Biochemical and Biophysical Research Communications 466 (2015) 46e51

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Biochemical and Biophysical Research Communications

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Acute reduction of neuronal RNA binding Elavl2 protein and Gap43

mRNA in mouse hippocampus after kainic acid treatment
Takafumi Ohtsuka a, Masato Yano a, b, *, Hideyuki Okano a, **
a
Department of Physiology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan
b
Division of Neurobiology and Anatomy, Graduate School of Medical and Dental Sciences, Niigata University, 757, Ichibancho, Asahimachidori, Chuo-ku,
Niigata-shi, Niigata, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Activity-dependent gene regulation in neurons has been hypothesized to be under transcriptional
Received 11 August 2015 control and to include dramatic increases in immediate early genes (IEGs) after neuronal activity. In
Accepted 24 August 2015 addition, several reports have focused on post-transcriptional regulation, which could be mediated by
Available online 30 August 2015
neuronal post-transcriptional regulators, including RNA binding proteins (RNABPs). One such protein
family is the neuronal Elavls (nElavls; Elavl2, Elavl3, and Elavl4), whose members are widely expressed in
Keywords:
peripheral and central nervous system. Previous reports showed that Elavl3 and 4 are up-regulated
Elavl2
following repeated stimulation such as during cocaine administration, a seizure, or a spatial discrimi-
RNA binding protein
Post-transcriptional control
nation task. In this study, we focused on Elavl2, a candidate gene for schizophrenia and studied its role in
Neuronal activity neuronal activity. First we found that Elavl2 has a cell-type specific expression pattern that is highly
expressed in hippocampal CA3 pyramidal neurons and hilar interneurons using Elavl2 specific antibody.
Second, unexpectedly, we discovered that the Elavl2 protein level in the hippocampus was acutely down-
regulated for 3 h after a kainic acid (KA)-induced seizure in the hippocampal CA3 region. In addition,
level of Gap43 mRNA, a target mRNA of Elavl2 is decreased 12 h after KA treatment, thus suggesting the
involvement of Elavl2 in activity-dependent RNA regulation.
© 2015 Elsevier Inc. All rights reserved.

1. Introduction as autism spectrum disorder (ASD), mental retardation, Alzheimer's

RNA binding proteins (RNABPs) are a major contributor of post-
transcriptional gene regulation through their direct binding to
specific RNA targets in vivo. This is particularly true for neurons, in
which neuronal, activity-dependent gene regulation, including
alternative splicing and translational control of target RNA by
RNABPs, has been reported to be associated with facilitating syn-
aptogenesis [1,2]. Therefore, activity-dependent, post-transcrip-
tional regulation is thought to be responsible both for
spatiotemporal gene expression and for the formation of complex
neural circuits that lead to higher ordered brain functions, such as
learning or memory. Furthermore, impairments of this type of
regulation are associated with various neurological disorders, such
* Corresponding author. Division of Neurobiology and Anatomy, Graduate School
of Medical and Dental Sciences, Niigata University, 757, Ichibancho, Asahimachidori,
Chuo-ku, Niigata-shi, Niigata, Japan.
** Corresponding author.
E-mail addresses: myano@med.niigata-u.ac.jp (M. Yano), hidokano@a2.keio.jp
(H. Okano).
disease (AD), and amyotrophic lateral sclerosis (ALS) [3e5].
