A priming role of local estrogen on exogenous estrogen-mediated synaptic plasticity and neuroprotection.

Abstract

The localization of estrogen (E2) has been clearly shown in hippocampus, called local hippocampal E2. It enhanced neuronal synaptic plasticity and protected neuron form cerebral ischemia, similar to those effects of exogenous E2. However, the interactive function of hippocampal and exogenous E2 on synaptic plasticity activation and neuroprotection is still elusive. By using hippocampal H19-7 cells, we demonstrated the local hippocampal E2 that totally suppressed by aromatase inhibitor anastrozole. Anastrozole also suppressed estrogen receptor (ER)β, but not ERα, expression. Specific agonist of ERα (PPT) and ERβ (DPN) restored ERβ expression in anastrozole-treated cells. In combinatorial treatment with anastrozole and phosphoinositide kinase-3 (PI-3K) signaling inhibitor wortmannin, PPT could not improve hippocampal ERβ expression. On the other hand, DPN induced basal ERβ translocalization into nucleus of anastrozole-treated cells. Exogenous E2 increased synaptic plasticity markers expression in H19-7 cells. However, exogenous E2 could not enhance synaptic plasticity in anastrozoletreated group. Exogenous E2 also increased cell viability and B-cell lymphoma 2 (Bcl2) expression in H(2)O(2)-treated cells. In combined treatment of anastrozole and H(2)O(2), exogenous E2 failed to enhance cell viability and Bcl2 expression in hippocampal H19-7 cells. Our results provided the evidence of the priming role of local hippocampal E2 on exogenous E2-enhanced synaptic plasticity and viability of hippocampal neurons.

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EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 44, No. 6, 403-411, June 2012
Copyright ⓒ 2012 by the Korean Society for Biochemistry and Molecular Biology
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
A priming role of local estrogen on exogenous
estrogen-mediated synaptic plasticity and neuroprotection
Siriporn Chamniansawat1,3 and
Sukumal Chongthammakun2
1Faculty of Allied Health Sciences
Burapha University
Chonburi 20131, Thailand
2Department of Anatomy and Center for Neuroscience
Faculty of Science
Mahidol University
Bangkok 10400, Thailand
3Corresponding author: Tel, 66-38-393-497;
Fax, 66-38-393-497; E-mail, siripornc@buu.ac.th
http://dx.doi.org/10.3858/emm.2012.44.6.046
Accepted 26 March 2012
Available Online 18 April 2012
Abbreviations: Arc, activity-regulated cytoskeleton associated
protein; Bcl2, B cell lymphoma 2; DPN, estrogen receptor β agonist;
E2, estrogen; ER, estrogen receptor; GnRH, gonadotrophin
releasing hormone; H2O2, hydrogen peroxide; OVX, ovarectomized;
PI-3K, phosphoinositide kinase-3; PPT, estrogen receptor α agonist;
PSD-95, postsynaptic density protein-95; Wort, wortmannin
Abstract
The localization of estrogen (E2) has been clearly
shown in hippocampus, called local hippocampal E2.
It enhanced neuronal synaptic plasticity and protected
neuron form cerebral ischemia, similar to those effects
of exogenous E2. However, the interactive function of
hippocampal and exogenous E2 on synaptic plasticity
activation and neuroprotection is still elusive. By using
hippocampal H19-7 cells, we demonstrated the local
hippocampal E2 that totally suppressed by aromatase
inhibitor anastrozole. Anastrozole also suppressed
estrogen receptor (ER)β β, but not ERα α, expression.
Specific agonist of ERα α (PPT) and ERβ β (DPN) restored
ERβ β expression in anastrozole-treated cells. In combi-
natorial treatment with anastrozole and phosphoinosi-
tide kinase-3 (PI-3K) signaling inhibitor wortmannin,
PPT could not improve hippocampal ERβ β expression.
On the other hand, DPN induced basal ERβ β trans-
localization into nucleus of anastrozole-treated cells.
