© 2015. Published by The Company of Biologists Ltd.
Interaction between the close homolog of L1 and serotonin receptor 2c regulates signal
transduction and behavior in mice
Ralf Kleene1, Harshita Chaudhary1, Nicole Karl1, Jelena Katic1, Agnieszka Kotarska1, Kathrin
Guitart1, Gabriele Loers1, and Melitta Schachner2,3*
1
Zentrum für Molekulare Neurobiologie, Universitätsklinikum Hamburg-Eppendorf,
Martinistr. 52, 20246 Hamburg, Germany
2
Keck Center for Collaborative Neuroscience and Department of Cell Biology and
Neuroscience, Rutgers University, 604 Allison Road, Piscataway, NJ 08854, USA
3
Center for Neuroscience, Shantou University Medical College, 22 Xin Ling Road,
Shantou, Guangdong 515041, China
*
Correspondence should be addressed to:
Melitta Schachner, Center for Neuroscience, Shantou University Medical College, 22 Xin
Ling Road, Shantou, Guangdong 515041, China; Tel: + 86 754 8890 0276; Fax: + 86 754
8890 0236; E-mail: [email protected]
Journal of Cell Science • Advance article
Keywords: CHL1, 5-HT2c receptor, exploratory behavior, signal transduction
JCS Advance Online Article. Posted on 2 November 2015
ABSTRACT
The serotonergic system plays important roles in multiple functions of the nervous system and
its malfunctioning leads to neurological and psychiatric disorders. Here, we show that the cell
adhesion molecule close homolog of L1 (CHL1), which has been linked to mental disorders,
binds to a peptide stretch in the third intracellular loop of the serotonin 2c (5-HT2c) receptor
via its intracellular domain. Moreover, we provide evidence that CHL1 deficiency in mice
leads to 5-HT2c receptor-related reduction in locomotor activity and reactivity to novelty and
that CHL1 regulates signaling pathways triggered by constitutively active isoforms of the
5-HT2c receptor. Furthermore, we found that 5-HT2c receptor and CHL1 co-localize in
striatal and hippocampal GABAergic neurons and that 5-HT2c receptor phosphorylation and
association of phosphatase and tensin homolog (PTEN) and β-arrestin 2 with the 5-HT2c
receptor is regulated by CHL1. Our results demonstrate that CHL1 regulates signal
transduction pathways via constitutively active 5-HT2c receptor isoforms, thereby altering
5-HT2c receptor functions and implicating CHL1 as a novel modulator of the serotonergic
Journal of Cell Science • Advance article
system.
INTRODUCTION
Cell adhesion molecules, such as the close homolog of L1 (CHL1) (Holm et al., 1996;
Hillenbrand et al., 1999), have been implicated in numerous functions in the developing and
adult nervous system, including neural cell migration and survival, neuritogenesis, axon
guidance and synaptogenesis as well as synaptic plasticity (for review, see Maness and
Schachner, 2007). In humans, mutations of CHL1 are associated with mental retardation,
epilepsy, schizophrenia and autism spectrum disorders characterized by deficits in behavior,
including cognition and social interactions (Angeloni et al., 1999a, b; Sakurai et al., 2002;
Frints et al., 2003; Chen et al., 2005; Chu and Liu, 2010; Tam et al., 2010; Cuoco et al., 2011;
Salyakina et al., 2011; Shoukier et al., 2013). Ablation of CHL1 in mice leads to impairments
in synaptic transmission, long-term potentiation, working memory, gating of sensorimotor
information and stress response as well as alterations in social and exploratory behavior
(Montag-Sallaz et al., 2002; Frints et al., 2003; Pratte et al., 2003; Demyanenko et al., 2004,
2011; Irintchev et al., 2004; Leshchyns´ka et al., 2006; Nikonenko et al., 2006; Morellini et
al., 2007; Wright et al., 2007; Kolata et al., 2008; Pratte and Jamon, 2009).
Being interested in molecules interacting with the intracellular domain of CHL1 with the
expectation to find bidirectionally acting modifiers of individual functions, we identified the
5-HT2c receptor as a CHL1 binding partner. The seven-transmembrane-spanning,
G-protein-coupled 5-HT2c receptor triggers different signal transduction pathways and is
involved in regulating different types of behavior (Werry et al., 2005, 2008; Berg et al., 2008).
different degrees of constitutive activity of the differentially edited receptors (Iwamoto et al.,
2009; O'Neil and Emeson, 2012). The non-edited isoform and the partially edited isoforms
exhibit pronounced or intermediate constitutive activity, while the fully edited isoform is not
constitutively active and requires binding of serotonin for activation (Burns et al., 1997;
Herrick-Davis et al., 1999; Niswender et al., 1999). Reduced reactivity to a novel environment
and sensitivity to locomotor stimulants as well as alterations in sleep, feeding and
reward-associated behaviors are observed in 5-HT2c receptor-deficient mice, while mice
expressing the non-edited or fully-edited isoforms show anxiety and depression-like symptoms,
respectively (for review and references, see Heisler et al., 2007; Iwamoto et al., 2009; Fletcher
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Adenosine-to-inosine RNA editing of this receptor alters its signaling characteristics leading to
et al., 2009; Mombereau et al., 2010; Pennanen et al., 2013). In humans, the 5-HT2c receptor
has been associated with obesity as well as neurological and psychiatric disorders, such as
schizophrenia, major depression, addiction, epilepsy, anxiety, sleep disorders, motor
dysfunction, and bipolar disorder (Berg et al., 2008; Lee et al., 2010).
In the present study we show that the interaction of CHL1 with the 5-HT2c receptor regulates
the receptor’s functions, 5-HT2c receptor-mediated signaling and behavior in mice, suggesting
that some interdependent functions of the two molecules are linked through their molecular
Journal of Cell Science • Advance article
associations.
