Neuroscience Letters 550 (2013) 9397

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Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet

Neuroprotective and procognitive effects of sertraline: In vitro and

in vivo studies
Michal Taler a, , Oded Miron a , Irit Gil-Ad a , Abraham Weizman a,b
a
Laboratory of Biological Psychiatry, Felsenstein Medical Research Center and Sackler Faculty of Medicine, Tel Aviv University, Israel
b
Research Unit, Geha Mental Health Center, Israel

h i g h l i g h t s

Sertraline induced the strongest neurotrophic activity in vivo in SHSY5Y cells.

Sertraline has pro-cognitive effect on young and aged mice.
Sertraline showed up regulation of brain BDNF, phospho-ERK and Bcl-2 in mice.
Sertraline may be the preferred drug in treatment of depressed elderly patients.

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

Article history: Selective serotonin reuptake inhibitors (SSRIs) stimulate synaptic plasticity and neurogenesis, most
Received 21 March 2013 likely via the MAP-kinase signal transduction pathway (by phosphorilation of ERK) and by stimulating
Received in revised form 6 June 2013 neurotrophic factors such as brain derived neurotrophic factor (BDNF) and the neuroprotective protein
Accepted 20 June 2013
(Bcl-2). Using human neuroblastoma cells (SHSY5Y), we found that sertraline and its derivative,
desmethylsertraline, at low concentrations (110 M), induced potent neurotrophic activity. Subse-
Keywords:
quently, we have treated for 21 days young and aged mice with sertraline. Sertraline in certain doses
Sertraline
improved signicantly spatial memory learning, in both young and old mice. Sertraline treatment resulted
Cognition
BDNF
in up-regulation of brain BDNF, phospho-ERK and Bcl-2 that may be involved in the pro-cognitive effect of
Selective serotonin reuptake inhibitors sertraline.
Neuroprotection 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction pivotal role in neurogenesis, synaptogenesis and plasticity of the

Depression is one of the most common psychiatric disorders,
affecting 1030% of women and 715% of men [14]. Antidepres-
sants have a dual effect: one being the immediate inhibition of the
monoamine reuptake and/or metabolism, and the second, a slower
process resulting in stimulation of neurotrophic factors release,
neurogenesis and neural plasticity [24,30]. It was reported that
there is a decrease in serum levels of brain derived neurotrophic
factor (BDNF) of depressed patients while antidepressants increase
BDNF levels [30].
The antidepressant-associated stimulation of monoamine sig-
nal transduction pathway, via cAMP, protein kinase A (PKA) and
the transcription factor cAMP response element binding protein
(CREB), lead to the synthesis and release of BDNF [12] that play a
Corresponding author. Tel.: +972 3 9376783; fax: +972 3 9211478.
E-mail address: michalt@post.tau.ac.il (M. Taler).
brain [24].
Another factor that might be regulated by monoamine signaling
is the antiapoptotic, neuroprotective protein, Bcl-2 [10,11]. The
time-course of transcription and translation of these factors cor-
relate with time frame required for response to antidepressants.
These cellular events are also attributed to the mitogen activated
protein kinase (MAPK) extra cellular kinase (ERK) signaling
pathway [28,33]. ERK pathway is activated by neurotrophins like
BDNF and is involved in the differentiation, survival, apoptosis,
structural and functional plasticity of neurons [6,29] as well as in
learning and memory [1]. In vivo study with knock-in mice that
lose the ability to phosphorylate and activate TrKB (BDNF receptor)
showed impaired spatial memory [16].
Antidepressants that activate BDNF may contribute to learning
and memory processes. In this study we evaluated the inuence
of the selective serotonin reuptake inhibitor (SSRI) sertraline treat-
ment on cognitive parameters and relevant protein expression in
young and old mice.

0304-3940/$ see front matter 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.neulet.2013.06.033
94 M. Taler et al. / Neuroscience Letters 550 (2013) 9397

2. Materials and methods
2.1. Cell line
Human neuroblastoma cells (SHSY5Y) (ATCC, Manassas, VA)
sustained in DMEM supplemented with 10% heat-inactivated fetal
calf serum (FCS;stress conditions did not include FCS), 1% penicillin
(10,000 U/ml), streptomycin (10 mg/ml) nystatin (1250 U/ml), and
1% glutamine. Conuent cultures were washed with phosphate-
buffered saline (PBS) and detached with trypsin (0.25%) all
purchased from Biological Industries, Beit Haemk, Israel.
membranes (Whatman, Dassel, Germany) and probed with either
pERK, ERK, BDNF, Bcl-2 (Santa Cruz Biotechnology, Santa Cruz,
CA) or -actin (Millipore, Temecula, CA). Detection was carried
out by horseradish peroxidase-conjugated goat-anti-mouse/rabbit
secondary antibodies (Jackson, Bar Harbor, ME, USA) and enhanced
chemiluminescence with super signal (Pierce, Rockford, IL). Den-
sitometry of electropherograms was carried out by means of a
soft laser-scanning densitometer (SLR-2D/1D; Biomed Instrument,
Ottawa, Canada).
3. Results

