J. Pineal Res.

2015; 58:7185

2014 John Wiley & Sons A/S.

Published by John Wiley & Sons Ltd

Molecular, Biological, Physiological and Clinical Aspects of Melatonin

Doi:10.1111/jpi.12194

Journal of Pineal Research

Melatonin attenuates D-galactose-induced memory impairment,

neuroinammation and neurodegeneration via RAGE/NF-KB/JNK
signaling pathway in aging mouse model
Abstract: Melatonin acts as a pleiotropic agent in various age-related
neurodegenerative diseases. In this study, we examined the underlying
neuroprotective mechanism of melatonin against D-galactose-induced memory
and synaptic dysfunction, elevated reactive oxygen species (ROS),
neuroinammation and neurodegeneration. D-galactose was administered
(100 mg/kg intraperitoneally (i.p.)) for 60 days. After 30 days of D-galactose
administration, vehicle (same volume) or melatonin (10 mg/kg, i.p.) was
administered for 30 days. Our behavioral (Morris water maze and Y-maze
test) results revealed that chronic melatonin treatment alleviated D-galactoseinduced memory impairment. Additionally, melatonin treatment reversed
D-galactose-induced synaptic disorder via increasing the level of memoryrelated pre-and postsynaptic protein markers. We also determined that
melatonin enhances memory function in the D-galactose-treated mice possibly
via reduction of elevated ROS and receptor for advanced glycation end
products (RAGE). Furthermore, Western blot and morphological results
showed that melatonin treatment signicantly reduced D-galactose-induced
neuroinammation through inhibition of microgliosis (Iba-1) and astrocytosis
(GFAP), and downregulating other inammatory mediators such as p-IKKb,
p-NF-KB65, COX2, NOS2, IL-1b, and TNFa. Moreover, melatonin lowered
the oxidative stress kinase p-JNK which suppressed various apoptotic
markers, that is, cytochrome C, caspase-9, caspase-3 and PARP-1, and prevent
neurodegeneration. Hence, melatonin attenuated the D-galactose-induced
memory impairment, neuroinammation and neurodegeneration possibly
through RAGE/NF-KB/JNK pathway. Taken together, our data suggest that
melatonin could be a promising, safe and endogenous compatible antioxidant
candidate for age-related neurodegenerative diseases such as Alzheimers
disease (AD).

Introduction
Aging is a major factor involved in gradual decline of brain
function and has been implicated in progressive memory
loss, dementia, and cognitive disorders [1, 2]. Numerous
studies have shown a key role of mitochondria in aging
process. Reactive oxygen species (ROS) and oxidative
stress are known to be associated with several age-associated neuronal disorders such as Alzheimers disease (AD)
[3, 4]. The over accumulation of ROS and oxidative stress
triggers cellular lipids, proteins, or DNA damage which
disturbed normal cellular activity and deregulates homeostatic system of neuron, ultimately leading to neuronal cell
death [5]. Hence, to prevent oxidative stress-induced neuronal degeneration could be a potential neurotherapeutic
approach to treat the age-associated neurodegenerative
disease such as AD.
It is well known that injection of D-galactose is a model
for brain aging which induces and accelerates senescence
in rodents to develop AD like symptoms [6]. Elevated

Tahir Ali*, Haroon Badshah*,

Tae Hyun Kim and Myeong
Ok Kim
Department of Biology and Applied Life Science
(BK 21), College of Natural Sciences (RINS),
Gyeongsang National University, Jinju, Korea

Key words: D-galactose, melatonin, memory

impairment, neurodegeneration,
neuroinflammation, reactive oxygen species
Address reprint requests to Myeong Ok Kim,
PhD, Head of Neuroscience Pioneer Research
Center, Department of Biology and Applied of
Life Science, College of Natural Sciences
(RINS), Gyeongsang National University, Jinju
660-701, Korea.
E-mail: mokim@gnu.ac.kr
*These authors contributed equally to this work.
Received October 29, 2014;
Accepted November 12, 2014.