RESULTS
CHL1 interacts with the 5-HT2c receptor
To find novel intracellular interaction partners of CHL1, we performed a phage display
screening of a random 12mer peptide library using the intracellular domain of CHL1
(CHL1-ICD) (Fig. 1A), which belongs to the L1 family of structurally related proteins. A
phage expressing a CHL1-ICD-binding peptide was obtained that showed similarity to a short
sequence stretch within the third intracellular loop of the 5-HT2c receptor comprising the
amino acids 292-304 (Fig. 1B). To ascertain that the peptide sequence expressed by the phage
binds to CHL1-ICD, a synthetic peptide with the phage sequence was probed by ELISA using
increasing concentrations of CHL1-ICD. As control we used increasing concentrations of
L1-ICD which shows homology to CHL1-ICD like the other members of the L1 family,
namely neurofascin and NrCAM (Fig. 1A). A concentration-dependent and saturable binding
of CHL1-ICD to the peptide was observed, while L1-ICD did not bind to the peptide (Fig.
1C). To substantiate the interaction between CHL1 and the 5-HT2c receptor, a synthetic
peptide comprising the amino acids 281-309 of the 5-HT2c receptor and carrying a biotin
moiety at its N-terminus was applied in increasing concentrations to substrate-coated
CHL1-ICD or to the control substrate L1-ICD. Using HRP-conjugated streptavidin, a
concentration-dependent and saturable binding of the 5-HT2c receptor-derived peptide to
CHL1-ICD, but not L1-ICD was observed (Fig. 1D).
were performed using mouse brain homogenate and CHL1-ICD. As controls, we used L1-ICD
and the intracellular domain of NCAM140 which belongs to the immunoglobulin superfamily
like CHL1. Western blot analysis of the precipitates revealed that the mouse 5-HT2c receptor
antibody recognized a double band of approximately 50 and 55 kDa in the CHL1-ICD
precipitate, while no immunopositive bands for the 5-HT2c receptor were detectable in the
L1-ICD and NCAM-ICD precipitates (Fig. 1E). These experiments show that the 5-HT2c
receptor specifically interacts with CHL1-ICD.
To further substantiate the interaction of CHL1 with the 5-HT2c receptor, we performed
immunoprecipitation experiments using detergent extracts of brain homogenate from
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As a further verification of the interaction between the two molecules, pull-down experiments
wild-type and CHL1-deficient mice using polyclonal rabbit CHL1 antibody. By Western blot
analysis, the mouse 5-HT2c receptor antibody recognized predominantly the 50 kDa band in
the CHL1 immunoprecipitates from wild-type brain, but not from CHL1-deficient brain (Fig.
1F), indicating that the 5-HT2c receptor and CHL1 are associated with each other in the intact
tissue.
To narrow down the 5-HT2c receptor binding site in CHL1-ICD, we performed ELISA with
substrate-coated 5-HT2c receptor-derived peptide and recombinant N- and C-terminal
truncations of CHL1-ICD fused to glutathione-S-transferase (GST) (Fig. 2A). CHL1-ICD
with C-terminal truncations bound to the 5-HT2c receptor-derived peptide with the shortest
CHL1-ICD construct showing the highest concentration-dependent binding to the peptide (Fig.
2B,C). In contrast, CHL1-ICD with the N-terminal truncation and GST did not bind to the
peptide (Fig. 2B,C).
The combined results indicate that CHL1 binds to a sequence stretch in the third intracellular
loop of the 5-HT2c receptor via the 29 N-terminal amino acids in its intracellular domain and
that the 5-HT2c receptor does not interact with L1 and NCAM which are structurally related
to CHL1.
CHL1 co-localizes with the 5-HT2c receptor in GABAergic neurons in the striatum and
hippocampus
The interaction between CHL1 and 5-HT2c receptor was further assayed by immunohistology
in the adult mouse brain. Double-labeling with goat antibody against CHL1 and mouse
the striatum (Fig. 3A), which contains a large number of inhibitory GABAergic medium spiny
neurons (90-95% of the total neuronal population) and GABAergic interneurons. In the CA3
region of the hippocampus, co-immunostaining of CHL1 and the 5-HT2c receptor was
observed in distinct cells (Fig. 4A), which likely represent GABAergic interneurons according
to their scarcity, position and cell body size. Co-immunostaining of CHL1 and the 5-HT2c
receptor in some cells was also observed in the CA1 region of the hippocampus (data not
shown). To test whether CHL1 and 5-HT2c are expressed in GABAergic interneurons, we
performed double immunostaining with antibodies against CHL1 or 5-HT2c receptor and
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antibody against the 5-HT2c receptor showed a considerable overlap of immunostaining in
parvalbumin, a marker protein for GABAergic interneurons (Houser, 2007; Tepper et al.,
2010). Co-stainings of 5-HT2c receptor or CHL1 with parvalbumin were observed in cells in
the putamen (Fig. 3B,C) and stratum pyramidale in the CA3 region of the hippocampus (Fig.
4B,C), indicating that the 5-HT2c receptor and CHL1 are associated with each other in
GABAergic interneurons in the hippocampus and striatum.
To investigate whether CHL1 and 5-HT2c receptor directly interact with each other in
hippocampal and striatal neurons of intact adult mouse brain, we used the antibodies against
CHL1 and 5-HT2c receptor for proximity ligation assay which allows the detection of close
protein interactions with high sensitivity and specificity by generation and amplification of
fluorescent signals from a pair of oligonucleotide-labeled secondary antibodies, when the
primary antibodies have bound to their antigens in close proximity (less than 40 nm).
Fluorescent signals were observed throughout the striatum (Fig. 5A) and in the CA3 and CA1
regions of the hippocampus (Fig. 5B,C), suggesting a close interaction of CHL1 and 5-HT2c
receptor in striatal GABAergic medium spiny neurons and in striatal and hippocampal
GABAergic interneurons.
When tissue from CHL1-deficient mice was used as negative control for proximity ligation
assay with antibodies against CHL1 and the 5-HT2c receptor, no fluorescent signals were
detectable, for example, in the hippocampal CA3 region (Fig. 5D), showing the specificity of
the close interaction between CHL1 and 5-HT2c receptor.
Since PTEN binds to the 5-HT2c receptor (Ji et al., 2006), a proximity ligation assay was
performed with antibodies against PTEN and the 5-HT2c receptor to verify this interaction
tissues with higher signal levels in the CHL1-deficient than wild-type brain regions in the
hippocampal CA3 region (Fig. 5E,F). This result not only confirms the close interaction
between PTEN and the 5-HT2c receptor, but also indicates that the level of this close
interaction is enhanced in the absence of CHL1.