Exogenous E2 increased synaptic plasticity markers
expression in H19-7 cells. However, exogenous E2
could not enhance synaptic plasticity in anastrozole-
treated group. Exogenous E2 also increased cell via-
bility and B-cell lymphoma 2 (Bcl2) expression in
H2O2-treated cells. In combined treatment of anas-
trozole and H2O2, exogenous E2 failed to enhance cell
viability and Bcl2 expression in hippocampal H19-7
cells. Our results provided the evidence of the priming
role of local hippocampal E2 on exogenous E2-enhanced
synaptic plasticity and viability of hippocampal
neurons.
Keywords: estrogen receptor beta; estrogens; hippo-
campus; neuronal plasticity; neurons; neuroprotective
agents
Introduction
E2 is mainly synthesized in the gonad, called gonadal
or exogenous E2, and reaches its target organ via
blood circulation. It has been reported to influence
memory function (Henderson, 2010; Gorenstein et
al., 2011) and neuroprotection (McCullough et al.,
2003) through estrogen receptor (ER), which highly
expressed in cerebral cortex and hippocampus
(Henderson, 2010). In menopausal women, E2
replacement therapy relieved memory impairment
(Gorenstein et al., 2011). Previous study demonstrated
the decreasing of hippocampal dendritic spine in
ovarectomized (OVX) rats that restored by an
administration of exogenous E2 (Gould et al., 1990).
During estrous cycle, the hippocampal spine density
is varied in response to fluctuating level of E2 in
female rats (Woolley and McEwen, 1992). Moreover,
our previous studies demonstrated that a variety of
synaptic marker proteins is up-regulated after
exogenous E2 application (Chamniansawat and
Chongthammakun, 2009; 2010), confirming the
positive role of exogenous E2 on synaptic plasticity
and memory consolidation. E2 also prevents
neuronal death from ischemic brain injury (Dubal et
al., 1999). It regulated neuronal viability through
Bcl2 expression, but not other members of Bcl2
family (Dubal et al., 1999).

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404 Exp. Mol. Med. Vol. 44(6), 403-411, 2012
Figure 1. Secreted local E2 in cul-
ture media of hippocampal H19-7
neurons. Secreted E2 of control
(open bars), GnRH-treated (gray
bars) and anastrozole-treated (black
bars) in H19-7 hippocampal cells
(A). *P ＜ 0.05, **P ＜ 0.01, ***P
＜ 0.001 vs day-matched control
group, †P ＜ 0.05, ††P ＜ 0.01, †††P
＜ 0.001 vs corresponding day 0
group. Percent maximum secreted
E2 of control (B), GnRH-treated (C)
and anastrozole-treated (D) in
H19-7 hippocampal cells. Dark line
represented the best-fitted sigmoid
line (non-liner regression) in B and
straight line (linear regression) in C
and D (n = 5).
In addition to gonad, endogenous E2 production
and secretion in hippocampus were well
demonstrated (Prange-Kiel and Rune, 2006). The
presence of all enzymes responsible for endogenous
E2 biosynthesis and a sixfold higher E2
concentration in hippocampus than that in plasma
(Hojo et al., 2004) strongly indicate the potential
roles of local hippocampal E2 on hippocampal
functions. Previous studies revealed that inhibition
of the local E2 biosynthesis by aromatase inhibitor
significantly reduced the density of hippocampal
synapses and down-regulated synaptic proteins,
including spinopholin and synaptophysin (Kretz et
al., 2004; Mukai et al., 2010; Zhou et al., 2010).
Local hippocampal E2 also regulated the
expression of ER (Murata et al., 2003; Prange-Kiel
et al., 2003; Oliveira et al., 2004). In addition to
synaptic plasticity, the neuroprotective role of local
hippocampal E2 had been demonstrated by using
aromatase knockout mice (McCullough et al., 2003).
Loss of endogenous or local E2 exhibited an
increase in the severity of ischemic injury compared
with the normal as well as OVX mice. These
evidences indicated that the neuronal functions are
mainly affected by local hippocampal E2. However,
the decreased in synaptic plasticity was presented
in OVX rats (Woolley and McEwen, 1992), which
local hippocampal E2 should be intact. Therefore,
hippocampal neuronal function is depended on both
endogenous and exogenous E2. We hypothesized in
the present study that the mechanism of exogenous
E2 action is an endogenous E2-dependent manner.
