Behavioural Brain Research 262 (2014) 35–41

Contents lists available at ScienceDirect

Behavioural Brain Research

journal homepage: www.elsevier.com/locate/bbr

Research report

Disturbance of endogenous hydrogen sulfide generation and

endoplasmic reticulum stress in hippocampus are involved in
homocysteine-induced defect in learning and memory of rats
Man-Hong Li a,b,1 , Ji-Ping Tang a,b,1 , Ping Zhang a , Xiang Li c , Chun-Yan Wang d ,
Hai-Jun Wei b , Xue-Feng Yang a , Wei Zou a,∗∗ , Xiao-Qing Tang a,b,∗
a
Department of Neurology, Nanhua Affiliated Hospital, University of South China, Hengyang, 421001 Hunan, PR China
b
Institute of Neuroscience, Medical College, University of South China, Hengyang, 421001 Hunan, PR China
c
Department of Anesthesiology, the First Affiliated Hospital, University of South China, Hengyang, 421001 Hunan, PR China
d
Department of Pathophysiology, Medical College, University of South China, Hengyang, 421001 Hunan, PR China

h i g h l i g h t s

• Exposure of rats to homocysteine (Hcy) leads to learning and memory dysfunctions.

• Hcy decreases the generation of endogenous H2 S in the hippocampus of rats.
• Hcy up-regulates the endoplasmic reticulum (ER) stress in the hippocampus of rats.
• Disturbed H2 S formation and ER stress involve in Hcy-caused memory disorder.

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

Article history: Homocysteine (Hcy) is a risk factor for Alzheimer’s disease (AD). Hydrogen sulfide (H2 S) acts as an endoge-
Received 11 August 2013 nous neuromodulator and neuroprotectant. It has been shown that endoplasmic reticulum (ER) stress is
Received in revised form 2 January 2014 involved in the pathological mechanisms of the learning and memory dysfunctions and that H2 S exerts its
Accepted 5 January 2014
neuroprotective role via suppressing ER stress. In the present work, we explored the effects of intracere-
Available online 11 January 2014
broventricular injection of Hcy on the formation of learning and memory, the generation of endogenous
H2 S, and the expression of ER stress in the hippocampus of rats. We found that intracerebroventricular
Keywords:
injection of Hcy in rats leads to learning and memory dysfunctions in the Morris water maze and novel of
Homocysteine
Hydrogen sulfide object recognition test and decreases in the expression of cystathionine-␤-synthase, the major enzyme
Endoplasmic reticulum stress responsible for endogenous H2 S generation, and the generation of endogenous H2 S in the hippocampus
Learning and memory of rats. We also showed that exposure of Hcy could up-regulate the expressions of glucose-regulated
Morris water maze protein 78 (GRP78), CHOP, and cleaved caspase-12, which are the major mark proteins of ER stress, in the
Novel object recognition test hippocampus of rats. Taken together, these results suggest that the disturbance of hippocampal endoge-
nous H2 S generation and the increase in ER stress in the hippocampus are related to Hcy-induced defect
in learning and memory.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Homocysteine (Hcy) is a thiol-containing amino acid formed by

∗ Corresponding author at: Institute of Neuroscience, Medical College, University
of South China, 28 W Changsheng Road, Hengyang, Hunan, PR China.
Tel.: +86 734 8281389; fax: +86 734 8281673.
∗∗ Corresponding author at: Department of Neurology, Nanhua Affiliated Hospital,
University of South China, 336 E Dongfeng Road, Hengyang, 421001 Hunan, PR China.
Tel.: +86 734 8358096; fax: +86 734 8358399.
E-mail addresses: zouw415@163.com (W. Zou), tangxq01001@foxmail.com,
txq01001@163.com (X.-Q. Tang).
1
These authors contributed equally to this work.
demethylation of methionine [1,2]. Elevated plasma homocysteine
levels were found to be associated with increased risk of neuronal
cells [2–9]. More than 40% Alzheimer’s disease (AD) patients were
found to have high Hcy levels in the plasma and the patients with
high plasma Hcy levels displayed more rapid neural atrophy than
those with normal levels of Hcy by epidemiological studies, which
has accumulated that elevated plasma Hcy is a strong, independent
risk factor of AD [10–14]. It has reported that treatment of rats
with Hcy for 2 weeks could induce the deficit in spatial memory
[15]. Therefore, preventing the neurotoxicity of Hcy may be a novel