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with a positive control. Fluorescent signals were found in wild-type and CHL1-deficient brain
Reduced reactivity of CHL1-deficient mice to novelty results from abnormal signaling
via constitutively active 5-HT2c receptor isoforms
Based on the finding that CHL1 and 5-HT2c receptor interact with each other predominantly
in inhibitory GABAergic neurons and the findings that CHL1 and the 5-HT2c receptor are
associated with mental disorders in humans and with abnormal novelty-seeking exploratory
behavior in mice, we assumed that binding of CHL1 to the 5-HT2c receptor is physiologically
relevant in the intact adult mouse brain to control behavior. Thus, we investigated whether the
altered exploratory behavior of CHL1-deficient mice resulted from alterations in
responsiveness or activity of serotonin-reactive and/or constitutively active 5-HT2c receptor
isoforms. To this aim, CHL1-deficient and wild-type mice were monitored for locomotor
activity in the open field test as a read-out of exploratory behavior immediately after
intraperitoneal
injection
of
the
serotonin
agonist
Ro60-0175
which
activates
serotonin-activatable receptor isoforms, of the antagonist/inverse agonist SB-206553 which
inactivates constitutively active receptor isoforms or of vehicle solution. CHL1-deficient
females moved less than wild-type females when vehicle solution was applied (Fig. 6A),
indicating that CHL1-deficient females show a pronounced and prolonged reduction of
reactivity to the novel environment in the open field. In contrast to CHL1-deficient females,
CHL1-deficient males showed reduced locomotor activity only within the first 5 min in the
open field after application of vehicle solution (Fig. 6B), indicating that CHL1-deficient male
mice show a less pronounced and short-term reduction of reactivity to novelty in the open
within 5 min in the open field was not altered by Ro60-0175 treatment (Fig. 6C,D). Wild-type
females and males did not respond to SB-206553, whereas the distance moved by
CHL1-deficient females within 30 min and males within 5 min increased after SB-206553
treatment and reached values observed for wild-type females and males treated with
SB-206553 or vehicle (Fig. 6C,D). Since rearing is a typical novelty-induced behavior in
males and since CHL1-deficient males show drastically reduced rearing within the first few
minutes in the open field (Morellini et al., 2007), we analyzed whether SB-206553 treatment
also affected the rearing behavior of CHL1-deficient males in the open field. CHL1-deficient
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field. The distance moved by wild-type and CHL1-deficient females within 30 min and males
males showed less rearing activity within the first 5 min in the open field after injection of
vehicle solution when compared to wild-type males, but this reduced rearing activity was not
observed after application of SB-206553 (Fig. 6E).
These results indicate that the behavioral changes caused by CHL1-deficiency are due to a
dysregulation of SB-206553-sensitive constitutively active 5-HT2c receptor isoforms and that
Ro60-0175-reactive serotonin-activatable 5-HT2c receptor isoforms are not affected by the
lack of CHL1. Moreover, the results suggest that an altered 5-HT2c receptor-mediated
signaling in CHL1-deficient mice is associated with reduced reactivity to novel stimuli and/or
abnormalities in exploratory behavior.
Lack of CHL1 alters phosphorylation of the 5-HT2c receptor and interaction of the
receptor with PTEN and β-arrestin 2
Since activity of the 5-HT2c receptor and downstream signaling pathways triggered by
activated 5-HT2c receptor are regulated by phosphorylation and dephosphorylation of the
5-HT2c receptor, we tested whether ablation of CHL1 affects the association of the receptor
with PTEN which dephosphorylates the 5-HT2c receptor (Ji et al., 2006).
Since PTEN and CHL1 bind directly to the amino acids 284-298 (Ji et al., 2006) and 281-309
(present study) of the 5-HT2c receptor, respectively, we first investigated whether PTEN
interferes with the binding of CHL1-ICD to the 5-HT2c receptor and performed pull-down
experiments using a detergent brain extract from wild-type mice and CHL1-ICD. In the
presence of a 5-fold molar excess of PTEN-GST or GST, as the negative control, levels of the
(12.2±5.8%) (Fig. 7A), indicating that PTEN does not compete with CHL1 for 5-HT2c
receptor binding. However, in the presence of the tat/281-309 peptide containing the 5-HT2c
receptor sequence stretch 281-309, levels of the 5-HT2c receptor in the CHL1-ICD precipitate
was reduced by more than 40% (41.4±3.4%) compared to the levels in the absence of the
peptide (Fig. 7A). This result indicates that the peptide which contains the CHL1-binding site
of the 5-HT2c receptor interferes with the interaction between the 5-HT2c receptor and
CHL-ICD.
To substantiate the notion that receptor-derived peptide tat/281-309 interferes with the
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5-HT2c receptor in the CHL1-ICD precipitate were reduced by approximately 10%
interaction between CHL1 and the 5-HT2c receptor and to investigate whether PTEN binds to
the receptor in the absence of CHL1, we performed immunoprecipitation experiments with
CHL1, PTEN and 5-HT2c receptor antibodies using brain extracts from wild-type and
CHL1-deficient mice in the absence or presence of the tat/281-309 peptide. Similar amounts
of PTEN were found in the PTEN immunoprecipitates of both genotypes, whereas
approximately three times (2.9±0.4) higher amounts of HT2c receptor were observed in the
PTEN immunoprecipitate from CHL1-deficient brain extracts than in the immunoprecipitates
from wild-type brain extracts (Fig. 7B), indicating that more PTEN associates with the
receptor in the absence of CHL1. No CHL1 was detectable in the PTEN immunoprecipitates
from extracts of either genotype (Fig. 7B), implying that CHL1 does not directly interact with
PTEN. In the presence of the tat/281-309 peptide, the levels of the 5-HT2c receptor in the
PTEN immunoprecipitates from wild-type mice were approximately two times (2.2±0.2)
higher than the levels in the immunoprecipitates obtained in the absence of the peptide, while
the amounts of 5-HT2c receptor in the PTEN immunoprecipitates from extracts of
CHL1-deficient mice were only slightly reduced in the presence of the tat/281-309 peptide
(12.8±5.4% less) (Fig. 7B). This result indicates that the receptor-derived peptide disturbs the
interaction between the 5-HT2c receptor and CHL1, but does not interfere with the interaction
between the receptor and PTEN in the absence of CHL1.