Neuronal ELAV-like proteins (nElavls) are neuron specific
RNABPs, which include Elavl2, Elavl3, and Elavl4 (also known as
HuB, HuC, and HuD). They have been identified as autoimmune
antigens in the sera of patients with paraneoplastic neurologic
disorder (PND) [6]. Many in vitro and in vivo studies have revealed
that nElavls play important roles in multiple steps of neuronal
development [7e9]. One such role during neurodevelopment is the
function of nElavl in post-transcriptional control. In vitro modeling
has revealed that Elavl4 stimulates Cap-dependent translation
through its direct binding to eIF4A and the poly-A sequence of
target mRNA [10]. Moreover, this function is mediated by the direct
binding of Elavl4 to active Akt1, which leads to the activation of
translational initiation via mammalian Target of Rapamycin (mTOR)
[11]. In contrast, the control of nElavl expression itself is also
associated with several brain functions. For example, nElavl pro-
teins are up-regulated in the hippocampus after learning in a radial
maze and after several neuronal activities [12,13]. Additionally,
previous experiments in which Elavl3 is knocked down in these
trained mice resulted in decreased mRNA expression of Gap43,

http://dx.doi.org/10.1016/j.bbrc.2015.08.103
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 T. Ohtsuka et al. / Biochemical and Biophysical Research Communications 466 (2015) 46e51
which is both a crucial component of axogenesis and an nElavl
target [13]. This same experimental group also showed that phos-
phokinase C (PKC a) directly binds to and phosphorylates nElavl
proteins and recruits them to the cytoplasm, which results in Gap43
translation [14]. Furthermore, after spatial learning using the
Morris water maze, Elavl4 expression increases, which induces the
up-regulation of Gap43, even one month after spatial training [15].
These reports suggest that nElavls could regulate the activity-
dependent, mRNA stability and translational activation of target
mRNAs by controlling their expression level and localization,
thereby manipulating the complex processes of synaptogenesis and
axogenesis in vivo.
Here, we show that Elavl2, a schizophrenia susceptibility gene in
Asian populations [16], has a region- and cell type-specific
expression pattern in the hippocampus. There, it is strictly
restricted to the CA3 pyramidal neurons and hilar interneurons,
which indicates that Elavl2 is differentially expressed and regulated
from other nElavl family members. Moreover, we find that Elavl2 is
acutely decreased at the protein-expression level in the CA3 after
kainic acid-induced neuronal activity, followed by the reduction of
Elavl2 target transcript, Gap43 mRNA.
2. Materials and methods
2.1. Animals and seizure induction using kainic acid
All animal care and treatment procedures were performed in
accordance with the institutional guidelines approved by the
Experimental Animal Care Committee of the Keio University School
of Medicine (approved No.09091). In all experiments, adult, male,
CD-1 mice (8 weeks of age) were used. Kainic acid (KA; Sigma,
K0250) was dissolved in sterile phosphate-buffered saline (PBS)
and administered intraperitoneally at 20 mg/kg body weight. Same
amounts of sterile PBS were administered to control (Sham) mice,
as above. After the KA administration, seizure activities, such as
lying motionless or adopting a rigid posture with tail extension,
myoclonic contracture of the neck, forelimb clonus, and rearing
were observed at each time point.
2.2. Quantitative RT-PCR
Total RNA was extracted from dissected hippocampi of three
sham and KA-treated mice, using Trizol reagent (Invitrogen) and a
RNeasy kit (QIAGEN). Then, 1 mg of total RNA was reverse tran-
scribed with a iScript cDNA synthesis kit (BioRad). The amount of
cDNA was quantified with SYBR Premix Ex Taq II (Takara) and
StepOnePlus real-time PCR system (Life technology).
2.3. Western blotting
Hippocampi sections were dissected and homogenized in
MAPK-lysis buffer that contained 10 mM TriseHCl (pH7.6), 50 mM
NaCl, 30 mM Na3PO4, 50 mM NaF, 20 mM b-glycerophosphoric acid,
1% Triton X-100, and Complete EDTA-free protease inhibitor
(Roche). Protein concentrations were measured with BCA assay kit
(Thermo Fisher Scientific), and 10 mg of protein was separated on
each lane by SDS-PAGE. Then, protein was electroblotted onto a
polyvinylidene difluoride membrane. The membranes were pre-
blocked with Tris-buffered saline containing 2% skim milk (wt/v)
for 1 h at room temperature. They were then incubated with pri-
mary antibodies at 4  C overnight, followed by an additional in-
cubation with an HRP-conjugated secondary antibody at room
temperature for 90 min. The protein amount was quantified as a
measure of intensity of chemiluminescence with ECL prime west-
ern blotting detection reagent (GE Healthcare) on the LAS4000
47
mini detector (GE Healthcare).