0166-4328/$ – see front matter © 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bbr.2014.01.001
36 M.-H. Li et al. / Behavioural Brain Research 262 (2014) 35–41
therapeutic strategy for AD. However, the underlying mechanisms
that Hcy-induced AD-like lesions were not investigated clearly.
Hydrogen sulfide (H2 S) is the third endogenous signaling gaso-
transmitter, besides nitric oxide and carbon monoxide [16,17].
H2 S plays an important role in brain functions, probably acting
as a neuromodulator as well as a neuroprotectant [18–20]. In the
mammalian brain, H2 S is formed from the amino acid cysteine
by the action of cystathionine beta-synthase (CBS) [16,17,21], the
key enzyme in the transsulfuration pathway that processes Hcy.
It has been observed that H2 S was decreased, but that Hcy was
increased in AD brains [22]. Our previous studies have shown that
Hcy-associated neurotoxicity is due to reduced endogenous gen-
eration of H2 S [23] and that H2 S directly antagonizes the toxicity
of Hcy to neuronal cells [24]. Therefore, we want to know whether
Hcy-impaired learning and memory involves in the disturbance of
H2 S generation.
The endoplasmic reticulum (ER) lumen is a unique oxidative
environment, critical for protein folding and formation of disul-
fide bonds [25]. Excessive and prolonged ER stress can trigger cell
death [26]. Glucose-regulated protein 78 (GRP78), C/EBP homolo-
gous protein (CHOP), and cleaved caspase 12 are molecular markers
of ER stress. ER stress is considered to be an important mecha-
nism involved in neurodegenerative diseases, including AD [27].
Recently, ER stress was proposed to explain the pathogenic effects
of Hcy [28–31], which may be a common pathway of injury of tis-
sues and cells induced by Hcy. Interestingly, Wei et al. has reported
that H2 S could antagonize cardiomyocytic ER stress in Hcy-induced
cardiomyocytic injury [31]. Given the importance of Hcy in the
pathogenesis of AD, altered endogenous production of H2 S in Hcy
exposure, and the antagonistic role of H2 S in Hcy-induced ER stress,
it is conceivable that Hcy-impaired learning and memory involves
the disturbance of H2 S generation, which in turn causes ER stress
in the hippocampus of rats.
In this study, we demonstrated that intracerebroventricular
exposure of brain to Hcy impairs the learning and memory in
Morris water maze and novel object recognition test, inhibits the
generation of hippocampal H2 S, and up-regulates the expressions
of GRP78, CHOP, and cleaved caspase12 in the hippocampus. Our
study implied that the disturbance of H2 S generation is involved
Hcy-induced deficit in learning and memory and that the ER stress
in the hippocampus may be a potential contributing mechanism.
2. Materials and methods
2.1. Reagents
Hcy [dissolved in phosphate-buffered saline (PBS)] was pur-
chased from Sigma Chemical Co (St. Louis, MO, USA). Specific
monoclonal anti-CBS was purchased from Sant Cruz Biotechnology,
Inc (Sant Cruz, CA, USA). Specific monoclonal anti-GRP78 and anti-
CHOP antibodies were purchased from Epitomic Inc (Burlingame,
UK). Specific monoclonal anti-Caspase-12 antibody was obtained
from Sigma Chemical (St. Louis, MO, USA).
2.2. Animals
The male Sprague-Dawley rats (250–280 g), supplied by the SJA
Lab Animal Center of Changsha (Changsha, China), were individ-
ually housed with free access to food and water under a 12:12 h
reversed light–dark cycle. After being housed, the rats were han-
dled for 1 week to habituate them to the experimenter. All animal
experiments were performed according to the National Institutes
of Health Guide for the Care and Use of Laboratory Animals and
were approved by the Animal Use and Protection Committee of
University of South China.
2.3. Intracerebroventricular injection
PBS or Hcy (0.2, 0.6, or 2 ␮mol) in 2.5 ␮l was injected into the
bilateral ventricle at the following coordinates: anterior/posterior
−1.4 mm; medial/lateral 1.8 mm; dorsal/ventral −3.0 mm, from
Bregma, with an injection rate of 0.5 ␮l per min under the control of
micropump, respectively [32]. In order to make sure that the entire
injection had been delivered, the injection cannula was allowed to
remain in place for an additional minute before being removed.
2.4. Spatial learning and memory tests
The water maze was a circular pool (diameter 180 cm, height
60 cm). The water temperature (23 ± 2 ◦ C), light intensity, external
cues in the room, and water opacity were rigorously reproduced.
A transparent Plexiglas non-slippery platform (diameter 12.5 cm)
was immersed under the water surface (2 cm) during acquisition
trials. Swimming was recorded using a camera capture, and ana-
lyzed using video track software. The software divided the pool
into four quadrants. Several landmarks were fixed to the walls of
the water maze room as the distal spatial extra-maze cues for the
rats to find the platform.
2.4.1. Acquisition trail
The place navigation training consisted of four swims per day
for 7 days, with about a 20-min intertrial time. Four start positions
were pseudo-randomly selected across the four trials each day, and
each animal was allowed a 120-s swim to find the platform. Once
the rat reached the platform, it was left 20 s on the platform; if an
animal did not reach the platform within 120 s, it was guided to the
platform to remain for 20 s. The path and the escape latencies were
recorded by an MT-200 Morris image motion system (Chengdu
Technology and Market Corp, Chengdu, China).
2.4.2. Probe trail
The probe test was performed 24 h after the last swim on day 7.
The platform was removed and each animal was allowed a free
120-s swim. The start position for each mouse corresponded to
one of two positions remote from the platform location in coun-
terbalanced order. The platform quadrant was termed the target
quadrant. The percentage of time spent in the target quadrant and
the number of times that the same animal crossed the former plat-
form area were determined.
2.4.3. Visible platform test
After the probe test, visual, motor, and motivation skills were
also tested with a visible platform to rule out the possible deficits
in sensorimotor processes. The platform was raised 2 cm above the
water surface. The platform was moved to a novel quadrant in the
pool at a fixed location for the four consisted trials, and the latency
to reach the platform and the average speed were recorded.
2.5. Novel Objects Recognition (NOR) test
The NOR test consisted of two sessions: a training session fol-
lowed by a retention trial 24 h later. Two days prior to training, rats
were habituated to the arena (50.0 × 50.0 × 60) for 5 min once a day