levels of D-galactose trigger ROS formation in brain

which induce oxidative stress and enhance advanced glycation end products (AGEs) via activating their receptor
RAGE [7, 8]. Numerous studies indicate that increase production of ROS and AGEs in brain lead to decits of spatial memory function and their related pre-and
postsynaptic proteins, and pathologically mediate astrocytes and other inammatory mediators [712]. Systemic
administration of D-galactose for the long-term activates
the mitochondrial apoptotic pathway via cytochrome C
release and enhanced levels of caspases [8]. A number of
studies demonstrated that oxidative stress and RAGE are
involved in the pathology of age-related diseases [13]. It
was recently reported that activated RAGE induces a
downstream pathological cascade that involves activation
of NF-kB and other inammatory mediators [14].
Melatonin (N-acetyl-5-methoxytryptamine) is a key
endogenous indole amine secreted and released by pineal
gland of mammals, including humans, melatonin plays a
critical role in the regulation of circadian rhythms and
71

Ali et al.
numerous other functions [15, 16]. Melatonin is amphiphilic in nature and acts as a potent free radical scavenger
and possesses antioxidant, anti-inammatory and antiapoptotic properties [1724]. Its role in protecting the
central nervous system from oxidative damage is well documented [25, 26]. Plasma melatonin levels are higher in
young people than that in old individuals [27]. The
reduced level of melatonin in aged person has been proposed one of the important factors in the development of
age-related neurodegenerative disorders such as AD [28
30]. Recently, Corrales et al. [31] showed that chronic melatonin treatment improved learning and memory in a
mouse model of brain deterioration. In this study, we
investigated the underlying neuroprotective mechanism of
melatonin against D-galactose-induced neurotoxicity. Our
results show that chronic melatonin treatment attenuates
D-galactose-induced memory impairment, synaptic dysfunction, ROS, oxidative stress, neuroinammation, and
neurodegeneration possibly through RAGE/NF-KB65/
JNK signaling pathway.

Materials and methods

The MWM test is a well parameter task to evaluate memory functions; we performed MWM as described previously
with some modication [8]. The experimental apparatus
consisted of a circular water tank (100 cm in diameter,
40 cm in height), containing water (23  1C) to a depth of
15.5 cm, which was rendered opaque by adding white ink.
A transparent escape platform (10 cm in diameter, 20 cm in
height) was hidden 1 cm below the water surface and placed
at the midpoint of one quadrant. Each mouse received training per day for six consecutive days using a single hidden
platform in one quadrant with three quadrants of rotational
starting. Latency to escape from the water maze (nding the
submerged escape platform) was calculated for each trial.
On day seven, probe test was performed for the evaluation
of memory consolidation. The probe test was carried out by
removing platform and allowing each mouse to swim freely
for 60 s. The mice spent time in the target quadrant (where
the platform was located during hidden platform training)
was measured. Time spent in the target quadrant is considered to represent the degree of memory consolidation, taken
place after learning. All data were recorded using videotracking software (SMART, Panlab Harward Apparatus;
Bioscience Company, Holliston, MA, USA).

Chemicals
D-galactose, Melatonin, and 20 70 -dichlorodihydrouorescein diacetate (DCFH-DA) were purchased from Sigma
Chemical Co. (St. Louis, MO, USA).
Animals
Male wild type C57BL/6N mice (2530 g, 8 weeks old)
were purchased from Samtako Bio (Osan, Korea). The
mice were acclimatized for 1 week in the university animal house under a 12-h/12-h light/dark cycle at 23C
with 60  10% humidity and provided with food and
water ad libitum. All eorts were made to minimize the
number of mice used and their suering. The experimental procedures were approved through the animal ethics
committee of the Division of Applied Life Sciences,
Department of Biology at Gyeongsang National University, South Korea.
Drug treatment protocols
Mice were divided into the following groups: (i) control
(C) mice treated with saline as a vehicle for 2 months, (ii)
mice treated with D-galactose (D-gal) (100 mg/kg) for
2 months, (iii) mice treated with D-galactose (100 mg/kg)
for 2 months and melatonin 10 mg/kg for 30 days
(D-gal+M), and (iv) mice treated with melatonin 10 mg/kg
alone for 30 days (M).
Melatonin was rst dissolved in 0.1% dimethyl sulfoxide (DMSO) and then makes the nal administered volume in saline. Melatonin (10 mg/kg) or saline was
administered (i.p.) for 30 days each 12 hr before evening.
Morris water maze (MWM) test
The behavioral study was performed on mice (n = 15/
group) using MWM and Y-maze test.
72