2.4. Immunofluorescence microscopy
Animals were perfused with 4% (w/v) paraformaldehyde in PBS
and brains were removed. The brains were post fixed in the same
fixative at 4  C overnight and were then sliced into 50-mm sections
using a Vibratome (Leica). The sections were incubated in HistoVT
One antigen retrieval solution (Nacalai tesque) for 30 min at 70  C
and preblocked with a Tris-NaCl blocking buffer (PerkinElmer) for
1 h at room temperature. Then, sections were incubated with pri-
mary antibodies at 4  C overnight, followed by incubation with an
Alexa-conjugated secondary antibody for 90 min at room temper-
ature. Slices were analyzed with a LSM700 Laser Scanning Confocal
Microscope (Carl Zeiss Microscopy).
2.5. Antibodies
The following primary antibodies were used in this study: rabbit
anti-Elavl2 (Sigma, H1538, 1:1000), mouse anti-actin (Sigma,
A1978, 1:1000) and human anti-Pan-nElavls (gift from Dr. Darnell
R. B., 1:2000) for immunoblotting; and rabbit anti-Elavl2 (Sigma,
H1538, 1:400), rabbit anti-c-Fos (Calbiochem, PC38, 1:400), mouse
anti-parvalbumin (Sigma, P3088, 1:400), mouse anti-Calbindin-D-
28K (Sigma, C9848, 1:400) and human anti-Pan-nElavls (gift from
Dr. Darnell R. B., 1:800) for immunohistochemistry.
3. Results
3.1. Elavl2 is specifically expressed in the CA3 pyramidal neurons
and hilar interneurons in the hippocampus
All neurons express sets of nElavl family mRNA and proteins;
however, each nElavl shows a different expression pattern in its
mRNA [17]. These different sets of expression pattern for the same
family member are thought to predicate the diversity and
complexity of neuronal development and the functions of the brain.
In a recent report, the ELAVL2 gene has been shown to be a
schizophrenia-related, top candidate gene in Asian populations
[16]. To discriminate Elavl2 and other Elav members, on the basis of
expression, we first analyzed detailed Elavl2 protein expression by
using an anti-Elavl2 specific antibody and pan-neuronal Elavl anti-
serum. Immunohistochemistry revealed that Elavl2 expression is
restricted to specific neurons, including the CA3 pyramidal neurons
of the hippocampus, but on the other hand the other nElavl pro-
teins are expressed in all neurons (Fig. 1AeC). This agrees with a
previous in situ hybridization analysis for Elavl2 mRNA [17]. In
addition, we observed that Elavl2 protein is expressed in
parvalbumin-positive interneurons and parvalbumin-negative
basket neurons by double immunostaining (Fig. 1D). This unique
expression indicates that Elavl2 is a useful marker for specific
neurons in hippocampus and implies an involvement of Elavl2 in
diversity and specificity of post-transcriptional regulation in
various neuron cell types. Moreover, Elavl2 protein localization is
predominantly cytoplasmic in any neuron (Fig. 1), which suggests
that the major function of Elavl2 is translational control or mRNA
stabilization rather than splicing control, which is generally a nu-
clear event.
3.2. Acute reduction of Elavl2 protein level depending on kainic
acid-derived neuronal activity
The unique expression of Elavl2 in the hippocampus is likely to
be involved in neuronal activity because both of the high-affinity
kainate receptors (GluK4, and GluK5) and their auxiliary subunits
48 T. Ohtsuka et al. / Biochemical and Biophysical Research Communications 466 (2015) 46e51

Fig. 1. Unique expression pattern of Elavl2 in adult hippocampus. (A) Immunolabeling for Elavl2 protein in coronal section of adult hippocampus were immunostained with pan-
nElavls (green) and Elavl2 (Magenta). (B) Calbindin (Green) and Elavl2 (Magenta) (C) High magnification of CA3 region shown in (B) (D) Dentate gyrus were immunostained
with Parvalbumin (PV: Green), Elavl2(Magenta) and Hoechst (blue). Arrow indicates PV and Elavl2 positive neurons and arrow head indicates PV negative Elavl2 positive neurons.