in the absence of objects. During the training session, two different
objects (A and B) were placed in the testing arena. Each animal was
allowed to explore the objects for 5 min. The rat was considered
to be exploring the object when the head of the animal was facing
the object, or the animal was touching or sniffing the object. The
total time spent exploring each object was recorded by a trained
observer blind to treatments, and expressed as percentage of total
exploration time. In the retention session, one identical and one
novel object (A and C) were used. A rat was allowed to explore
 M.-H. Li et al. / Behavioural Brain Research 262 (2014) 35–41
the objects for 5 min, and the time spent exploring each object
was recorded. The discrimination index (= (novel object − familiar
object)/(novel object + familiar object)) was used to measure the
cognitive function of rats [32,33].
2.6. Western blot analysis
The hippocampus tissues were homogenized in ice-cold homog-
enizing buffer (50 mM Tris–Cl, pH 7.4, 150 mM NaCl, 5 mM EDTA,
0.1% sodium dodecyl sulfate, 1% NP-40, 1% deoxycholate, 1% Triton
X-100, 10 mM PMSF, 50 mM sodium vanadate, and 0.1% protease
inhibitors cocktail) (Roche, Switzerland). After centrifugation at
14,000 × g for 30 min at 4 ◦ C, the supernatant was collected and
the protein content was subsequently assayed by using a BCATM
Protein Assay Kit (Beyotime, Shanghai, China). Equal quantities of
total protein (25 ␮g/lane) were separated by SDS-PAGE, followed
by transfer to a PVDF. The membranes were then blocked with 5%
bovine serum albumin in TBS-T buffer (50 mM Tris–HCl, pH 7.5,
150 mM NaCl, 0.05% Tween-20, pH 7.6) for at least 2 h at room
temperature. The primary antibody was diluted in TBS-T buffer
containing 5% bovine serum albumin. Membranes were incubated
with the primary antibody (anti-CBS, 1:2000; anti-CHOP, 1:500;
anti-GRP78, 1:2000; and anti-cleaved caspase12, 1:2000) at room
temperature for 2 h or at 4 ◦ C overnight. The membranes were
washed three times for 20 min each with TBS-T, and incubated
with secondary antibody for 2 h. The membranes were then washed
three times with TBS-T for 20 min and once with TBS for 5 min and
the electrogenerated chemiluminescence reaction solutions were
added (solution 1: 0.1 M Tris–HCl, luminol, p-coumaric acid; solu-
tion 2: 0.1 M Tris–HCl, hydrogen peroxide) for 2 min. The signal of
the immunoblots was visualized using an image analysis system
equipped with a software BIO-ID (Vilber Lourmat, France) and the
integrated optical density for the protein band was calculated by
Image-J software.
2.7. Assay of H2 S generation
Hippocampus was homogenized in 50 mM ice-cold potassium
phosphate buffer (pH 6.8). The reaction mixture contained 100 mM
potassium phosphate buffer (pH 7.4), l-cysteine (20 ␮l, 10 mM),
pyridoxyal 5 -phosphate (20 ␮l, 2 mM), saline (30 ␮l), and 11%
(w/v) tissue homogenate (430 ␮l). The reaction was performed in
tightly stoppered cryovial test tubes and initiated by transferring
the tubes from ice to a shaking water bath at 37 ◦ C. After incuba-
tion for 30 min, 1% (w/v) zinc acetate (250 ␮l) was added to trap
evolved H2 S followed by 10% (v/v) trichloroacetic acid (250 ␮l) to
denature the protein and stop the reaction. Subsequently, NNDPD
(20 ␮M; 133 ␮l) in 7.2 M HCl was added, immediately followed by
Fig. 1. Effect of Hcy on the performance of spatial memory acquisition phase in the Morris water maze. After 7-day intracerebroventricular administration of homocysteine
(Hcy, 0.2, 0.6, 2.0 ␮mol), the rats were tested in the Morris water maze task. (A) Acquisition profiles: rats were submitted to acquisition of an invisible platform placed in
a fixed location (target quadrant) with four swims per day during 7 days. (B) Representative swimming tracks of rats searching for the underwater platform at 1st and 7th
training day. Values are the mean ± S.E.M. (n = 8–12). *P < 0.05; **P < 0.01, vs nontreated control group.
37
FeCl3 (30 ␮M; 133 ␮l) in 1.2 M HCl. The absorbance of the resulting
solution at 670 nm was measured by spectrophotometry. The H2 S
concentration was calculated against a calibration curve of NaHS
and H2 S synthesizing activity is expressed as ␮mol H2 S formed
from g protein (determined using a BCATM Protein Assay Kit) per
minute (nmol/min/mg protein).
2.8. Statistical analysis
Data are expressed as mean ± S.E.M. The significance of inter-
group differences was evaluated by one-way analyses of variance
(Dunnett’s test). Differences were considered significant at two
tailed <0.05.
3. Results
3.1. Hcy impairs spatial learning and memory in rats
The Morris water maze was used to analyze the effect of Hcy
on spatial learning and memory. In the acquisition trail, after intra-
cerebroventricular administration of Hcy (0.2, 0.6, 2.0 ␮mol) for 7
days, rats had to learn the location of an invisible platform set at
a fixed position, by performing 4 swimming/day for seven consec-
utive days. The latency traveled to find the platform was shown
in Fig. 1A. All of four groups over the seven training days exhib-
ited significant, substantial reductions in their times to find the
platform. However, compared to the control group, Hcy (0.2, 0.6,
2.0 ␮mol) groups took significantly longer to find the platform in a
concentration-dependent manner, implying a significant impair-
ment of spatial learning process, and this impairment occurred
from training day 4 onward (p < 0.05). Fig. 1B shows the representa-
tive swimming tracks of rats searching for the underwater platform
at 1st and 7th training days. The distance swam in searching for the
hidden platform became longer in the rats treated with Hcy (0.2,
0.6, 2.0 ␮mol).
In the probe trial, the platform was removed and the rats were
allowed to swim freely for 120 s. The crossing platform times in Hcy
(0.6, 2.0 ␮mol)-treated rats was significantly less than that of the
control group (Fig. 2A). The rats treated with Hcy (0.6, 2.0 ␮mol)
spent lower percentage of time in the target quadrant than that of
the control group (Fig. 2B). However, Hcy (0.2 ␮mol) group has no
difference from with the control group (Fig. 2A and B).
The aforementioned results demonstrated that spatial learning
and memory in rats is impaired by treatment of Hcy. To exclude
the possibility that the results are due to the impairments of vision
and motor ability in the rats, we examined the escape latency and
the average swimming speeds of rats by performing a visible plat-
form test after finishing the probe trials. There was no statistical
38 M.-H. Li et al. / Behavioural Brain Research 262 (2014) 35–41