The aims of this study were to investigate the effects
and mechanisms of endogenous E2 action on the
modulation of synaptic plasticity and neuroprotection
in hippocampal H19-7 cells.
Results
Local E2 production in hippocampal H19-7 cells
To determine the production and secretion of E2 in
hippocampal H19-7 cells, we measured E2
concentration in culture media of untreated,
gonadotrophin releasing hormone (GnRH)-treated
or anastrozole-treated hippocampal H19-7 cells by
using ELISA kit. At day 0, basal E2 levels of
untreated, GnRH-treated, and anastrozole-treated
cells were 5.46 ± 0.51, 5.11 ± 0.25, and 4.89 ±
0.25 pg/ml, respectively (Figure 1A). In untreated
hippocampal H19-7 cells, secreted E2 significantly
increased from days 5 through 12 (37.52 ± 4.58
pg/ml to 80.04 ± 7.40 pg/ml) (Figure 1A). Maximum
secreted E2 level in untreated hippocampal H19-7
cells was comparable to those report in hippocampal
slice cultures (Kretz et al., 2004). GnRH increased
hippocampal E2 secretion from days 3 through 12
(26.81 ± 4.55 to 148.72 ± 12.47 pg/ml) (Figure
1A). Moreover, when compared with the untreated
group, GnRH significantly increased E2 levels from
days 2 through 12 (Figure 1A). However, when the
activity of aromatase was inhibited by anastrozole,
the E2 levels were not changed throughout 12 days
of experimental period.
The hippocampal E2 secretion profile of control,
GnRH-treated, and anastrozole-treated hippocampal
H19-7 cells were demonstrated by the percent

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Estrogen-mediated neuroprotection and synaptic plasticity 405
Figure 2. Endogenous H19-7 hip-
pocacampal E2 regulates ERβ
expression. Representative
munofluorescent images of ERα
and ERβ expression of control, and
anastrozole-treated H19-7 hippo-
campal cells (A). Red signal repre-
sented nuclear-staining and green
signal represented ERα or ERβ
(scale bars = 10 μm). The number
of ERα or ERβ positive green signal
was counted and presented (B).
Quantitative immunoblot analysis of
ERα and ERβ proteins expression
in control and anastorzole-treated
H19-7 hippocampal cells (C, D).
Representative qRT-PCR of ERα
and ERβ mRNA expression in
H19-7 hippocampal neurons (E).
β-actin was the housekeeping
protein. *P ＜0.05, ***P ＜0.001
vs control group,
anastrozole-treated group (n = 5).
im-
†P ＜ 0.05 vs
maximum secreted E2-experimental period relation-
ship. The data of control cells were best fitted with
the non-liner regression equation that revealed the
sigmoidal line (r2= 0.90, Figure 1B). In the first three
days, E2 levels slowly increased, while it rapidly
increased from 4th day and to the maximum level in
8th day (Figure 1B). The half maximum E2 secretion
was presented in 5th day to 6th day and the
maximum E2 secretion with the plateau phase was
showed in day 8 though 12 (Figure 1B). Therefore,
we selected 8th day as a suitable culture condition in
next experiments for studied the effect of local E2 on
hippocampal H19-7 neuron. GnRH changed the
secretory profile form sigmoid function to linear
function (r2= 0.97, Figure 1C). The E2 levels of
GnRH-treated group rapidly increased from day 1
through 12 (Figure 1C), whereas E2 levels of
anastrozole-treated group showed relatively constant
throughout 12 days of experimental period (Figure
1D).
Local E2 regulates ERβ β expression in hippocampal
H19-7 cells
It is well known that ER, including ERα and ERβ,
expression is upregulated by E2 (Murata et al., 2003;
Oliveira et al., 2004). We therefore investigated the
effects of the local E2 on ER expression by using the
confocal immunocytochemistry, qRT-PCR, and
Western blot techniques. To determine those effects,
hippocampal H19-7 cells were cultured in either
steroid-deprived media with or without anastrozole
for 8 days. In control group, ERβ expression was
significantly higher than ERα (Figures 2A-2D). In the
presence of anastrozole, the level of ERβ
expression was significantly decreased, whereas
ERα expression was not changed (Figures 2A-2E).