In the CHL1 immunoprecipitates from wild-type mice, similar levels of CHL1 were found in
the absence and presence of the tat/281-309 peptide, while no CHL1 was detectable in the
immunoprecipitates from CHL1-deficient mice (Fig. 7C). In the presence of the peptide,
immunoprecipitates from wild-type mice, confirming that the peptide interferes with the
interaction between CHL1 and the receptor. PTEN was not detectable in the CHL1
immunoprecipitates from wild-type mice (Fig. 7C), supporting the view that a direct
interaction between CHL1 and PTEN can be excluded.
After immunoprecipitation with the rabbit 5-HT2c receptor antibody, reduced levels (by
31.5±5.5%) of the 50 kDa receptor form were detectable by Western blot analysis with the
goat antibody against the receptor in the presence of the tat/281-309 peptide relative to the
levels in its absence (Fig. 7D). Relative to the receptor levels in the immunoprecipitates, the
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reduced levels (by 60.6±6.3%) of the 5-HT2c receptor were found the CHL1
PTEN levels were approximately 1.5-fold (1.6±0.1) higher in immunoprecipitates from CHL1
deficient extracts and from wild-type extracts in the presence of the peptide, when compared
to the levels of PTEN in immunoprecipitates from wild-type extracts in the absence of peptide
(Fig. 7D). These results show that association of PTEN with the receptor is enhanced in the
absence of CHL1 and that the peptide interferes with the interaction between CHL1 and the
5-HT2c receptor, thereby allowing PTEN to associate with the receptor in the absence of
CHL1.
It has been reported that phosphorylation of the 5-HT2c receptor promotes binding of
β-arrestin 2 to the 5-HT2c receptor and that an enhanced interaction of β-arrestin 2 with
SB-206553-sensitive constitutively active 5-HT2c receptor isoforms modulates the activity of
these isoforms (Marion et al., 2004). Since we observed that the behavioral changes of
CHL1-deficient mice are due to dysregulation of SB-206553-sensitive constitutively active
5-HT2c receptor isoforms, we analyzed whether binding of β-arrestin 2 to the 5-HT2c
receptor was altered in the absence of CHL1. Relative to the β-arrestin 2 levels in the
immunoprecipitates from wild-type brains without peptide, the levels in immunoprecipitates
from CHL1-deficient brains were reduced by approximately 70% (69.5±4.5), while the levels
in the immunoprecipitates from wild-type brains were decreased by approximately 35%
(36.8±3.5) in the presence of the tat/281-309 peptide (Fig. 7D). This result indicates that the
association of β-arrestin 2 with the 5-HT2c receptor is reduced in the absence of CHL1,
suggesting that CHL1 regulates the phosphorylation of the receptor and the consecutive
association of β-arrestin 2 with the receptor.
used wild-type and CHL1-deficient brain extracts for immunoprecipitation with the rabbit
antibody against the 5-HT2c receptor and subjected the immunoprecipitates to Western blot
analysis with a phospho-serine antibody followed by a goat antibody against the 5-HT2c
receptor. Similar amounts of the 5-HT2c receptor were immunoprecipitated from brains of
both genotypes (Fig. 7E), while 5-fold (4.7±0.7) lower levels of phosphorylated 5-HT2c
receptor were observed in the immunoprecipitates from CHL1-deficient extracts when
compared to immunoprecipitates from wild-type brains (Fig. 7E). These results indicate that
CHL1 regulates the phosphorylation of the 5-HT2c receptor and suggest that increased
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To test whether phosphorylation of the 5-HT2c receptor is altered in the absence of CHL1, we
association of PTEN with the 5-HT2c receptor in the absence of CHL1 maintains the receptor
in a dephosphorylated state.
CHL1 regulates signaling via constitutively active 5-HT2c receptor isoforms
The results from behavioral experiments suggest that CHL1 regulates the activity of
constitutively active SB-206553-sensitive 5-HT2c receptor isoforms. To confirm the
interaction of CHL1 with these isoforms, we performed immunoprecipitation experiments
with transfected COS-7 cells which either expressed the non-edited constitutively active INI
isoform alone or co-expressed the INI isoform with CHL1. Pronounced levels of the INI
isoform were co-immunoprecipitated by a CHL1-antibody from cells co-expressing CHL1,
while low levels were non-specifically immunoprecipitated from CHL1-negative cells (Fig.
8A), indicating that CHL1 associates with the constitutively active 5-HT2c receptor isoforms.
Next, the transfected cells were taken to analyze whether CHL1 regulates signaling of the
constitutively active 5-HT2c receptor isoforms. Since the non-edited INI isoform can activate
phospholipase D (PLD) (McGrew et al., 2004) and since SB-206553 inhibits Erk
phosphorylation triggered by the INI isoform (Labasque et al., 2010), we asked whether
CHL1 affects PLD activity and Erk phosphorylation by regulating the constitutively active
INI isoform. Transfected COS-7 cells expressing the INI isoform alone showed similar levels
of PLD activity and Erk phosphorylation as cells co-expressing the INI isoform and CHL1
(Fig. 8B,C). Treatment with SB-206553 reduced PLD activity and phospho-Erk levels in cells
expressing the INI isoform alone, while SB-206553 had no effect on PLD activity and Erk
indicate that binding of SB-206553 to the INI isoform triggers signaling pathways leading to
reduction in phospho-Erk levels and PLD activity when CHL1 is absent, indicating that CHL1
controls signal transduction pathways mediated by the constitutively active INI isoform.