Y-maze test
The Y-maze was made of black-painted wood. The each
arm of the maze was 50 cm long, 20 cm high and 10 cm
wide at the bottom and 10 cm wide at the top. Each
mouse was placed at the center of the apparatus and
allowed to move freely through the maze for three 8-min
sessions. The series of arm entries was visually observed.
Spontaneous alteration was dened as the successive entry
of the mice into the three arms in overlapping triplet sets.
Alteration behavior (%) was calculated as [successive triplet sets (entries into three dierent arms consecutively)/
total number of arm entries-2] 9 100.
Protein extraction from mouse brain
After behavioral analysis, the mice were killed. The brains
were immediately removed, and hippocampus and cortex
tissue were dissected carefully, frozen on dry ice, and
stored at 80C. The hippocampus tissue was homogenized in 0.2 M PBS with phosphatase inhibitor and protease inhibitor cocktail. The samples were then centrifuged
at 10,000 g at 4C for 25 min. The supernatants were collected and stored at 80C.
Western blot analysis
The protein concentration was measured (Bio-Rad protein
assay kit, Bio-Rad Laboratories, CA, USA). Equal
amounts of protein (1530 lg) were electrophoresis using
412% BoltTM Mini Gels (Novex; Life Technologies, Kiryat Shmona, Israel). The membranes were blocked in 5%
(w/v) skim milk to reduce nonspecic binding and incubated with primary antibodies overnight at 4C at a
1:1000 dilution. After reaction with a horseradish peroxidase-conjugated secondary antibody, as appropriate, the
proteins were detected using an ECL detection reagent

Melatonin prevents D-galactose neurotoxicity

according to the manufacturers instructions (Amersham
Pharmecia Biotech, Uppsala, Sweden). The X-ray lms
were scanned, and the optical densities of the bands were
analyzed through densitometry using the computer-based
Sigma Gel program, version 1.0 (SPSS Inc., Chicago, IL,
USA).
Antibodies
The following antibodies were used. Anti-SNAP25, antiphospho-AMPARs (GluR1) Ser845), anti-RAGE, antiIba-1, anti-NF-KB 65, total anti-p-JNK, anti-TNFa, IL-1b,
NOS2, anti-caspase-3, anti-cytochrome C, anti-PARP-1,
anti-GFAP, and anti-b-Actin from Santa Cruz Biotechnology; antisynaptophysin, PSD95, anti-p-CREB (Ser 133),
and anticaspase-9 from Cell Signaling and 8-oxoguanine
from Millipore, Billerica, MA, USA.
ROS assay
ROS was assessed as described previously with some modication [13]. The assay was based on the oxidation of
20 70 -dichlorodihydrouorescein diacetate (DCFH-DA) to
20 70 -dichlorouorescein (DCF). Shortly, brain homogenate
was diluted with ice-cold Locks buer at 1: 20 times to
make the nal concentration 2.5 mg tissue/500 lL. The
reaction mixture Locks buer (1 mL, pH = 7.4), 0.2 mL
homogenate, and 10 mL of DCFH-DA (5 mM) was incubated at room temperature for 15 min to convert DCFHDA to the uorescent product DCF. Conversion of
DCFH-DA to the DCF was assessed using a spectrouorimeter with excitation at 484 nm and emission at 530 nm.
For background uorescence (conversion of DCFH-DA
in the absence of homogenate), we run a parallel blanks.
Quantication analysis of ROS was expressed as pmol
DCF formed/mg protein.
Tissue collection and sample preparation
For the tissue analysis (N = 5 mice per group), the mice
were perfused transcardially with 4% ice-cold paraformaldehyde, and the brains were postxed for 72 hr in 4%
paraformaldehyde and transferred to 20% sucrose for
72 hr. The brains were frozen in O.C.T compound (A.O,
USA), and 14-lm coronal sections were cut using a CM
3050C cryostat (Leica, Germany). The sections were thawmounted on probe-on plus charged slides (Fisher, Rockford, IL, USA).
Immunofluorescence staining
The immunouorescence was performed with some modication as we described previously [32]. Briey, slides were
washed twice for 10 min in 0.01 M PBS, followed by incubation for 1 hr in blocking solution containing 2% normal
serum according to the antibody treatment and 0.3% Triton
X-100 in PBS. After blocking, the slides were incubated
overnight at 4C in the primary antibodies (p-JNK, IL-1b,
TNFa, NF-KB65, GFAP from Santa Cruz Biotechnology,
mouse monoclonal 8-Oxoguanine from Millipore, diluted
1:100 in blocking solution. Following incubation with pri-