Fig. 2. Effect of Hcy on the performance of spatial memory probe phase in the Morris water maze. One day after finishing the place navigation task (day 8), the platform was
removed and the rats intracerebroventricularly exposed to homocysteine (Hcy, 0.2, 0.6, 2.0 ␮mol) were submitted to the probe trial. The number of times that the animal
crossed the platform area (A) and the percentage of time spent in the target quadrant (B) were analyzed. Values are the mean ± S.E.M. (n = 8–12). **P < 0.01; ***P < 0.001, vs
nontreated control group.

Fig. 3. Effect of Hcy on the motor function and vision of rats. After the probe test, the rats intracerebroventricularly exposed to homocysteine (Hcy, 0.2, 0.6, and 2.0 ␮mol)
were submitted to the visible platform test. The latency to reach the platform (A) and the average swimming speeds (B) were recorded.

difference in the escape latencies (Fig. 3A) and there was no sig-
nificant difference in swimming speeds (Fig. 3B) among all groups
in the visible platform test, demonstrating that the alteration of all
parameters in the hidden platform tests and probe trials does not
result from changes in visual or motor abilities of rats.
3.2. Hcy induces learning and memory impairment in the novel
object recognition test
We performed an additional experiment to extend our obser-
vation to a nonspatial form of cognition. As shown in Fig. 4,
compared to control groups, the discrimination index was signif-
icantly decreased in the rats treated with Hcy (0.2, 0.6, 2.0 ␮mol)
(Fig. 4A), however, Hcy did not change the total object exploration
(Fig. 4B), also implying that Hcy could impair learning and memory
process.
3.3. Hcy causes ER stress in the hippocampus of rats
To explore whether ER stress is involved in Hcy-induced impair-
ment in learning and memory, the expression levels of CHOP,
GRP78, and cleaved caspase-12 in the hippocampus of rats treated
with Hcy were measured by Western blot analysis. As illustrated
in Fig. 5, after 7-day exposure of Hcy (0.2, 0.6, 2 ␮mol), the
amount of CHOP (Fig. 5A), GRP78 (Fig. 5B), and cleaved caspase-
12 (Fig. 5C) in the hippocampus were significantly increased. These
data indicated that ER stress participates in Hcy-induced cognitive
deficit.

Fig. 4. Effect of Hcy on learning and memory in the novel object recognition test. After 7-day intracerebroventricular administration of homocysteine (Hcy, 0.2, 0.6, 2.0 ␮mol),
rats were tested in the novel object recognition test. The discrimination index (A) and total object exploration (B) are recorded. Values are the mean ± S.E.M. (n = 8–12).
**P < 0.01; ***P < 0.001, vs nontreated control group.
 M.-H. Li et al. / Behavioural Brain Research 262 (2014) 35–41 39

Fig. 5. Effect of Hcy on the expressions of CHOP, GRP78 and cleaved caspase-12 in the hippocampus of rats. After 7-day intracerebroventricular administration of homocysteine
(Hcy, 0.2, 0.6, and 2 ␮mol), the hippocampus of rats were homogenized. The levels of CHOP (A), GRP78 (B), and cleaved caspase-12 (C) expression in hippocampus were
detected by Western blot using anti-CHOP, -GRP78, and -cleaved caspase-12 antibody, respectively. In all blots, staining for ␤-actin was used as a loading control. Values are
the mean ± S.E.M. (n = 3–5). *P < 0.05; **P < 0.01; ***P < 0.001, vs nontreated control group.