Scale bar: A and B 100-mm, C and D 20-mm.

Neto1 are highly expressed and subsequently accumulate in the
hippocampus CA3 region [18,19]. To address whether kainate
stimulation regulates Elavl2 expression, we administered kainic
acid (KA) intraperitoneally to adult mice, to quantify the abundance
of Elavl2 according to the neuronal activity in the hippocampus
(Fig. 2A). First, we measured the amount of Elavl2 protein via
Western blotting for each time point after KA treatment. Unex-
pectedly, the level of Elavl2 protein was acutely and drastically
reduced to half of the level of its control at 3 h after KA treatment.
However, there was no significant change in the amounts of Elavl2
protein at other time points (Fig. 2BeC). We also analyzed the
amount of nElavl whole protein using anti-sera of a patient with
PND, which detects all isoforms of nElavls [20]. In contrast, we
found few differences in nElavl whole protein level between the
control and KA groups at any time point, which indicated a specific
regulation of Elavl2 (Fig. 2C). Next to address whether the acute
Elavl2 protein reduction that was observed at 3 h after KA treat-
ment correlated with a specific expression pattern of Elavl2
compared with other nElavl proteins, we performed an immuno-
histochemical analysis of mice brains after KA treatment. Inter-
estingly, we found that acute Elavl2 protein reduction was
specifically observed in the KA treated CA3 region in contrast to
accumulation of cFos protein (Fig. 2DeE). Alternatively, the Elavl2
fluorescence intensity was not significantly changed in the hilus,
though Elavl2 protein is co-expressed with cFos protein. Finally, we
concluded that an acute reduction of Elavl2 expression by KA
stimulation predominantly occurs in the CA3 region.
3.3. Elavl2 is involved in RNA metabolism during KA induced
neuronal activity
Next to see RNA metabolism at several time points, 30 min, 3 h
and 12 h after KA administration, we isolated mRNA from mice that
were exhibiting seizure activities and then analyzed them with
quantitative RT-PCR (Fig. 3). We observed drastic increases in the
expression of immediate early genes (IEGs), such as cFos and Bdnf
mRNA with hierarchical expression, of which peaked at 30 min and
3 h, respectively and returned to basal level to 12 h after KA induced
seizure (Fig. 3). At these time points, we did not find any significant
changes in the levels of Elavl2 mRNA (Fig. 3). Given that acute
reduction of Elavl2 protein appeared to peak 3 h after KA induced
neuronal activity, thus we hypothesized that Elavl2 dependent RNA
metabolism could occur in the following time course. We did
quantitative RT-PCR assay to look at the expression of Elavl2 RNA
target Gap43 mRNA previously used to be an in vivo RNA target of
nElavls [20]. In this result, we found that Gap43 mRNA was reduced
in 12 h after KA administration, which could be occur in concert
with Elavl2 reduction (Fig. 3). Finally, these results suggest that
Elavl2 might be involved in region-specific and neuronal activity-
dependent RNA metabolism.
4. Discussion
In this work, we focused on Elavl2, a member of the nElavl
neuron-specific RNA-binding protein family in the mouse
 T. Ohtsuka et al. / Biochemical and Biophysical Research Communications 466 (2015) 46e51 49

Fig. 2. Acute reduction of Elavl2 protein and Gap43 mRNA after Kainic-acid treatment. (A) Scheme of KA administration and sampling time points (B) Western blotting analysis of
Elavl2 (upper), Pan-nElavls (midlle) and Actin (lower) in hippocampus using 3 time points after KA induction (C) Quantification of relative protein expression levels of pan-nElavls
(top) and Elavl2 (bottom) normalized with Actin from data in (A). Each point represents the average of three biological replicates. (D and E) Immunofluorescent double staining in
the hippocampus 3 h after KA treatment. cFos protein (green) is highly expressed and accumulated in KA treated mice hippocampus CA region shown in (D) Intensity of Elavl2
immunofluorescence (magenta) is reduced in KA treated CA3 region shown in (E). Scale bar: D 50-mm, E 100-mm. Error bars indicates mean ± SEM. *p < 0.05.