mary antibody, the sections were incubated for 2 hr in the

secondary antibodies tetramethyl rhodamine isothiocyanate
(TRITC)/uorescein isothiocyanate (FITC)-labeled antibodies (1:50) (Santa Cruz Biotechnology, Dallas, TX,
USA). Subsequently, after the incubation of the TRITC/
FITC-labeled, the sections were incubated overnight in
another primary antibody, following incubation with primary antibody, the sections were incubated for 2 hr FITC/
TRITC-labeled antibody (1:50) (Santa Cruz Biotechnology)
under the same conditions. The slides were mounted with 40 ,
60 -diamidino-2-phenylindole (DAPI) and Prolong Anti-fade
Reagent (Molecular Probe, Eugene, OR, USA). Staining
patterns of the immunouorescence were examined using a
confocal laser-scanning microscope (Flouview FV 1000).
Immunohistochemical analysis
The slides were washed twice for 10 min in 0.01 M PBS,
followed by quenching for 10 min in a solution of methanol containing 30% hydrogen peroxidase and incubated
for 1 hr in blocking solution containing 5% normal goat
serum and 0.3% Triton X-100 in PBS. After blocking, the
slides were incubated overnight in rabbit anticaspase-3
antibody (Cell Signaling Technology, Beverly, MA, USA)
diluted 1:100 in blocking solution. Following incubation
with primary antibody, the sections were incubated for
1 hr in biotinylated goat anti-rabbit secondary antibody
diluted 1:500 in PBS and subsequently incubated with
ABC reagents (Standard Vectastain ABC Elite Kit; Vector
Laboratories, Burlingame, CA, USA) for 1 hr in the dark
at room temperature. The sections were washed twice with
PBS and incubated in 3, 30 -Diaminobenzidine tetra hydrochloride (DAB), and sections were washed with distilled
water, dehydrated in graded ethanol (70%, 95%, and
100%), placed in xylene and coverslipped using mounting
medium. An examiner, blinded to the treatment conditions, counted the active caspase-3 positive cells in the
DG, CA1, and CA3 regions of the hippocampus using
computerized ImageJ program analysis.
Fluoro-jade B (FJB) staining
FJB (Millipore, cat# AG310, lot # 2159662) staining was
performed as previously we described [33].
Nissl staining
Nissl staining was used for the histological examination
and measurement of neuronal loss. The slides with 14-lm
sections were washed twice for 15 min in 0.01 M PBS and
stained with a 0.5% cresyl violet solution (containing few
drops glacial acetic acid) for 1015 min. The sections were
washed with distilled water and dehydrated in graded ethanol (70%, 95%, and 100%), placed in xylene and coverslipped using mounting medium. The cells in the Cortex
and CA1 and CA3 regions of hippocampus were counted
using computerized image analysis.
Statistical analysis
The Western blot bands were scanned and analyzed
through densitometry using the Sigma Gel System (SPSS
73

Ali et al.
Inc.). The density values were expressed as the
means  SEM. One-way analysis of variance (ANOVA)
followed by a two-tailed independent Students t-test was
used for comparisons among the treated groups and the
control. The ImageJ software was used for immunohistological quantitative analysis. P values <0.05 (P < 0.05)
were considered to be statistically signicant.