3.4. Hcy reduces the expression of CBS and the generation of H2 S
in the hippocampus of rats
It has been confirmed that H2 S modulates learning and mem-
ory [34,35]. To explore whether the distribution of H2 S generation
involves in Hcy-induced impairment in learning and memory, the
expression of CBS and the generation of H2 S in the hippocampus of
rats treated with Hcy were investigated. As shown in Fig. 6A, after
7-day exposure of Hcy (0.2, 0.6, and 2 ␮mol), the expression of CBS
in the hippocampus was significantly decreased. Simultaneously,
after administration of Hcy (0.2, 0.6, and 2 ␮mol), the generation
of H2 S in the hippocampus was significantly inhibited (Fig. 6B).
These data demonstrated that Hcy could disturb the synthesis of
endogenous H2 S in the hippocampus of rats.
4. Discussion
The present study investigates the role of Hcy in learning and
memory and the underlying mechanisms. We demonstrated that
intracerebroventricular injection of Hcy leads to (1) impairment
in learning and memory, (2) disturbance of H2 S generation, and
(3) upregulation of ER stress in the hippocampus. Our results sug-
gest that Hcy-exerted disturbance of hippocampal H2 S generation
and ER stress in the hippocampus may be involved in the deficit in
learning and memory under Hcy exposure.
Increasing evidence suggests that a moderate elevation of
plasma Hcy is a potential risk factor for AD [10,14]. However, the
underline mechanisms have not been fully clarified [36]. In order
to directly investigate the aversive effect of Hcy on learning and
memory, we intracerebroventricularly injected Hcy to rats for 7
days and the functions of learning and memory of rats were tested
in Morris water maze (MWM) and novel object recognition test.
The MWM, described 20 years ago as a device to investigate
spatial learning and memory in laboratory rats, has become one
of the most frequently used laboratory tools in behavioral neuro-
science [32,37]. In MWM, the spatial learning of rats is evaluated
through the hidden-platform acquisition test as well as the spatial
memory is investigated in the probe trial test. In MWM test, we
showed that the rats treated with Hcy increases the escape latency
in hidden-platform acquisition training and decreases the crossing
platform times and the percentage of time elapsed in the target
quadrant in the probe trail, which indicated that Hcy decreases
spatial learning ability and memory of rats. The novel object recog-
nition test is based on the differential exploration of familiar and
new objects [38], and it is used to study short-term, declarative
memory and attention. In the present study, we showed that in
novel object recognition test, the discrimination index of rats was
significantly decreased by intracerebroventricular injection of Hcy
for 7 days, which also indicates the harmful role of Hcy in learning
and memory.
It has been reported that Hcy induces the learning and memory
deficit [12,39]. Other studies have demonstrated that homocysteine
perturbs the methionine cycle [40], fosters aberrant calcium influx
[41], alters phosphatase, kinase and methylation activities [4,41],
and induces oxidative damage to brain tissue [42], which could
be the cause of cognitive decline. However, the underline mecha-
nisms have not been fully elucidated. The endoplasmic reticulum
(ER) is susceptible to various type of injury and the destructive
40 M.-H. Li et al. / Behavioural Brain Research 262 (2014) 35–41

Fig. 6. Effect of Hcy on the expression of CBS and the generation of H2 S in the hippocampus of rats. After 7-day intracerebroventricular administration of homocysteine
(Hcy, 0.2, 0.6, and 2 ␮mol), the hippocampus of rats were homogenized. (A) The level of CBS expression was detected by Western blot using an anti-CBS antibody. In all
blots, staining for ␤-actin was used as a loading control. (B) The generation of H2 S was measured by the N,N-dimethyl-p-phenylenediamine sulphate (NNDPD) method as
described in Section 2. Values are the mean ± S.E.M. (n = 5). **P < 0.01; ***P < 0.001, vs nontreated control group.