Journal of Cell Science • Advance article
phosphorylation in cells co-expressing CHL1 and the INI isoform (Fig. 8B,C). These results
DISCUSSION
The 5-HT2c serotonin receptor is essential for normal nervous system functions. Several
human mental disorders have been linked to abnormalities of the 5-HT2c receptor and
disturbance of 5-HT2c receptor-regulated physiological processes. Because many different
5-HT2c receptor isoforms exist, receptor-triggered signal transduction pathways and cellular
responses are complex. Different 5-HT2c receptor isoforms are generated by A-to-I RNA
editing: the non-edited isoform is constitutively active, the partially edited isoforms exhibit
intermediate constitutive activity and the fully edited isoform becomes active only in the
presence of its ligand serotonin. Thus, elucidation of the molecular mechanisms underlying
regulation of 5-HT2c receptor-mediated signaling in the context of interaction partners as
possible modulators of the 5-HT2c receptor isoform activities is of interest and thereby may
contribute to a better understanding of 5-HT2c receptor functions in the normal brain as well as
in the pathogenesis of 5-HT2c receptor-related mental disorders, such as addiction, obesity,
epilepsy, anxiety, sleep disorders, schizophrenia, major depression and bipolar disorder (Berg
et al., 2008; Lee et al., 2010). Here, we identified CHL1 as a novel binding partner of the
5-HT2c receptor in vitro and in vivo. In humans, CHL1 has been linked to neurological and
psychiatric disorders, such as mental retardation, schizophrenia, epilepsy and autism, but so far
has not been associated with addiction, obesity, anxiety or sleep disorders (Angeloni et al.,
1999a, b; Sakurai et al., 2002; Frints et al., 2003; Chen et al., 2005; Chu and Liu, 2010; Tam et
al., 2010; Cuoco et al., 2011; Salyakina et al., 2011; Shoukier et al., 2013). CHL1-deficient
(Montag-Sallaz et al., 2002; Pratte et al., 2003; Morellini et al., 2007; Pratte and Jamon, 2009).
Mice expressing only the non-edited or the fully-edited isoform of the 5-HT2c receptor display
anxiety- or depression-like symptoms and 5-HT2c receptor-deficient mice display reduced
reactivity to a novel environment or external stimuli as well as alterations in sleep, feeding and
reward-associated behaviors (for review and references, see Heisler et al., 2007; Fletcher et al.,
2009; Iwamoto et al., 2009; Mombereau et al., 2010; Pennanen et al., 2013). Alterations in
sleep, feeding and reward-associated behaviors have so far not reported for CHL1-deficient
mice.
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mice showed reduced reactivity to novelty or external stimuli, but no anxiety-related behavior
Here, we show that the 29 N-terminal amino acids in the intracellular domain of CHL1 mediate
binding to a sequence stretch in the third intracellular domain of the 5-HT2c receptor and
provide evidence that the interaction of CHL1 with the receptor effectively reduces the binding
of PTEN to the receptor and enhances the interaction of the receptor with β-arrestin 2 which is
involved in regulating the activity of constitutively active 5-HT2c receptors (Marion et al.,
2004). Since PTEN dephosphorylates the 5-HT2c receptor (Ji et al., 2006), enhanced
interaction between the 5-HT2c receptor and PTEN in the absence of CHL1 leads to prolonged
dephosphorylation
of
5-HT2c
receptor
in
CHL1-deficient
mice,
and
sustained
dephosphorylation of the 5-HT2c receptor at the plasma membrane by PTEN may affect the
activity of the 5-HT2c receptor and the signal transduction pathways downstream of the
5-HT2c receptor. Furthermore, reduced interaction of the dephosphorylated receptor with
β-arrestin 2 may affect receptor activity and receptor-dependent downstream signaling. The
results from behavioral analysis indicate that CHL1 predominantly controls the activity of
SB-206553-sensitive constitutively active isoforms of the 5-HT2c receptor and regulates the
signaling via these isoforms, while CHL1 does not appear to regulate expression of the
serotonin-activatable 5-HT2c receptor isoforms. Application of SB-206553, which specifically
reduces the activity of the constitutively active 5-HT2c receptors isoforms, increases locomotor
activity of CHL1-deficient, but not wild-type mice, and reverted the phenotype of
CHL1-deficient mice to that of wild-type mice. This finding strongly suggests that the functions
of constitutively active 5-HT2c receptor isoforms are abnormal in the absence of CHL1 and that
hypolocomotion of CHL1-deficient mice may be due to inhibition of locomotion mediated by
constitutively active 5-HT2c receptor isoforms regulates these receptor isoforms’ activities and
controls downstream signal transduction pathways: Erk phosphorylation and PLD activity is
reduced after SB-206553 treatment of cells expressing the constitutively active INI isoform but
not CHL1, while cells co-expressing the INI isoform and CHL1 did not respond to SB-206553.
On the basis of these observations, we propose that ablation of CHL1 reduces the activity of
constitutively active 5-HT2c receptor isoforms resulting in an enhancement of tonic inhibition
of locomotor activity. It is likely that absence of CHL1 leads to enhancement of inhibitory
transmission as seen in the CA1 region of juvenile CHL1-deficient mice (Nikonenko et al.,
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the constitutively active 5-HT2c receptor isoforms. Indeed, association of CHL1 with the
2006). Of note, CHL1 and 5-HT2c receptor are co-expressed in GABAergic interneurons
(Serrats et al., 2005; Liu et al., 2007; Ango et al., 2008; Jakovcevski et al., 2009) and
co-localize in GABAergic neurons of the putamen and hippocampal CA1 and CA3. Enhanced
activity of constitutively active 5-HT2c receptor isoforms in GABAergic neurons, e.g.
medium spiny neurons and/or interneurons, in the absence of CHL1 may result in increased
inhibition of neurons innervated by these GABAergic neurons, resulting in reduced locomotor
activity. Binding of SB-206553 to dysregulated constitutively active 5-HT2c receptor
isoforms in CHL1-deficient GABAergic interneurons may trigger signaling pathways which,
for example, reduce PLD activity and Erk phosphorylation, abolishing enhanced tonic
inhibition of locomotion by GABAergic neurons. Further investigations of CHL1´s role in
serotonergic and GABAergic transmission and/or in 5-HT2c receptor-mediated signaling
pathways in different brain areas should provide novel insights into the functions of the
serotonergic system under normal and pathological conditions with hope for therapy (see, for
Journal of Cell Science • Advance article
instance, Giorgetti and Tecott, 2004; Meltzer et al., 2012).