2.2. In vitro cell viability 3.1. In vitro study

Neutral red (Sigma, Rehovot, Israel) staining was used for mea- 3.1.1. The effects of antidepressants on the viability of SHSY5Y
surements of cell viability [2]. Quantitative analysis was performed cells
by a colorimetric assay (ELISA reader at 550 nm). Viability was Fig. 1 describes the effect of different antidepressants (at low
tested in cells treated with different agents compared to control concentration range 110 M) on the viability of the human
(untreated cells). Results were expressed as a percent of control. neuroblastoma cells (SHSY5Y) after 24 h of drug exposure. The
antidepressant sertraline and its derivative desmethylsertraline
2.3. In vivo animal studies (both SSRIs) at concentrations ranging between 1 and 10 M,
stimulated the cell survival rate by >50% compared to controls
C57BL/6J black young (46 weeks) and old (1214 weeks) [results are shown as a percentage of controls; F(16,59) = 0.846,
female mice were purchased from Harlen, Jerusalem, Israel. Ani- p = 0.63. Scheffes post hoc test revealed: sertraline vs. control
mals were housed in a temperature-controlled room with food p < 0.05, and desmethylsertraline vs. control p < 0.01]. Paroxetine
and water ad libitum and under 12 hL:12 hD cycles. Animals were (SSRI) also stimulated viability by 40% at 1 M, whereas other
divided into 4 treatment groups (8 animals/group).According to antidepressants tested including the SSRIs uoxetine and citalo-
our experience 3 weeks of treatment with antidepressants are suf-
cient to affect neurotransmission and cognition in rodents [13],
thus mice were treated daily for 3 weeks sertraline at 3 doses:
1, 5, and 10 mg/kg or vehicle (saline). After 3 weeks of treatment,
animals were submitted to Morris water maze (MWM) behavioral
test [25] and were sacriced 24 h after the last MWM test. Imme-
diately after decapitation, brains were removed, and the frontal
cortex (FC) and hippocampus (HC) were dissected and frozen at
70 C for further biochemical analysis.

2.4. Morris water maze (MWM)

Spatial learning and memory was measured using MWM. This

test assesses the ability of the animals to locate an underwater hid-
den platform, using surrounding visual cues. The water maze is a
circular pool 1.2 m in diameter 35 cm in height lled with water
(21 1 C). A circular platform (10 cm diameter) was placed 1 cm
under the water surface.
Mice were given six trials per day, for three consecutive days, up
to 60 s each trial, to nd the platform (acquisition phase). The time
taken to reach the platform (seconds) was recorded. If the mouse
did not nd the platform within 60 s, it was manually placed on it for
10 s. Day after, the platform was removed and mice were subjected
to the pool for four trails (60 s each)-extinction phase. Next phase
is the reacquisition; the platform was placed in an opposite loca-
tion from its location in the acquisition phase. Animals were given
six trails of 60 s for two days. The three phases of the MWM test
(acquisition, extinction and reacquisition) were monitored in both
groups. Data were recorded using an automated tracking system Fig. 1. The in vitro effects of the different antidepressants (paroxetine, sertraline
(Ethovision 3.1 Noldus Information Technology B.V., Wageningen, and desmethylsertraline) and chemotherapy (doxorubicin and cisplatin) agents
The Netherlands). (150 M) on SHSY5Y cells, (A) 24 h and (B) 48 h post drug treatments. Results
are expressed as a percent of controls. Each point represents the mean SEM of 3
determinations. (A) Two-way ANOVA test revealed a signicant variance among the
2.5. Western blot analysis effects of the agents on cell viability F(20,65) = 4.405, p < 0.01. All treatments differed
signicantly from the controls follows Scheffes post hoc test for multiple compar-
Brain tissues were directly resuspended in lysis buffer isons; sertraline vs. control and desmethylsertraline vs. control, p < 0.05. Paroxetine,
(150mMNaCl, 200 mM HEPES, 5 mM EDTA, 50 mM NaF, 1 mM doxorubicin and cisplatin vs. control, p < 0.01. (B) A signicant variance between
treatments and concentration and their effects on cell viability: F(20,65) = 11.077,
Na2 VO4 , 1%NP-40 and 0.5%DOC) for 1 hour. Samples were then
p < 0.01, was noted using the two-way ANOVA test. Scheffes post hoc test for mul-
subjected to SDS-PAGE under reducing conditions using 7.512.5% tiple comparisons showed that all treatments were signicantly different from the
polyacrylamide gels. Proteins were transferred to nitrocellulose controls, p < 0.01.
 M. Taler et al. / Neuroscience Letters 550 (2013) 9397 95