hippocampus. Histological analysis revealed that Elavl2 has unique mRNA reduction. These phenomena strongly suggest the involve-
expression pattern in the hippocampus compared with other nElavl ment of Elavl2 in activity-dependent, spatiotemporal, post-
proteins. Additionally, we found KA treatment induced down- transcriptional regulations in vivo.
regulation of Elavl2 protein in a specific region followed by Gap43 nElavls have been shown to be both neuronal gene regulators
50 T. Ohtsuka et al. / Biochemical and Biophysical Research Communications 466 (2015) 46e51
Fig. 3. Altered mRNA expression after Kainic-acid induction. Quantitative RT-PCR analysis at each time points for various mRNAs, cfos, bdnf, Elavl2 and Gap43 in the hippocampus.
Quantification was calculated using the DD Ct method, and is presented as normalized expression by control (sham). Error bars indicates mean ± SEM. *p < 0.05, **p < 0.01.
and neuronal markers through the use of pan-nElavl antibodies.
However, these members show different distribution and expres-
sion levels in various brain regions [17], which suggests that their
different series generate molecular diversity and complexity of the
brain via RNA regulation. The redundancy of expression and the
lack of specific antibodies against each nElavl protein have hin-
dered progress in research on nElavls. In this study, we used the
antibody specific for Elavl2 to determine the Elavl2-specific
expression pattern in the brain. Previous reports have indicated
that Elavl3 is most highly expressed among nElavls in the hippo-
campus [17,20], but our data show that Elavl2 protein is also highly
expressed in the hippocampus and the CA3 pyramidal neurons and
hilar interneurons in particular. We also observed that it was not
expressed in granule neurons and most of CA1/2 pyramidal neu-
rons (Fig. 1). This expression pattern of nElavls and Elavl2 might
enable individual neurons to control their own transcriptome and
translatome.
Several reports have shown that the neuronal Elavl3 and Elavl4
levels are up-regulated by neuronal activity [12e15]. This finding
suggests that the control of their expression level by several stimuli
might play an important role in the molecular complexity of the
brain and the behavior of the individual. In contrast, we found that
Elavl2 protein is reduced at an early time point following neuronal
activation without change of its mRNA level (Figs. 2 and 3).
Although Elavl3 or Elavl4 up-regulation has been shown to occur
after at least 24 h of neural activity, our model shows a decrease in
Elavl2 protein expression occurring in CA3 pyramidal neurons
following drastic changes in gene regulation, including acute
activity-dependent, RNA regulation, without a change in mRNA
level. Interestingly, given that the Elavl2 expression pattern was
similar to that of the kainic acid receptors (KARs) and their cellular
components, including Neto1, this region-specific protein reduc-
tion of Elavl2 may be dependent on the KA-KARs signaling axis. For
example, the translational efficiency of Elavl2 mRNA might be
reduced by miRNA or Elavl2 protein might be acutely degradated
using ubiquitin-proteasome pathway through KA-KARs signaling.
In fact, several miRNAs are up-regulated after hippocampal
neuronal activity [21]. For another example, the p97-UBXD8 com-
plex directly binds to the RNA recognition motif (RRM) 3 region of
another Elav family protein, Elavl1, thereby leading to the ubiq-
uitination of Elavl1 [22]. This finding suggests that the p97-UBXD8
complex also strongly binds to Elavl2 and Elavl3 in vitro [22, sup-
plemental data]. Although we did not find any evidence to support
this prediction, the need to identify the key molecule that leads to
acute down-regulation of the Elavl2 protein level warrants the
further analysis to verify this prediction in vivo.
Further, the ELAVL2 gene has been reported to be a
schizophrenia-related, top candidate gene in Asian populations
[16]. Interestingly, its SNP site (SNP ID; rs10491817) is located in the
ELAVL2, non-coding first intron region, which could likely contain
regulatory elements of gene expression. Therefore, it is important
to understand expression mechanism of Elavl2 gene itself and
Elavl2 dependent RNA regulation during neuronal activity. In the
present study, we found unique expression pattern and controlled
expression level of Elavl2, which they might help us to understand
the complex disease. So far, we do not yet know the role of con-
trolling Elavl2 levels in the regulation of higher ordered brain
functions linked to the symptoms of schizophrenia. Nevertheless,
improved molecular, genetic, and transcriptome-wide strategies
will be able to elucidate these precise mechanisms and biological
roles in vivo.