Results
To investigate the eect of D-galactose and melatonin on
mice behavior and memory function, Morris water maze
(MWM) and Y-maze tasks were performed. MWM training tests were performed for six consecutive days and the
escape latencies (time to reach the hidden platform) was
recorded. Mice treated with D-galactose showed more
escape latency as compared to the vehicle-treated mice.
Melatonin treatment (10 mg/kg, i.p. for 30 days) to
D-galactose-treated mice showed less latency time than
that of D-galactose-treated mice (Fig. 1A).
After trial session on day 7, the hidden platform
removed and probe test was performed. The number of
platform crossings was signicantly increased by melatonin treatment in the D-galactose-treated mice compared to
the D-galactose-treated-alone mice (Fig. 1B). In addition,
melatonin-treated mice spent more time in the target
quadrant than that of D-galactose alone (Fig. 1C), showing that melatonin reduced D-galactose-induced memory
impairment.
Following the MWM test, we performed a Y-maze task to
analyze the spatial working memory using spontaneous
alteration behavior percentage (%). A higher percentage of
spontaneous alteration behavior was considered to be
enhanced cognitive performance. Measurements of the spontaneous alternation rate from the center of the maze, which
is the percentage of the total number of arm entries that can
(A)

(C)

74

be counted as successive nonrepetitive triplets, show that the

D-galactose-treated mice had a lower percentage of alternation percent than vehicle-treated mice, demonstrating less
working memory. Melatonin treatment signicantly
increased the spontaneous alteration behavior % in the
D-galactose-treated mice compared to the D-galactose alone
mice (Fig. 1D), showing that melatonin attenuated memory
decit in the D-galactose inducing aged mice.
Western blot analysis also revealed that memory-related
presynaptic proteins synaptophysin and synaptosomal
associated protein 25 (SNAP-25), postsynaptic density proteins (PSD95), and phosphorylated a-amino-3-hydroxy5-methylisoxazol-4-propionic acid (AMPA) receptors
(AMPAR1s) [GluR1 Ser 845] were decreased in the
D-galactose-treated mice compared to vehicle-treated mice.
Melatonin treatment (10 mg/kg, i.p. for 30 days) signicantly increased the levels of the presynaptic proteins
synaptophysin and SNAP-25; and postsynaptic protein
PSD95, and GluR1 (Ser 845) compared with D-galactose
treated mice (Fig. 2).
Phosphorylated cAMP response element binding protein
p-CREB (Ser 133) level was also decreased in the hippocampus of adult mice as compared to vehicle-treated
group. Treatment with melatonin signicantly increased
the level of p-CREB Ser 133 in the D-galactose-treated
mice as compared to the D-galactose-treated-alone mice
(Fig. 2).
To determine the eect of melatonin on D-galactoseinduced elevated level of ROS, the ROS assay was performed. Our results showed that D-galactose signicantly
increased ROS level as compared to the vehicle-treated
mice, while D-galactose treatment along with melatonin
(10 mg/kg, i.p. for 30 days) signicantly reduced the ROS
level than that of D-galactose-treated-alone mice (Fig. 3A).
The 8-oxoguanine is an oxidative stress marker, highly
expressed in the brain of patients with AD and dementia,

(B)

(D)

Fig. 1. Melatonin improves memory

impairment in D-galactose-treated mice.
For behavioral study, the number of mice
(N = 15) per group was used. (A) Mean
escape latency time (sec) to reach hidden
platform during training session of
MWM. (B) The number of platform
crossing over the previous platform place
during the probe test of MWM. (C) Time
spent in the target quadrant (where the
platform was located during the
hidden
platform
training
session)
during the probe test of MWM. (D)
The spontaneous alteration behavior
percentage during the Y-maze task.
* Signicantly dierent from the vehicle
treated; # signicantly dierent from
D-galactose
treated.
Signicance =
**P < 0.01, ##P < 0.01.

Ali et al.
Inc.). The density values were expressed as the
means  SEM. One-way analysis of variance (ANOVA)
followed by a two-tailed independent Students t-test was
used for comparisons among the treated groups and the
control. The ImageJ software was used for immunohistological quantitative analysis. P values <0.05 (P < 0.05)
were considered to be statistically signicant.