MATERIAL AND METHODS
Animals
CHL1-deficient mice (Montag-Sallaz et al., 2002) which have been back-crossed onto the
C57BL/6J background for more than eight generations and their age-matched wild-type
littermates as well as C57BL/6J mice were bred and maintained at the Universitätsklinikum
Hamburg-Eppendorf. Animals were housed at 25°C on a 12 h light/12 h dark cycle with ad
libitum access to food and water. Adult 10- to 12-week-old mice of either sex were used. All
animal experiments were approved by the local authorities of the State of Hamburg and
conform to the guidelines set by the EU.
Antibodies and reagents
Polyclonal antibodies against the extracellular domain of CHL1 (Chen et al., 1999; Rolf et al.,
2003) was used for Western blot analysis and a polyclonal goat antibody against intracellular
CHL1 epitopes from R&D systems (AF-2147) (Minneapolis, MN, USA) was used for
immunoprecipitation, immunostaining and proximity ligation assay. Polyclonal antibodies
against 5-HT2c receptor from Santa Cruz Biotechnology (goat N-19, sc-15081; mouse D-12,
sc-17797) (Heidelberg, Germany) were used for Western blot analysis, immunostaining and
proximity ligation assay and a polyclonal rabbit antibody (Pierce, PA1-18069) from Life
Technologies (Darmstadt, Germany) was used for immunoprecipitation. PTEN antibody
(clone 6H2.1) from Merck Chemicals (Millipore; Schwalbach, Germany) was used for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and total Erk were from Santa Cruz
Biotechnology, pErk antibody (phospho-p44/42 (Erk1/2) (T202/Y204) antibody) was from
New England Biolabs (Frankfurt, Germany) and monoclonal parvalbumin antibody was from
Sigma-Aldrich (p-3088) (Taufkirchen, Germany). Cloning and production of recombinant
His-tagged or GST-tagged intracellular domains of CHL1 (CHL1-ICD), His-tagged L1
(L1-ICD) and His-tagged NCAM140 (NCAM-ICD) have been described (Xiao et al., 2009;
Andreyeva et al., 2010). The plasmid pGEX3X-GST-PTEN (Addgene, Cambridge, MA, USA)
was used for the expression of recombinant PTEN tagged with GST. Production of
PTEN-GST was as described (Xiao et al., 2009). Horseradish peroxidase (HRP)-coupled and
Journal of Cell Science • Advance article
immunoprecipitations and Western blot analysis. Antibodies against β-arrestin 2,
fluorescent dye-coupled secondary antibodies were from the Jackson Laboratory (Bar Harbor,
ME, USA). The synthetic phage peptide (KKDRRPRSLHPH), the 5-HT2c receptor-derived
peptide (NPNPDQKPRRKKKEKRPRGTMQAINNEKK) and the peptide tat/281-309
containing the HIV tat sequence YGRKKRRQRRR (Schwarze and Dowdy, 2000) and the
amino
acids
281-309
of
the
murine
5-HT2c
receptor
(YGRKKRRQRRRNPNPDQKPRRKKKEKRPRGTMQAINNEKK) were from Schafer-N
(Copenhagen, Denmark). Ro60-0175 fumarate (CAS 169675-09-6) and SB-206553
hydrochloride, (CAS 158942-04-2) were from Biozol (Eching, Germany). For the
determination of the PLD activity, the Amplex® Red Phospholipase D Assay Kit (A12219)
from Molecular Probes (LifeTechnologies) was used.
Phage display
The Ph.D.-12TM Phage Display Peptide Library (New England Biolabs) displaying 108-1010
random 12-mer peptides at the pili of M13-like phage particles in fusion with the N-terminus
of the pVIII major coat protein and the intracellular domain of CHL1 (CHL1-ICD) were used
for screening of peptides binding to CHL1-ICD. All selection steps were performed according
to the Ph.D.-12TM Phage Display Peptide Library Kit instruction manual version 2.0 (New
England Biolabs) and have been described (Wang et al., 2011).
Western blot analysis, immunoprecipitation, biochemical crosslinking, pull-down assay
and ELISA
biochemical crosslinking have been described in detail (Makhina et al., 2009; Xiao et al.,
2009). For preparation and immunoprecipitation of phosphorylated proteins PhosSTOP
Phosphatase Inhibitor Cocktail Tablets (Roche, Mannheim, Germany) were used and TBS
buffers were used for detection. For visualization of immunopositive bands by Western blot
analysis the chemiluminescent substrate with extended duration (Pierce, Bonn, Germany) was
used.
Band
intensities
(http://imagej.nih.gov/ij/).
were
densitometrically quantified
using
ImageJ
software
Journal of Cell Science • Advance article
Western blot analysis, immunoprecipitation, pull-down assay and ELISA as well as
Tissue preparation, immunohistochemistry and proximity ligation assay
Brains from 3-month-old mice were fixed with 4% formaldehyde in 0.1 M cacodylate buffer,
pH 7.3, at room temperature, post-fixed overnight at 4°C in the formaldehyde solution,
incubated in 15% sucrose solution in 0.1 M cacodylate buffer, pH 7.3, for 2 days at 4°C,
frozen by immersion for 2 min in 2-methyl-butane (isopentane) precooled to -80°C and stored
in liquid nitrogen. Serial coronal 25-μm-thick sections were cut in a cryostat (Leica CM3050,
Leica Instruments, Wetzlar, Germany) and collected on SuperFrost Plus glass slides (Carl
Roth, Karlsruh, Germany). Antigen retrieval was performed by incubating the sections in 10
mM sodium citrate solution (pH 9.0) for 30 min at 80°C, followed by blocking of
non-specific binding sites with phosphate buffered saline, pH 7.3 (PBS) containing 0.2%
Triton X-100, 0.02 % sodium azide, and 5% normal donkey serum for 1 h at room
temperature. The sections were incubated with primary antibodies diluted in PBS overnight at
4°C in a humidified chamber. For immunohistochemistry, the sections were washed in PBS (3
× 15 min at room temperature), incubated at room temperature with donkey Cy3- or
Cy-2-conjugated secondary antibodies diluted in PBS for 2 h, washed in PBS, and incubated
for 10 min at room temperature with bis-benzimide solution (Hoechst dye 33258, 5 μg/ml in
PBS, Sigma-Aldrich) to stain cell nuclei. Finally, the sections were washed again and
mounted with Fluoromount G (Southern Biotechnology Associates, Birmingham, AL, USA)
and microphotographs were taken on a Leica confocal laser scanning microscope (Leica) at a
1024 × 1024 digital resolution. For proximity ligation assay, the sections were incubated with
a pair of secondary antibodies conjugated with oligonucleotides (Duolink anti-goat PLA
RED (Sigma-Aldrich) was used. Coverslips were mounted with Roti-Mount FluorCare DAPI
(Carl Roth, Karlsruhe, Germany) and confocal images were taken with an Olympus Fluoview
FV1000 confocal laser scanning microscope (Hamburg, Germany) in sequential mode with a
60x objective.