stimulus may impair the ER function and leads to the activation
of unfolded-protein-response-ER stress [43–45]. It has been shown
that ER stress is implicated in the pathogenic effects of Hcy on car-
diovascular disease [31], insulin resistance of adipose tissue [46],
apoptosis of osteoblastic cells [47], type 2 diabetes mellitus [48],
and hepatic steatosis [30], which indicates that ER stress is a com-
mon pathway of the injury of tissues and cells induced by Hcy.
Thus, we hypothesized that ER stress could be one of the patholog-
ical mechanisms related to the disorder of learning and memory
in Hcy-treated rats. In order to confirm the hypothesis, the expres-
sion levels of GRP78, CHOP and cleaved caspase-12 in hippocampus
were tested by Western blot analysis. In the present work, we
demonstrated that intracerebroventricular injection of Hcy leads to
upregulations of GRP78, CHOP and cleaved caspase-12 expression
in the hippocampus of rats. Increasing evidence suggests signif-
icant relationships between ER stress and disorders of memory
and cognitive. Chen et al. observed that the long time exposure of
sevoflurane could induce neuronal ER stress and cognitive impair-
ment in aging rats, suggesting a potential role of ER stress in the
sevoflurane-induced memory impairment in aging rats [49]. It also
has been demonstrated that impaired ER stress response could be
one of the pathological mechanisms related to diabetic cognitive
impairment [50], age-related spatial memory deterioration [51]
and the memory disorders in post-traumatic stress disorder [52].
Considering the involvement of ER stress in cognitive and memory
disorder, our data suggested that Hcy-induced ER stress response in
the hippocampus of rats plays an important role in its impairment
of learning and memory.
Interestingly, Hcy and H2 S are metabolites of methionine [53],
but they exert entirely opposite effects on the viability of neu-
rocyte: Hcy causes reactive oxygen species (ROS) formation and
stimulates neurotoxicity [54,55], while H2 S scavenges ROS forma-
tion and prevents oxidative stress-induced neuron death [56–59]. It
has been reported that H2 S serves as a protective gaseous signaling
molecule by preventing ER stress [31,60–62] and acts as a neuro-
modulator by facilitating the formation of hippocampal long-term
potentiation (LTP) [63], the major cellular mechanisms underlying
learning and memory. Therefore, we proposed that the disturbance
of hippocampal H2 S generation is involved in Hcy-induced hip-
pocampal ER stress and impairment of learning and memory in rats.
In our study, we tested the expression of CBS and the generation of
H2 S in the hippocampus of rats treated with Hcy. Our data demon-
strated that intracerebroventricular injection of Hcy reduced the
expression of CBS and inhibited the generation of H2 S in hippocam-
pus. The present data provided a comprehensive picture of the
disturbance of hippocampal H2 S generation under Hcy exposure
involved in hippocampal ER stress and cognition impairment.
In conclusion, the present study demonstrated that Hcy induces
ER stress in the hippocampus and produces a deficit in learning
and memory. We also found the reduction of endogenous H2 S gen-
eration in hippocampus. Based on the role of H2 S in regulating ER
stress and the function of learning and memory, our data suggested
that Hcy-induced loss of learning and memory is involved in the
disturbance of endogenous H2 S and the increase in ER stress in
hippocampus.
Acknowledgements
This study was supported by Natural Science Foundation of
China (81071005 and 81200985), Natural Science Foundation of
Hunan Province, China (11JJ3117, 12JJ9032), the Scientific Research
Foundation for the Returned Overseas Chinese Scholars, State Edu-
cation Ministry ([2010]508), and the construct program of the key
discipline in Hunan province.
References
[1] Prudova A, Bauman Z, Braun A, Vitvitsky V, Lu SC, et al. S-adenosylmethionine
stabilizes cystathionine beta-synthase and modulates redox capacity. Proc Natl
Acad Sci U S A 2006;103:6489–94.
[2] Selhub J. Homocysteine metabolism. Annu Rev Nutr 1999;19:217–46.
[3] Baydas G, Reiter RJ, Akbulut M, Tuzcu M, Tamer S. Melatonin inhibits neural
apoptosis induced by homocysteine in hippocampus of rats via inhibition of
 M.-H. Li et al. / Behavioural Brain Research 262 (2014) 35–41
cytochrome c translocation and caspase-3 activation and by regulating pro-
and anti-apoptotic protein levels. Neuroscience 2005;135:879–86.
[4] Ho PI, Ortiz D, Rogers E, Shea TB. Multiple aspects of homocysteine neuro-
toxicity: glutamate excitotoxicity, kinase hyperactivation and DNA damage. J
Neurosci Res 2002;70:694–702.
[5] Kim JP, Koh JY, Choi DW. L-homocysteate is a potent neurotoxin on cultured
cortical neurons. Brain Res 1987;437:103–10.
[6] Kruman II, Culmsee C, Chan SL, Kruman Y, Guo Z, et al. Homocysteine elicits a
DNA damage response in neurons that promotes apoptosis and hypersensitiv-