Results
To investigate the eect of D-galactose and melatonin on
mice behavior and memory function, Morris water maze
(MWM) and Y-maze tasks were performed. MWM training tests were performed for six consecutive days and the
escape latencies (time to reach the hidden platform) was
recorded. Mice treated with D-galactose showed more
escape latency as compared to the vehicle-treated mice.
Melatonin treatment (10 mg/kg, i.p. for 30 days) to
D-galactose-treated mice showed less latency time than
that of D-galactose-treated mice (Fig. 1A).
After trial session on day 7, the hidden platform
removed and probe test was performed. The number of
platform crossings was signicantly increased by melatonin treatment in the D-galactose-treated mice compared to
the D-galactose-treated-alone mice (Fig. 1B). In addition,
melatonin-treated mice spent more time in the target
quadrant than that of D-galactose alone (Fig. 1C), showing that melatonin reduced D-galactose-induced memory
impairment.
Following the MWM test, we performed a Y-maze task to
analyze the spatial working memory using spontaneous
alteration behavior percentage (%). A higher percentage of
spontaneous alteration behavior was considered to be
enhanced cognitive performance. Measurements of the spontaneous alternation rate from the center of the maze, which
is the percentage of the total number of arm entries that can
(A)

(C)

74

be counted as successive nonrepetitive triplets, show that the

D-galactose-treated mice had a lower percentage of alternation percent than vehicle-treated mice, demonstrating less
working memory. Melatonin treatment signicantly
increased the spontaneous alteration behavior % in the
D-galactose-treated mice compared to the D-galactose alone
mice (Fig. 1D), showing that melatonin attenuated memory
decit in the D-galactose inducing aged mice.
Western blot analysis also revealed that memory-related
presynaptic proteins synaptophysin and synaptosomal
associated protein 25 (SNAP-25), postsynaptic density proteins (PSD95), and phosphorylated a-amino-3-hydroxy5-methylisoxazol-4-propionic acid (AMPA) receptors
(AMPAR1s) [GluR1 Ser 845] were decreased in the
D-galactose-treated mice compared to vehicle-treated mice.
Melatonin treatment (10 mg/kg, i.p. for 30 days) signicantly increased the levels of the presynaptic proteins
synaptophysin and SNAP-25; and postsynaptic protein
PSD95, and GluR1 (Ser 845) compared with D-galactose
treated mice (Fig. 2).
Phosphorylated cAMP response element binding protein
p-CREB (Ser 133) level was also decreased in the hippocampus of adult mice as compared to vehicle-treated
group. Treatment with melatonin signicantly increased
the level of p-CREB Ser 133 in the D-galactose-treated
mice as compared to the D-galactose-treated-alone mice
(Fig. 2).
To determine the eect of melatonin on D-galactoseinduced elevated level of ROS, the ROS assay was performed. Our results showed that D-galactose signicantly
increased ROS level as compared to the vehicle-treated
mice, while D-galactose treatment along with melatonin
(10 mg/kg, i.p. for 30 days) signicantly reduced the ROS
level than that of D-galactose-treated-alone mice (Fig. 3A).
The 8-oxoguanine is an oxidative stress marker, highly
expressed in the brain of patients with AD and dementia,

(B)

(D)

Fig. 1. Melatonin improves memory

impairment in D-galactose-treated mice.
For behavioral study, the number of mice
(N = 15) per group was used. (A) Mean
escape latency time (sec) to reach hidden
platform during training session of
MWM. (B) The number of platform
crossing over the previous platform place
during the probe test of MWM. (C) Time
spent in the target quadrant (where the
platform was located during the
hidden
platform
training
session)
during the probe test of MWM. (D)
The spontaneous alteration behavior
percentage during the Y-maze task.
* Signicantly dierent from the vehicle
treated; # signicantly dierent from
D-galactose
treated.
Signicance =
**P < 0.01, ##P < 0.01.

Ali et al.
(A)

(B)

Fig. 3. Melatonin treatment ameliorates

ROS and oxidative stress in the Dgalactose-treated mice. (A) A representative histogram showing a comparative
ROS level in the cortex and hippocampus
of mice. N = 5. (B) A representative
image of immunouorescence staining of
8-Oxoguanine in the DG, CA1, and CA3
region of the hippocampus. N = 5. Magnication 109. Scales bar = 100 lm.*
signicantly dierent from the vehicle
treated; # signicantly dierent from
D-galactose
treated.
Signicance =
**P < 0.01, ***P < 0.001, ##P < 0.01,
###P < 0.001.