These results indicated that the local E2 up-regulated
ERβ, but not ERα, expression in hippocampal
H19-7 cells.
Local E2-increased ERβ β expression is a basal ERα α-
and ERβ β-dependent mechanism
We further examined the underlying mechanism of
local E2-induced ERβ expression by using qRT-PCR
and Western blotting analysis. Hippocampal H19-7
cells exposed to anastrozole for 8 days had
significantly lower ERβ expression than control
group (Figures 3A-3C). The level of ERβ expression
could be restored by administration of specific
agonist of ERα (100 nM PTT) and ERβ (100 nM
DPN) in day 7th of experimental period. This result
indicates that the local E2-induced ERβ expression
is a basal ERα and ERβ-dependent mechanisms.
Wortmannin (200 nM Wort.), a specific PI-3K
inhibitor, had no additive effect on anastrozole-
suppressed ERβ expression (Figures 3A-3C).
Wortmannin attenuated PPT-, but not DPN-, induced
ERβ expression. Therefore, local hippocampal E2

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406 Exp. Mol. Med. Vol. 44(6), 403-411, 2012
Figure 3. The mechanism of local hippocampal E2 regulated ERβ
expression. The quantitative immunoblot analysis of ERβ in control
and 8 days-anastrozole exposed neuron (A). 100 nM PPT, 100 nM DPN,
or 200 nM Wortmannin (Wort.) was added to the culture media of anas-
trozole-treated neuron in 7-8 days prior to harvested cells.
Representative densitometric analysis of ERβ expression in H19-7 cells
(B). Representative immunofluorescent image of ERβ (green signal) and
nucleus (red signal) of anastrozole and anastrozole plus DPN-treated
cells (C). Scale bar = 10 uM. β-actin was the housekeeping protein. ***P
＜ 0.001 vs control group.
Figure 4. The effect of endogenous E2 on exogenous E2-induced H19-7
hippocampal synaptic plasticity. Representative quantitative qRT-PCR (A,
B) and Western blotting analysis (C, D, E) of Arc and PSD-95 expression
in H19-7 hippocampal neurons. Densitometric analysis of Arc (D) and
PSD-95 (E) protein in H19-7 cells. GAPDH was the housekeeping mRNA
in real-time PCR. β-actin was the housekeeping protein. *P ＜ 0.05, **P
＜ 0.01, ***P ＜ 0.001 vs control group (n = 5).
enhanced ERβ expression in basal ERα-PI-3K
dependent mechanism.
We further examined the effect of local E2 on ERβ
activation by determining the translocation of basal
ERβ in hippocampal H19-7 cells by using laser
confocal microscopy (Figure 4D). In anastrozole
treated group, the basal ERβ localized in cytoplasmic
and membranous regions of hippocampal H19-7
cells. After DPN treatment, basal membranous and
cytoplasmic ERβ decreased, but the nuclear ERβ
increased. This findings suggest that the membranous
and cytoplasmic ERβ translocalize into nucleus in
ligand-dependent mechanism, indicating a charac-
teristic of classical genomic action.
Local E2 mediates exogenous E2-induced synaptic
plasticity
Although local hippocampal E2- and exogenous
E2-enhanced hippocampal synaptic plasticity had
been explained (Prange-Kiel and Rune, 2006), the
role of the local E2 on exogenous E2-mediated
synaptic plasticity is still unclear. By using synaptic
plasticity markers, Arc and PSD-95, we observed
the interactive effect of local and exogenous E2 on
synaptic plasticity using qRT-PCR and Western blot
analysis in hippocampal H19-7 cells. As shown in
Figures 3A and 3B, exogenous E2 treatment for 30
min and 6 h significantly increased Arc and PSD-95
mRNA expression, respectively. Anastrozole unaltered
the basal Arc and PSD-95 mRNA expression.
Interestingly, exogenous E2 could not enhance Arc
and PSD-95 mRNA expression when hippocampal
H19-7 cells were cultured in anastrozole-containing
media (Figures 3A and 3B).