Disclosure of conflicts of interests
H.O. is a paid Scientific Advisory Board of San Bio co. Ltd.
Acknowledgments
We are grateful to Dr. Robert B. Darnell for a kind gift of anti-sera
against Pan-nElavl, to all the members of Okano laboratory,
particularly I. Koya and S. Suyama for technical advice and helpful
discussions and Y. Hayakawa-Yano for critical comments. This work
was supported by grants from the SIL Research Fund from the
Takeda Pharmaceutical Company, Ltd. (to M. Y and H. O, J14K0123),
and a Grant-in-Aid for Encouragement of Young Medical Scientists
and the Keio University Doctorate Student Grant-in-Aid Program
from Keio University and the Japan Society for the Promotion of
Science (to T. O, 05-045-0233).
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.bbrc.2015.08.103.
 T. Ohtsuka et al. / Biochemical and Biophysical Research Communications 466 (2015) 46e51 51

References [12] D.M. Tiruchinapalli, M.G. Caron, J.D. Keene, Activity-dependent expression of
ELAV/Hu RBPs and neuronal mRNAs in seizure and cocaine brain,
J. Neurochem. 107 (2008) 1529e1543, http://dx.doi.org/10.1111/j.1471-
[1] T. Iijima, K. Wu, H. Witte, Y. Hanno-Iijima, T. Glatter, S. Richard, et al., SAM68
4159.2008.05718.x.
regulates neuronal activity-dependent alternative splicing of neurexin-1, Cell.
[13] a Quattrone, a Pascale, X. Nogues, W. Zhao, P. Gusev, a Pacini, et al., Post-
147 (2011) 1601e1614, http://dx.doi.org/10.1016/j.cell.2011.11.028.
transcriptional regulation of gene expression in learning by the neuronal
[2] J.D. Richter, N. Sonenberg, Regulation of cap-dependent translation by eIF4E
ELAV-like mRNA-stabilizing proteins, Proc. Natl. Acad. Sci. U. S. A. 98 (2001)
inhibitory proteins, Nature 433 (2005) 477e480, http://dx.doi.org/10.1038/
11668e11673, http://dx.doi.org/10.1073/pnas.191388398.
nature03205.
[14] A. Pascale, M. Amadio, G. Scapagnini, C. Lanni, M. Racchi, A. Provenzani, et al.,
[3] D.H. Ebert, M.E. Greenberg, Activity-dependent neuronal signalling and
Neuronal ELAV proteins enhance mRNA stability by a PKCalpha-dependent
autism spectrum disorder, Nature 493 (2013) 327e337, http://dx.doi.org/
pathway, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 12065e12070, http://
10.1038/nature11860.
dx.doi.org/10.1073/pnas.0504702102.
[4] J.C. Darnell, E. Klann, The translation of translational control by FMRP: ther-
[15] A. Pascale, P. a Gusev, M. Amadio, T. Dottorini, S. Govoni, D.L. Alkon, et al.,
apeutic targets for FXS, Nat. Neurosci. 16 (2013) 1530e1536, http://dx.doi.org/
Increase of the RNA-binding protein HuD and posttranscriptional up-
10.1038/nn.3379.
regulation of the GAP-43 gene during spatial memory, Proc. Natl. Acad. Sci.
[5] H. Jung, C.G. Gkogkas, N. Sonenberg, C.E. Holt, Remote control of gene function
U. S. A. 101 (2004) 1217e1222, http://dx.doi.org/10.1073/pnas.0307674100.
by local translation, Cell 157 (2014) 26e40, http://dx.doi.org/10.1016/
[16] K. Yamada, Y. Iwayama, E. Hattori, K. Iwamoto, T. Toyota, T. Ohnishi, Genome-
j.cell.2014.03.005.
