphorylated andinactivated by the Raf/MEK/ERK pathway (Saito et al.
1994; Kaidanovich-Beilin and Woodgett 2011). Interestingly, the Raf/MEK/ERK pathway is activated by DGKg
(Yasuda et al. 2009). Therefore, it is possible that DGKg
modiﬁes GSK3b activity through the regulation of the Raf/
MEK/ERK pathway.
Taken together, it is strongly suggested that DGKg is oneof the key enzymes of the etiology of BPD and that the
DGKg-KO mice would represent a bona ﬁde model ofhuman mania. Our ﬁndings facilitate the design and© 2016 International Society for Neurochemistry, J. Neurochem. (2016) 138, 448--456

Diacylglycerol kinase g and bipolar disorderdevelopment of novel therapeutic strategies for BPD. For
example, DGKg-speciﬁc inhibitors may be valuable as novel
drugs for depressive episodes of BPD. However, themolecular mechanism underlying the behavioral effects of
DGKg deﬁciency remains to be elucidated. Further studies
will be necessary to explore this issue.

Acknowledgments and conflict of interest
disclosureThis work received support from MEXT/JSPS KAKENHI Grant
Numbers 22370047 (Grant-in-Aid for Scientiﬁc Research (B)),
23116505 (Grant-in-Aid for Scientiﬁc Research on Innovative
Areas), 25116704 (Grant-in-Aid for Scientiﬁc Research on Innovative Areas), 26291017 (Grant-in-Aid for Scientiﬁc Research (B)),
and 15K14470 (Grant-in-Aid for Challenging Exploratory
Research); the Japan Science and Technology Agency(AS221Z00794F, AS231Z00139G, and AS251Z01788Q); the Naito
Foundation; the Hamaguchi Foundation for the Advancement ofBiochemistry; the Daiichi-Sankyo Foundation of Life Science; theTerumo Life Science Foundation, the Futaba Electronic Memorial
Foundation; the Daiwa Securities Health Foundation, the Ono
Medical Research Foundation; the Japan Foundation for Applied
Enzymology, the Food Science Institute Foundation; the Skylark
Food Science Institute; and Asahi Group Foundation (FS). The
authors declare that they have no conﬂicts of interest to disclose.
All experiments were conducted in compliance with the ARRIVE
guidelines.

Supporting information
Additional Supporting Information may be found online in the
supporting information tab for this article:
Figure S1. Generation of DGKg-deﬁcient mice by homologous
recombination.
Figure S2. Expression of DGK isozymes in WT and DGKg-KO
mouse brains.

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