Fig. 3. The in vivo effect of sertraline treatment at different doses (1, 5, and
10 mg/kg/day) on mouse visuo-spatial memory (acquisition and re-acquisition
Fig. 2. The in vitro effects of sertraline (A) and desmethylsertraline (B) 48 h post
phases in the Morris water maze) of young mice. Results are expressed as mean SE.
treatment on SHSY5Y cells exposed to stress (starvation) vs. non-stress conditions.
(A) Signicant difference was observed on day 2 of re-acquisition with sertraline
Cells were exposed to sertraline for 48 h while maintaining in normal or starvation
1 mg/kg treatment vs. Controls (p < 0.01). Sert = sertraline.
media (see Section 2 for more details). Results are expressed as a percent of controls.
Each point represents the mean SEM of 3 determinations. Using a two-sample
Students t-test yielded a signicant higher cell viability under starvation compared
to non-starvation conditions at concentrations of 2.5, 5, 8, 10 M. (B) Cells were
exposed to desmethylsertraline for 48 h while maintaining in normal or starvation
media. Results are expressed as a percent of controls. Each point represents the
mean SEM of 3 determinations. Using a two-sample Students t-test revealed sig-
nicant higher cell viability under starvation compared to non-starvation conditions
(at desmethylsertraline concentrations of 8 and 10 M).

pram, the norepinephrine reuptake inhibitor (NRI) reboxetine,

the serotoninnorepinephrine reuptake inhibitor venlafaxine, the
tricyclic antidepressant clomipramine and the atypical antidepres-
sant mirtazapine did not show a signicant effect.
In the next stage, we showed the protective effect of sertra-
line and desmethylsertraline (110 M) 48 h after drug exposure
of neuroblastoma cell line exposed to stress conditions (FCS depri-
vation) as compared to cells in standard conditions [Fig. 2(AB)].

3.2. In vivo study

3.2.1. The effect of sertraline treatment on spatial memory in

young and aged mice
Young (46 weeks) and aged (1214 months) mice were treated
daily with sertraline (1, 5, and 10 mg/kg) for 3 weeks and during
the behavioral test. Young mice (Fig. 3), treated with sertraline
1 mg/kg/day exhibited better performance in the reacquisition
phase. Noteworthy, in aged mice the score in the reacquisition
phase was the largest when treated with srtraline 10 mg/kg/day
(Fig. 4). No differences in performance were observed in the acqui-
sition and extinction phases (data not shown) in both young and
aged mice.