ity to excitotoxicity. J Neurosci 2000;20:6920–6.
[7] Linnebank M, Lutz H, Jarre E, Vielhaber S, Noelker C, et al. Binding of copper is a
mechanism of homocysteine toxicity leading to COX deficiency and apoptosis
in primary neurons, PC12 and SHSY-5Y cells. Neurobiol Dis 2006;23:725–30.
[8] Lipton SA, Kim WK, Choi YB, Kumar S, D’Emilia DM, et al. Neurotoxicity associ-
ated with dual actions of homocysteine at the N-methyl-d-aspartate receptor.
Proc Natl Acad Sci U S A 1997;94:5923–8.
[9] Parsons RB, Waring RH, Ramsden DB, Williams AC. In vitro effect of the cysteine
metabolites homocysteic acid, homocysteine and cysteic acid upon human
neuronal cell lines. Neurotoxicology 1998;19:599–603.
[10] Clarke R, Smith AD, Jobst KA, Refsum H, Sutton L, et al. Folate, vitamin B12, and
serum total homocysteine levels in confirmed Alzheimer disease. Arch Neurol
1998;55:1449–55.
[11] Dwyer BE, Raina AK, Perry G, Smith MA. Homocysteine and Alzheimer’s disease:
a modifiable risk. Free Radic Biol Med 2004;36:1471–5.
[12] Miller JW. Homocysteine and Alzheimer’s disease. Nutr Rev 1999;57:126–9.
[13] Seshadri S, Beiser A, Selhub J, Jacques PF, Rosenberg IH, et al. Plasma homo-
cysteine as a risk factor for dementia and Alzheimer’s disease. N Engl J Med
2002;346:476–83.
[14] Van Dam F, Van Gool WA. Hyperhomocysteinemia and Alzheimer’s disease: a
systematic review. Arch Gerontol Geriatr 2009;48:425–30.
[15] Zhang CE, Wei W, Liu YH, Peng JH, Tian Q, et al. Hyperhomocysteinemia
increases beta-amyloid by enhancing expression of gamma-secretase and
phosphorylation of amyloid precursor protein in rat brain. Am J Pathol
2009;174:1481–91.
[16] Wang R. Two’s company, three’s a crowd: can H2 S be the third endogenous
gaseous transmitter. FASEB J 2002;16:1792–8.
[17] Qu K, Lee SW, Bian JS, Low CM, Wong PT. Hydrogen sulfide: neurochemistry
and neurobiology. Neurochem Int 2008;52:155–65.
[18] Kimura H. Hydrogen sulfide as a neuromodulator. Mol Neurobiol
2002;26:13–9.
[19] Tan BH, Wong PT, Bian JS. Hydrogen sulfide: a novel signaling molecule in the
central nervous system. Neurochem Int 2010;56:3–10.
[20] Zhou CF, Tang XQ. Hydrogen sulfide and nervous system regulation. Chin Med
J (Engl) 2011;124:3576–82.
[21] Moore PK, Bhatia M, Moochhala S. Hydrogen sulfide: from the smell of the past
to the mediator of the future. Trends Pharmacol Sci 2003;24:609–11.
[22] Eto K, Asada T, Arima K, Makifuchi T, Kimura H. Brain hydrogen sulfide is
severely decreased in Alzheimer’s disease. Biochem Biophys Res Commun
2002;293:1485–8.
[23] Tang XQ, Shen XT, Huang YE, Chen RQ, Ren YK, et al. Inhibition of endoge-
nous hydrogen sulfide generation is associated with homocysteine-induced
neurotoxicity: role of ERK1/2 activation. J Mol Neurosci 2011;45:60–7.
[24] Tang XQ, Shen XT, Huang YE, Ren YK, Chen RQ, et al. Hydrogen sulfide
antagonizes homocysteine-induced neurotoxicity in PC12 cells. Neurosci Res
2010;68:241–9.
[25] Xu C, Bailly-Maitre B, Reed JC. Endoplasmic reticulum stress: cell life and death
decisions. J Clin Invest 2005;115:2656–64.
[26] McCullough KD, Martindale JL, Klotz LO, Aw TY, Holbrook NJ. Gadd153 sen-
sitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and
perturbing the cellular redox state. Mol Cell Biol 2001;21:1249–59.
[27] Lin JH, Walter P, Yen TS. Endoplasmic reticulum stress in disease pathogenesis.
Annu Rev Pathol 2008;3:399–425.
[28] Dickhout JG, Sood SK, Austin RC. Role of endoplasmic reticulum calcium dise-
quilibria in the mechanism of homocysteine-induced ER stress. Antioxid Redox
Signal 2007;9:1863–73.
[29] Oka Y, Hirabayashi Y, Ishii T, Takahashi R, Sasaki T. A monoclonal antibody
against human homocysteine-induced endoplasmic reticulum protein (Herp):
a useful tool for evaluating endoplasmic reticulum stress. Tohoku J Exp Med
2007;212:431–7.
[30] Werstuck GH, Lentz SR, Dayal S, Hossain GS, Sood SK, et al. Homocysteine-
induced endoplasmic reticulum stress causes dysregulation of the cholesterol
and triglyceride biosynthetic pathways. J Clin Invest 2001;107:1263–73.
[31] Wei H, Zhang R, Jin H, Liu D, Tang X, et al. Hydrogen sulfide attenu-
ates hyperhomocysteinemia-induced cardiomyocytic endoplasmic reticulum
stress in rats. Antioxid Redox Signal 2010;12:1079–91.
[32] Tang XQ, Zhuang YY, Zhang P, Fang HR, Zhou CF, et al. Formaldehyde impairs
learning and memory involving the disturbance of hydrogen sulfide generation
in the hippocampus of rats. J Mol Neurosci 2013;49:140–9.
[33] D’Agostino G, Russo R, Avagliano C, Cristiano C, Meli R, et al. Palmi-
toylethanolamide protects against the amyloid-beta25-35-induced learning
and memory impairment in mice, an experimental model of Alzheimer disease.
Neuropsychopharmacology 2012;37:1784–92.
41
[34] Eto K, Kimura H. The production of hydrogen sulfide is regulated by
testosterone and S-adenosyl-l-methionine in mouse brain. J Neurochem
2002;83:80–6.
[35] Eto K, Ogasawara M, Umemura K, Nagai Y, Kimura H. Hydrogen sulfide is pro-