Drug administration and measurement of locomotor activity
The 5-HT2c receptor-specific agonist Ro60-0175 or inverse agonist SB-206553 were
dissolved in saline and injected intraperitoneally before behavioral testing. All injections (1
Journal of Cell Science • Advance article
probe MINUS and Duolink anti-rabbit PLA probe PLUS) and the Duolink detection reagent
mg/kg body weight) were given in a volume of 0.1 ml. Saline injection served as vehicle
control. Locomotion was evaluated by the open field test. The open field consisted of a
wooden box (50×50×40 cm) laminated with rough, matted, light-gray resin, and illuminated
by a white bulb (100 Lux). After injection, each mouse was gently introduced into a cylinder
of opaque Plexiglas placed at one corner of the box for 5 sec. As the cylinder was lifted, the
mouse could move freely in the arena for 30 min. Locomotor activity was measured at 5 min
intervals and cumulative counts were taken for data analysis with the software EthoVision
(Noldus, Wageningen, Netherlands).
Cloning of expression vectors and transfection of COS-7 cells
For the single expression of the INI isoform and the co-expression of the INI isoform and
CHL1, COS-7 cells (ATCC® CRL-1651™; tested for contamination every 3 months) were
transfected with INI or INI/CHL1 coding bicistronic mammalian expression vectors using
Turbofect™ (Thermo Fisher Scientific, Darmstadt, Germany). For cloning, InFusion Cloning
Kit (Clontech) and PCR amplifications with Phusion Polymerase (New England Biolabs)
were used. The product resulting from PCR using INI-coding plasmid (Herrick-Davis et al.,
1999) and the primers pAG/HT2c fw (5´-GTT GCC TTC GCC CCG ATG GTG AAC CTT
GGC AAC G-3´; start codon in bold) and HT2c/IRES rev (5´-TCG CAC GAT TAC CAT TTA
CAC ACT ACT AAT CCT CTC-3´; stop codon is underlined) were used for cloning of
INI-coding cDNA. The INI-coding plasmid was a kind gift from Katharine Herrick-Davis
(Albany Medical College, Albany, NY, USA). For cloning of CHL1-coding cDNA, the
(5´-ATG ATG GAA TTG CCA TTA TGT-3´) and pCAG/Chl1 rev (5´-TGG CGG CCG GCC
GCT TCA TGC CCG GAG TGG GAA-3´) were used. For amplification of the IRES element,
the pCAG-GFP (Addgene, Cambridge, MA, USA) and the primers IRES fw (5´-ATG GTA
ATC GTG CGA GAG G-3´) and IRES/CHL1 rev (TGG CAA TTC CAT CAT GGT TGT
GGC CAT ATT ATC AT) were used. The products from PCR with pCAG-GFP and the primer
pAG fw (5´-AGC GGC CGG CCG CCA GCA CAG TGG-3´) and pCAG rev (5´-CGG GGC
GAA GGC AAC GCA GCG ACT-3´) or IRES/pCAG rev (5´-TGG CGG CCG GCC GCT GGT
TGT GGC CAT ATT ATC AT-3´) were used for cloning of INI-coding or of INI- and
Journal of Cell Science • Advance article
product from PCR with CHL1-coding vector (Holm et al., 1996) and the primers Chl1 fw
CHL1-coding PCR products. The vector resulting from the fusion of the pCAG PCR product
with 5-HT2c receptor-, IRES- and CHL1-coding PCR products was used to co-express the
5-HT2c receptor and CHL1 and the vector resulting from the fusion of the pCAG PCR
product with the 5-HT2c receptor- and IRES-coding PCR products was used for single
Journal of Cell Science • Advance article
expression of the receptor.
Acknowledgements
We are grateful to Eva Kronberg for excellent animal care, to Jeanette Reinshagen
(Fraunhofer IME ScreeningPort), Ute Bork and Emanuela Szpotowicz for excellent technical
assistance, Igor Jakovcevski for technical support and fruitful discussions and Katharine
Herrick-Davis for plasmids.
Competing interests
The authors declare no competing or financial interests.
Author contributions
R.K. and M.S. conceived the study; R.K. and G.L. designed experiments; H.C., N.K., J.K.,
A.K., K.G., G.L. and R.K. performed experiments and analyzed the data together with M.S.;
R.K. and M.S. wrote the manuscript.
Funding
Melitta Schachner is supported by the Li Kashing Foundation at Shantou University Medical
Journal of Cell Science • Advance article
College.
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Figures
the 5-HT2c receptor. (A) Alignment of the intracellular domains of the L1 subfamily
members CHL1, L1, neurofascin (Nfc) and NrCAM from mouse. Numbers designate amino
acid positions in CHL1-ICD. Conserved (light grey) and non-conserved (dark grey) amino
acids relative to the CHL1-ICD sequence are indicated. (B) The sequence of a peptide
identified in a phage display analysis using CHL1-ICD as bait shows significant similarity to
a sequence in the third intracellular domain (ICD3) of the 5-HT2c receptor. Numbers
designate amino acid positions. Identical (|), highly conserved (:) and weakly conserved (.)