3.2.2. Effect of sertraline treatment on protein expression in the

hippocampus of young and aged mice
BDNF was up-regulated in the HC of both aged and young mice
treated with 5 and 10 mg/Kg sertraline compared to the untreated
controls (Figs. 5A and 6A).
Fig. 4. The in vivo effect of sertraline treatment at different doses (1, 5, and
10 mg/kg/day) on mouse visuo-spatial memory (acquisition and re-acquisition
phases in the Morris water maze) of aged mice. Results are expressed as mean SEM.
Signicant difference was observed on day 1 of re-acquisition with sertraline
10 mg/kg/day treatment vs. controls (p < 0.01). Sert = sertraline.
96 M. Taler et al. / Neuroscience Letters 550 (2013) 9397
Fig. 5. The effect of sertraline treatment at different doses (1, 5, and 10 mg/kg/day)
on protein expression in hippocampus of young mice. Proteins were resolved in
7.5% SDS-PAGE and then reacted with antibodies. (A) Anti-BDNF (upper panel) and
anti-actin (lower panel) as a reference. Quantication of BDNF/actin ratio levels in
the blots-bar graph. (B) Anti-phspho-ERK (upper panel) and anti-ERK (lower panel).
Quantication of pERK/ERK ratio levels in the blots represent in a bar graph. (C)
Anti-BCL2 (upper panel) and anti-actin (lower panel) as a reference. Quantication
of BCL2/actin ratio levels in the blots represent in a bar graph. *p < 0.05 vs. control
Sert = sertraline.
Posphorylated ERK was also up-regulated in the HC of the
young mice treated with 5 mg/kg sertraline (Fig. 5B). Treatment
didnt affect hippocampal ERK phosphorylation levels in the aged
mice (Fig. 6B). Hippocampal Bcl-2 expression was increased after
treatment with 5 mg/kg sertraline in young mice (Fig. 5C) and
remained unaltered in the aged mice (Fig. 6C).
4. Discussion
Antidepressants in general and SSRIs in particular have pre-
viously been characterized by enabling neural plasticity and
neurogenesis, especially in the hippocampus [20]. Noteworthy, in
our present study the neuroprotective effect of the SSRIs was more
pronounced under stress conditions, as manifested in vitro by the
signicantly higher viability of cells exposed to serum decient
conditions (starvation condition).
Fig. 6. The effect of sertraline treatment at different doses (1,5,and 10 mg/kg/day)
on protein expression in hippocampus of old mice. Proteins were resolved in 7.5%
SDS-PAGE and then reacted with antibodies. (A) Anti-BDNF (upper panel) and anti-
actin (lower panel) as a reference. Quantication of BDNF/actin ratio levels in the
blots-bar graph. *p < 0.05 vs. control. (B) Anti-phspho-ERK (upper panel) and anti-
ERK (lower panel). Quantication of pERK/ERK ratio levels in the blots represent
in a bar graph. (C) Anti-BCL2 (upper panel) and anti-actin (lower panel) as a refer-
ence. Quantication of BCL2/actin ratio levels in the blots represent in a bar graph.
Sert = sertraline.
Our in vitro experiments showed neuroprotective effect of
various antidepressants. The most potent neuroprotective agent
was the SSRI sertraline. When using sertraline in stress conditions
the neuroprotection was even greater. Animal study showed
increase in extracellular dopamine concentrations in addition to
the increase in serotonin levels in the nucleus accumbens and
striatum [15]. These in vitro results encouraged us to examine
the potential benecial effect of sertraline on cognition and mem-
ory task in young and aged mice. Sertraline treatment elevates
both serotonin and dopamine extracellular concentrations in the
nucleus accumbens and striatum [19], such a dual effect may
improve the performance in the MWM test.
Adequate cognitive functioning and memory are maintained by
both brain neurogenesis and neuroprotection capacity [7,32]. From
middle age onward, age-related cognitive decits begin to appear
[19]. The cognitive decline is more prominent in old age [18]. In
rodents age-related decits in spatial navigation tasks, such as the
MWM occur earlier in females than males [10,23]. Therefore, in
this study, we used female mice in order to evaluate the effect of
sertraline on cognition.
As expected, results from the MWM test showed a decrease in
the escape latency to nd the platform in both young and old mice.
The maximal pro-cognitive effect was achieved with 1 mg/kg in the
 M. Taler et al. / Neuroscience Letters 550 (2013) 9397
young mice and 10 mg/kg in the old mice. Noteworthy, sertraline
improved attentional and executive functions in depressed patients
[4].
Park at al. [33] demonstrated that increase in BDNF levels in the
hippocampus is associated with better performance in the MWM
[27]. Previous studies have suggested that BDNF in the brain is
dependent on the MAPK (ERK) signaling pathway, namely, the
downstream targets in this pathway are required for the effects of
BDNF to occur [5,20]. Meta-analysis showed low serum BDNF lev-
els in depressed patients. In addition, BDNF levels were elevated
following a course of antidepressant treatment [31].
Postmortem brain studies of depressed suicide patients exhib-
ited low hippocampal and cerebral cortex pERK levels, contributing
to their volume reduction [8]. Our previous study in stressed rats
showed up-regulatory effect of reboxetine treatment on ERK phos-
phorylation [9]. In addition, the antiapoptotic protein Bcl-2, which
is another target activated by the MAPK signaling pathway, was
also suggested to have a role in neural cells protection [21,22]. In
present study we found that ERK and Bcl-2 were up-regulated in
the sertraline-treated young mice, indicating activation of this sig-
nal transduction pathway in this age group. No such up-regulatory
effect on ERK and Bcl-2 expression was obtained in old mice. In con-
trast, BDNF protein levels were up-regulated after treatment with
sertraline, in both age groups parallel to the successful achieve-
ments in the MWM test.
Carlini et al. [3] found that antidepressant treatment (uoxetine
and venlafaxine) in mice signicantly decrease memory perfor-
mance in the novel object recognition test and down-regulate ERK
signaling. However, Lyons et al. [17] showed that pre-treatment
with uoxetine can attenuate the cognitive decit induced by 5-
Fluorouraci (5-FU). In contrast, study sertraline improved memory
parallel to ERK phosphorylation, suggesting a differential effect on
signaling pathway of sertraline compared to uoxetine and ven-
lafaxine.
5. Conclusions
In conclusion, it seems that sertraline, due to it pro-cognitive
properties, may be the preferred antidepressant in treatment
for depressed/anxious elderly patients, especially those with sec-
ondary mild cognitive decline. However, sertraline is unable to
restore cognitive loss in depressed Alzheimer disease patients [26].
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