IL-1b and TNFa analysis also determined that the immunouorescence reactivity of IL-1b (Fig. 6B) and TNFa
(Fig. 6C) signicantly increased in cortex and hippocampus of D-galactose-treated mice as compared to vehicletreated group. Melatonin treatment (10 mg/kg, i.p. for
30 days) signicantly reduced the immunouorescence
reactivity of the IL-1b and TNFa in cortex, CA1 and CA3
region of hippocampus in the D-galactose-treated mice
compared to that of D-galactose-treated-alone mice
(Fig. 6B and C).
Diverse research data demonstrated that phospho-c-Jun
N-terminal Kinase 1 [p-JNK1] (T183/Y185) known as
stress-activated protein kinase (SAPK) overexpressed in
the oxidative stress condition and involved in the mediation of apoptotic signaling [41, 42]. Therefore, we analyzed
the p-JNK level through Western blot and immunouorescence analysis. Earlier study reported activated p-JNK in
the D-galactose mouse model [43]. Our Western blot
results showed that D-galactose-treated mice also indicated overexpressed p-JNK, while melatonin treatment
(10 mg/kg, i.p. for 30 days) signicantly reduces p-JNK
level compared to the D-galactose-treated mice (Fig. 7A).
76

Additionally, p-JNK immunouorescence reactivity

increased in the D-galactose-treated mice as compared to
the vehicle-treated mice. Treatment with melatonin signicantly reduced immunouorescence reactivity of p-JNK in
the cortex (Fig. 7B) and CA1, CA3, and DG region of
hippocampus in the D-galactose-treated mice as compared
to D-galactose-treated-alone group (Fig. 7C).
It has been investigated that activated JNK has direct
impact and released cytochrome C (Cyt.C), which further
activates caspases, resulting neuronal apoptosis [41, 44].
Our results revealed that D-galactose treatment signicantly elevated protein expression level of Cyt.C, caspase9, and caspase-3 in the hippocampus compared with
vehicle-treated group. Treatment with melatonin (10 mg/
kg, i.p. for 30 days) reverses this activation and signicantly reduced the expression level of Cyt.C, caspase-9,
and caspase-3 in the D-galactose-treated mice as compared
to the D-galactose-treated-alone mice (Fig. 8A). Moreover, the activated caspase-3 was also examined through
immunohistochemistry. The results (Fig. 8B) showed that
in the D-galactose-treated group, the caspase-3-positive
cells were signicantly increased in the DG, CA, and CA3

Melatonin prevents D-galactose neurotoxicity

(A)

(B)

Fig. 4. Melatonin
reduces
the
microgliosis, astrocytosis, and RAGE
expression in the D-galactose-treated
mice. (A) The Western blot analysis of
RAGE, GFAP, and Iba-1 in the
hippocampus of mice. The bands were
quantied using Sigma Gel software, and
the dierences are represented by a
histogram. b-Actin was used as a loading
control. The density values are expressed
in arbitrary units (A.U) as the
means  SEM
for
the
respective
indicated protein (N = 10 mice/group).
(B) Representative images showing
immunouorescence analysis of the
GFAP.
The
D-galactose-treated
increased the GFAP indicating activated
astrocytes in CA1, DG, and CA3 regions
of
hippocampus.
Treatment
with
melatonin ameliorated the D-galactose
eects and signicantly decreased the
immunoreactivity
of
GFAP.
Magnication 409, Scale bar = 50 lm.
(C) Representative images showing
immunouorescence analysis of the
GFAP in the cortex. Magnication 40 X,
Scale bar = 50 lm. * signicantly
dierent from the vehicle treated; #
signicantly dierent from D-galactose
treated.
Signicance = ***P < 0.01,
###P < 0.001.

(C)

region of the hippocampus as compared to the vehicletreated group. Melatonin treatment signicantly reduced
the immunoreactivity of caspase-3 in the DG, CA1, and
CA3 region of the hippocampus, showing a decreased
number of active caspase-3-positive cells in the melatonin
plus D-galactose-treated group as compared to the
D-galactose-treated-alone group (Fig. 8B). Next, we examined the level of cleaved poly (ADP-ribose) polymerase-1
(PARP-1). Cleaved PARP-1 is responsible for DNA

damage which leads to neurodegeneration [45]. Our Western blot results indicated increased cleaved PARP-1 level
in the D-galactose-treated mice while treatment with melatonin signicantly reduced cleaved PARP-1 level in the
D-galactose-treated mice as compared to D-galactose-treated-alone mice (Fig. 8A). FJB is one of the key markers
for the indication of neurodegeneration [46]. In the
D-galactose-treated group, FJB-positive neuronal cells
were signicantly increased in the DG and CA3 region of
77