Wide Assoc. Study Schizophrenia Jpn. Popul. 6 (2011), http://dx.doi.org/
[6] a Szabo, J. Dalmau, G. Manley, M. Rosenfeld, E. Wong, J. Henson, et al., HuD, a
10.1371/journal.pone.0020468.
paraneoplastic encephalomyelitis antigen, contains RNA-binding domains and
[17] H.J. Okano, R.B. Darnell, A hierarchy of Hu RNA binding proteins in developing
is homologous to Elav and Sex-lethal, Cell 67 (1991) 325e333, http://
and adult neurons, J. Neurosci. 17 (1997) 3024e3037.
dx.doi.org/10.1016/0092-8674(91)90184-Z.
[18] B. a. Copits, G.T. Swanson, Dancing partners at the synapse: auxiliary subunits
[7] W. Akamatsu, H. Fujihara, T. Mitsuhashi, M. Yano, S. Shibata, Y. Hayakawa, et
that shape kainate receptor function, Nat. Rev. Neurosci. 13 (2012), http://
al., The RNA-binding protein HuD regulates neuronal cell identity and
dx.doi.org/10.1038/nrn3335.
maturation, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 4625e4630, http://
[19] M.S. Wyeth, K. a Pelkey, R.S. Petralia, M.W. Salter, R.R. Mcinnes, C.J. Mcbain,
dx.doi.org/10.1073/pnas.0407523102.
Neto Auxiliary Protein Interactions Regulate Kainate and NMDA Receptor
[8] L. Bronicki, B. Jasmin, Emerging complexity of the HuD/ELAVl4 gene; impli-
Subunit Localization at Mossy Fiber e CA3 Pyramidal, Cell Syn. 34 (2014)
cations for neuronal development, function, and dysfunction, Rna (2013)
622e628, http://dx.doi.org/10.1523/JNEUROSCI.3098-13.2014.
1019e1037, http://dx.doi.org/10.1261/rna.039164.113.1.
[20] G. Ince-Dunn, H.J. Okano, K.B. Jensen, W.Y. Park, R. Zhong, J. Ule, et al.,
[9] A. Ratti, C. Fallini, L. Cova, R. Fantozzi, C. Calzarossa, E. Zennaro, et al., A role for
Neuronal Elav-like (Hu) Proteins Regulate RNA Splicing and Abundance to
the ELAV RNA-binding proteins in neural stem cells: stabilization of Msi1
Control Glutamate Levels and Neuronal Excitability, Neuron 75 (2012)
mRNA, J. Cell Sci. 119 (2006) 1442e1452, http://dx.doi.org/10.1242/jcs.02852.
1067e1079, http://dx.doi.org/10.1016/j.neuron.2012.07.009.
[10] A. Fukao, Y. Sasano, H. Imataka, K. Inoue, H. Sakamoto, N. Sonenberg, et al.,
[21] S.M. Eacker, M.J. Keuss, E. Berezikov, V.L. Dawson, T.M. Dawson, Neuronal
The ELAV Protein HuD Stimulates Cap-Dependent Translation in a Poly(A)-
activity regulates hippocampal miRNA expression, PLoS One 6 (2011), http://
and eIF4A-Dependent Manner, Mol. Cell. 36 (2009) 1007e1017, http://
dx.doi.org/10.1371/journal.pone.0025068.
dx.doi.org/10.1016/j.molcel.2009.11.013.
[22] H.L. Zhou, C. Geng, G. Luo, H. Lou, The p97-UBXD8 complex destabilizes mRNA
[11] T. Fujiwara, A. Fukao, Y. Sasano, H. Matsuzaki, U. Kikkawa, H. Imataka, et al.,
by promoting release of ubiquitinated HuR from mRNP,, Genes Dev. 27 (2013)
Functional and direct interaction between the RNA binding protein HuD and
1046e1058, http://dx.doi.org/10.1101/gad.215681.113.
active Akt1, Nucleic Acids Res. 40 (2012) 1944e1953, http://dx.doi.org/
10.1093/nar/gkr979.