duced in response to neuronal excitation. J Neurosci 2002;22:3386–91.
[36] Pacheco-Quinto J, Rodriguez de Turco EB, DeRosa S, Howard A, Cruz-Sanchez
F, et al. Hyperhomocysteinemic Alzheimer’s mouse model of amyloidosis
shows increased brain amyloid beta peptide levels. Neurobiol Dis 2006;22:
651–6.
[37] D’Hooge R, De Deyn PP. Applications of the Morris water maze in the study of
learning and memory. Brain Res Brain Res Rev 2001;36:60–90.
[38] Ennaceur A, Delacour J. A new one-trial test for neurobiological studies of
memory in rats. 1: Behavioral data. Behav Brain Res 1988;31:47–59.
[39] Selley ML. Increased homocysteine and decreased adenosine formation in
Alzheimer’s disease. Neurol Res 2004;26:554–7.
[40] Miller AL. The methionine-homocysteine cycle and its effects on cognitive dis-
eases. Altern Med Rev 2003;8:7–19.
[41] Chan AY, Alsaraby A, Shea TB. Folate deprivation increases tau phosphoryla-
tion by homocysteine-induced calcium influx and by inhibition of phosphatase
activity: alleviation by S-adenosyl methionine. Brain Res 2008;1199:133–7.
[42] Obeid R, Herrmann W. Mechanisms of homocysteine neurotoxicity in neu-
rodegenerative diseases with special reference to dementia. FEBS Lett
2006;580:2994–3005.
[43] Paschen W, Frandsen A. Endoplasmic reticulum dysfunction–a common
denominator for cell injury in acute and degenerative diseases of the brain.
J Neurochem 2001;79:719–25.
[44] Breckenridge DG, Germain M, Mathai JP, Nguyen M, Shore GC. Regulation of
apoptosis by endoplasmic reticulum pathways. Oncogene 2003;22:8608–18.
[45] Rao RV, Ellerby HM, Bredesen DE. Coupling endoplasmic reticulum stress to
the cell death program. Cell Death Differ 2004;11:372–80.
[46] Li Y, Zhang H, Jiang C, Xu M, Pang Y, et al. Hyperhomocysteinemia promotes
insulin resistance by inducing endoplasmic reticulum stress in adipose tissue.
J Biol Chem 2013;288:9583–92.
[47] Park SJ, Kim KJ, Kim WU, Oh IH, Cho CS. Involvement of endoplasmic reticulum
stress in homocysteine-induced apoptosis of osteoblastic cells. J Bone Miner
Metab 2012;30:474–84.
[48] Zbidi H, Redondo PC, Lopez JJ, Bartegi A, Salido GM, et al. Homocysteine induces
caspase activation by endoplasmic reticulum stress in platelets from type 2
diabetics and healthy donors. Thromb Haemost 2010;103:1022–32.
[49] Chen G, Gong M, Yan M, Zhang X. Sevoflurane induces endoplasmic reticu-
lum stress mediated apoptosis in hippocampal neurons of aging rats. PLoS One
2013;8:e57870.
[50] Zhang X, Xu L, He D, Ling S. Endoplasmic reticulum stress-mediated hippocam-
pal neuron apoptosis involved in diabetic cognitive impairment. Biomed Res
Int 2013;2013:924327.
[51] Yaguchi T, Nagata T, Nishizaki T. 1,2-dilinoleoyl-sn-glycero-3-
phosphoethanolamine ameliorates age-related spatial memory deterioration
by preventing neuronal cell death. Behav Brain Funct 2010;6:52.
[52] Liu H, Han F, Shi Y. Effect of calreticulin on Ca2+/CaM kinaseIIalpha and endo-
plasmic reticulum stress in hippocampal in a rat model of post-traumatic stress
disorder. Neurochem Res 2013;38:1407–14.
[53] Stipanuk MH. Sulfur amino acid metabolism: pathways for production and
removal of homocysteine and cysteine. Annu Rev Nutr 2004;24:539–77.
[54] White AR, Huang X, Jobling MF, Barrow CJ, Beyreuther K, et al. Homocysteine
potentiates copper- and amyloid beta peptide-mediated toxicity in primary
neuronal cultures: possible risk factors in the Alzheimer’s-type neurodegener-
ative pathways. J Neurochem 2001;76:1509–20.
[55] Ho PI, Collins SC, Dhitavat S, Ortiz D, Ashline D, et al. Homocysteine poten-
tiates beta-amyloid neurotoxicity: role of oxidative stress. J Neurochem
2001;78:249–53.
[56] Kimura Y, Kimura H. Hydrogen sulfide protects neurons from oxidative stress.
FASEB J 2004;18:1165–7.
[57] Tang XQ, Yang CT, Chen J, Yin WL, Tian SW, et al. Effect of hydrogen sulphide
on beta-amyloid-induced damage in PC12 cells. Clin Exp Pharmacol Physiol
2008;35:180–6.
[58] Whiteman M, Armstrong JS, Chu SH, Jia-Ling S, Wong BS, et al. The novel
neuromodulator hydrogen sulfide: an endogenous peroxynitrite ‘scavenger’.
J Neurochem 2004;90:765–8.
[59] Whiteman M, Cheung NS, Zhu YZ, Chu SH, Siau JL, et al. Hydrogen sulphide: a
novel inhibitor of hypochlorous acid-mediated oxidative damage in the brain.
Biochem Biophys Res Commun 2005;326:794–8.
[60] Chen ZF, Zhao B, Tang XY, Li W, Zhu LL, et al. Hydrogen sulfide regulates vascular
endoplasmic reticulum stress in apolipoprotein E knockout mice. Chin Med J
(Engl) 2011;124:3460–7.
[61] Wang XY, Yang CT, Zheng DD, Mo LQ, Lan AP, et al. Hydrogen sulfide protects
H9c2 cells against doxorubicin-induced cardiotoxicity through inhibition of
endoplasmic reticulum stress. Mol Cell Biochem 2012;363:419–26.
[62] Xie L, Tiong CX. Bian JS Hydrogen sulfide protects SH-SY5Y cells against 6-
hydroxydopamine-induced endoplasmic reticulum stress. Am J Physiol Cell
Physiol 2012;303:C81–91.
[63] Abe K, Kimura H. The possible role of hydrogen sulfide as an endogenous neu-
romodulator. J Neurosci 1996;16:1066–71.