Similar to those mRNA, administration of exogenous

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Estrogen-mediated neuroprotection and synaptic plasticity 407
Figure 5. The effect of endogenous E2 on neuroprotective effect of
exogenous E2. Representative cell viability data from MTT assay (A).
Anti apoptotic Bcl2 expression in H19-7 hippocampal neurons (B and C).
β-actin was the housekeeping protein. *P ＜ 0.05, ***P ＜ 0.001 vs con-
trol group (n = 5).
E2 (1 h and 48 h for Arc and PSD-95, respectively)
also induced Arc and PSD-95 proteins expression
(Figures 3C-3E). Anastrozole had no effect on basal
Arc and PSD-95 proteins expression. Exogenous
E2 failed to enhance Arc and PSD-95 protein
expression in anastrozole-treated neurons (Figures
3D and 3E). These findings indicated that
hippocampal E2 was prerequisited for exogenous
E2-enhanced hippocampal synaptic plasticity.
Local E2 mediates neuroprotective effect of
exogenous E2
The neuroprotective effects of E2 have been well
demonstrated. However, the involvement of local E2
on neuroprotective effect of exogenous E2 is still
elusive. As demonstrated in Figure 5A, 1 h of 200 μM
H2O2 exposure significantly decreased hippocampal
H19-7 cell viability. H2O2-induced cell death was
enhanced by anastrozole. Preincubation of the cells
with exogenous E2 for 24 h prior to addition of H2O2
attenuated H2O2-induced cell death. However,
therapeutic effect of exogenous E2 on H2O2-induced
H19-7 cell death was absent when cells were
exposed to anastrozole (Figure 5A).
We also observed the effect of local E2 and
exogenous E2 on anti-apoptotic protein Bcl2, the
specific target of E2 (Dubal et al., 1999). While 24 h
of exogenous E2 preincubation significantly enhanced
Bcl2 expression, H2O2 significantly suppressed its
expression (Figures 5B and 5C). Additional effect of
anastrozole on H2O2-supressed Bcl2 expression
was demonstrated. Similar to those cell viability
studies, exogenous E2 totally rescued Bcl2 expression
in H2O2-treated H19-7 cells. Exogenous E2 could
not restore H2O2-supressed Bcl2 expression in
anastrozole treated neurons (Figures 5B and 5C).
These findings suggest the prerequisite role of local
hippocampal E2 on neuroprotective function of
exogenous E2.
Discussion
De novo E2 production and secretion in hippocampus
has been demonstrated in both primary hippo-
campal neurons (Prange-Kiel et al., 2003) and
hippocampal slice cultures (Kretz et al., 2004). In
the present study, we showed the production and
secretion of local E2 in hippocampal H19-7 cells.
Our findings demonstrate the sigmoidal secretory
profile of H19-7 hippocampal E2. In first 3 days after
seeding the secreted E2 levels relatively constant
probably due to the preparation of intracellular
machinery that responsible for production and
secretion of E2, we named this period as “the
preparation state”. In days 4 to 7, secreted E2
rapidly increased form 16.2% to 90.6% of maximum
secreted E2; it was named “the active state”. Finally,
secreted E2 reached the maximum plateau state in
days 8 to 12. However, secreted E2 levels were
relatively unaltered when hippocampal H19-7 cells
were incubated with anastrozole. This finding
indicates that E2 production and secretion in
hippocampal H19-7 cells required aromatase
activity similar to those reported in primary
hippocampal neurons (Prange-Kiel et al., 2003) and
hippocampal slice cultures (Kretz et al., 2004). In
addition, the maximum secreted E2 levels in
hippocampal H19-7 cells were comparable to that
report in hippocampal slice cultures (Kretz et al.,
2004). Therefore, H19-7 hippocampal cells were a
suitable model for studying the effect and
mechanism of hippocampal E2 on hippocampal
neuronal functions.
It has clearly been demonstrated that GnRH
receptor expressed in hippocampus (Chu et al.,
2008; Schang et al., 2011), thus, hippocampal
neuron can selectively respond to GnRH treatment.
In the present study, we reported the activating
effect of GnRH on local hippocampal E2 level,
indicating that GnRH enhanced endogenous E2
biosynthesis in hippocampal H19-7 cells. Similar to
previous study, that demonstrated the activating