amino acids are indicated and a schematic representation of the 5-HT2c receptor membrane
Journal of Cell Science • Advance article
Fig. 1: Binding of CHL1-ICD to a sequence stretch in the third intracellular domain of
topology and the position of ICD3 within the 5-HT receptor is shown. (C,D) Substrate-coated
phage peptide (C), CHL1-ICD or L1-ICD (D) were incubated with increasing concentrations
of soluble His-tagged CHL1-ICD and L1-ICD (C) or a biotin-carrying 5-HT2c
receptor-derived peptide (D). Binding was determined by ELISA using His-tag antibody and
HRP-conjugated secondary antibody (C) or HRP-conjugated streptavidin (D). Mean values +
standard deviation from three independent experiments carried out in triplicates are shown. (E)
CHL1-ICD (CHL1), L1-ICD (L1) and NCAM-ICD (NCAM) were incubated with mouse
brain detergent extracts followed by pull-down with Ni-NTA beads. Brain extracts (input) and
precipitates were probed by Western blot (WB) analysis with a mouse 5-HT2c receptor
antibody. (F) Brain extracts from CHL1-deficient mice (-/-) and wild-type littermates (+/+)
(input) were subjected to immunoprecipitation (IP) with rabbit CHL1 antibody and Western
blot analysis (WB) with mouse 5-HT2c receptor antibody and GAPDH antibody to control for
loading. (E,F) Representative Western blots out of three independent experiments with
Journal of Cell Science • Advance article
identical results are shown.
Fig. 2: The 29 N-terminal amino acids of CHL1-ICD mediate binding to the 5-HT2c
receptor. (A) Schematic representation of full-length CHL1-ICD (FL) and of CHL1-ICD with
C- and N-terminal truncations. Numbers designate amino acid positions in CHL1-ICD as
represented in Fig. 1. (B,C) Substrate-coated 5-HT2c receptor-derived peptide was incubated
constructs or GST (C). Binding was determined by ELISA using GST antibody and
HRP-conjugated secondary antibody. Mean values + standard deviation from three
independent experiments carried out in triplicates are shown.
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with 100, 250 and 400 µg/ml (B) or with increasing concentrations of GST-tagged CHL1-ICD
striatum. Representative images of double immunofluorescence for CHL1 and 5-HT2c
receptor (5-HT2c) (A) or for parvalbumin (PV) and CHL1 (B) or for the 5-HT2c receptor (C)
in the putamen from three mice are shown. Superimposition indicates co-localizations in
yellow (arrows). Scale bars: 15 µm.
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Fig. 3: Co-localization of CHL1 and 5-HT2c receptor in GABAergic neurons of the
Fig. 4: Co-localization of CHL1 and 5-HT2c receptor in GABAergic interneurons of the
hippocampus. Representative images of double immunofluorescence for CHL1 and 5-HT2c
receptor (5-HT2c) (A) or for parvalbumin (PV) and CHL1 (B) or 5-HT2c receptor (C) in the
co-localizations in yellow (arrows). Scale bars: 15 µm.
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CA3 region of the hippocampus from three mice are shown. Superimposition indicates
Fig. 5: Close interaction between CHL1 and 5-HT2c receptor in striatum and
hippocampus. Striatal (A) and hippocampal (B,C-F) slices from wild-type (A-C,E) and
CHL1-deficient (D,F) mice were used for proximity ligation assay with antibodies against
CHL1 and 5-HT2c receptor (5-HT2c) (A-D) or with antibodies against CHL1 and PTEN
(E,F). Representative confocal fluorescent images from the putamen (A) and from the CA3
(B,D-F) and CA1 (C) regions from three mice per group are shown. Nuclei are stained with
DAPI (blue) and spots of intense fluorescent signals (red) indicate close protein interactions.
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Scale bars: 15 µm.
Fig. 6: Behavioral responses to inverse serotonin agonist application in CHL1-deficient
mice. CHL1-deficient female (A,C) or male (B,D,E) mice (CHL1-/-) and wild-type female
and male littermates (CHL1+/+) were placed into the open field immediately after injection of
vehicle solution, SB-206553 or Ro60-0175. Locomotor activity after injection of vehicle
solution was monitored for 30 min at 5 min intervals (A,B). After injection of vehicle solution
(NaCl), SB-206553 (SB) or Ro60-0175 (Ro), distance moved by females for a period of 30
min (C) and distance moved by males for a period of 5 min (D) as well as numbers of rearing
by males within 5 min (E) were measured. (A-E) Mean values ± standard deviation are shown
variance followed by Bonferroni post-hoc test and significant differences (*p<0.001) between
wild-type and CHL1-deficient mice and between untreated and treated mice are indicated.
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(n=10 females, n=14 males per group). The groups were analyzed by two-way analysis of
receptor. (A) CHL1-ICD was incubated with brain extracts in the absence (-) or presence of
GST or PTEN-GST (PTEN) and the tat/281-309 peptide (tat) and subjected to pull-down of
His-tagged CHL1-ICD by Ni-NTA beads followed by Western blot (WB) analysis with the
goat 5-HT2c receptor antibody. (B-E) Detergent extracts of brains from CHL1-deficient (-/-)
and wild-type mice (+/+) (input) were subjected to immunoprecipitation (IP) with PTEN,
CHL1 or 5-HT2c receptor antibody in the absence or presence of the tat/281-309 peptide (tat)
and to Western blot analysis (WB) with antibodies against 5-HT2c receptor (5-HT2c), PTEN,
phospho-serine (pSer) or β-arrestin 2 (arrestin). (A-E) Representative Western blot out of
three independent experiments with identical results are shown.
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Fig. 7: CHL1 regulates the association of PTEN and β-arrestin 2 with the 5-HT2c
Fig. 8: CHL1 regulates signaling via constitutively active 5-HT2c receptor isoforms.
COS-7 cells were transfected with a vector coding only for the non-edited INI isoform of the
5-HT2c receptor (I) or coding for the INI isoform and CHL1 (I/C). Cell lysates (input) were
subjected to immunoprecipitation (IP) with CHL1 and to Western blot analysis (WB) with
antibodies against 5-HT2c receptor (5-HT2c) (A), to quantification of PLD activity (B), or to
quantification of Erk phosphorylation by Western blot analysis with antibodies against pErk
and total Erk (C). (A,C) Representative Western blots from 3 independent experiments are
shown. (B,C) Mean values ± standard deviation for PLD activity and for pERK levels relative
to total Erk levels from 6 independent experiments in comparison to untreated cells
expressing the INI isoform (set to 100%) are shown. The groups were analyzed by two-way
analysis of variance followed by Bonferroni post-hoc test and significant differences
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(*p<0.001) between groups are indicated.