Ali et al.
(A)

(B)

Fig. 3. Melatonin treatment ameliorates

ROS and oxidative stress in the Dgalactose-treated mice. (A) A representative histogram showing a comparative
ROS level in the cortex and hippocampus
of mice. N = 5. (B) A representative
image of immunouorescence staining of
8-Oxoguanine in the DG, CA1, and CA3
region of the hippocampus. N = 5. Magnication 109. Scales bar = 100 lm.*
signicantly dierent from the vehicle
treated; # signicantly dierent from
D-galactose
treated.
Signicance =
**P < 0.01, ***P < 0.001, ##P < 0.01,
###P < 0.001.

IL-1b and TNFa analysis also determined that the immunouorescence reactivity of IL-1b (Fig. 6B) and TNFa
(Fig. 6C) signicantly increased in cortex and hippocampus of D-galactose-treated mice as compared to vehicletreated group. Melatonin treatment (10 mg/kg, i.p. for
30 days) signicantly reduced the immunouorescence
reactivity of the IL-1b and TNFa in cortex, CA1 and CA3
region of hippocampus in the D-galactose-treated mice
compared to that of D-galactose-treated-alone mice
(Fig. 6B and C).
Diverse research data demonstrated that phospho-c-Jun
N-terminal Kinase 1 [p-JNK1] (T183/Y185) known as
stress-activated protein kinase (SAPK) overexpressed in
the oxidative stress condition and involved in the mediation of apoptotic signaling [41, 42]. Therefore, we analyzed
the p-JNK level through Western blot and immunouorescence analysis. Earlier study reported activated p-JNK in
the D-galactose mouse model [43]. Our Western blot
results showed that D-galactose-treated mice also indicated overexpressed p-JNK, while melatonin treatment
(10 mg/kg, i.p. for 30 days) signicantly reduces p-JNK
level compared to the D-galactose-treated mice (Fig. 7A).
76

Additionally, p-JNK immunouorescence reactivity

increased in the D-galactose-treated mice as compared to
the vehicle-treated mice. Treatment with melatonin signicantly reduced immunouorescence reactivity of p-JNK in
the cortex (Fig. 7B) and CA1, CA3, and DG region of
hippocampus in the D-galactose-treated mice as compared
to D-galactose-treated-alone group (Fig. 7C).
It has been investigated that activated JNK has direct
impact and released cytochrome C (Cyt.C), which further
activates caspases, resulting neuronal apoptosis [41, 44].
Our results revealed that D-galactose treatment signicantly elevated protein expression level of Cyt.C, caspase9, and caspase-3 in the hippocampus compared with
vehicle-treated group. Treatment with melatonin (10 mg/
kg, i.p. for 30 days) reverses this activation and signicantly reduced the expression level of Cyt.C, caspase-9,
and caspase-3 in the D-galactose-treated mice as compared
to the D-galactose-treated-alone mice (Fig. 8A). Moreover, the activated caspase-3 was also examined through
immunohistochemistry. The results (Fig. 8B) showed that
in the D-galactose-treated group, the caspase-3-positive
cells were signicantly increased in the DG, CA, and CA3

Melatonin prevents D-galactose neurotoxicity

Fig. 5. Melatonin inhibits the p-IKKb/p-NF-KB65 in the D-galactose-treated mice. (A) The Western blot analysis of p-IKKb and
p-NF-KB65 in the hippocampus of mice. The bands were quantied using Sigma Gel software, and the dierences are represented by a
histogram. b-Actin was used as a loading control. The density values are expressed in arbitrary units (A.U) as the means  SEM for the
respective indicated protein (N = 10 mice/group). (B) A representative image of p-NF-KB65 immunouorescence showing nuclear translocation in the cortex and CA1 region of hippocampus in mice.* signicantly dierent from the vehicle treated; # signicantly dierent from
D-galactose treated. Signicance = **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.001, ###P < 0.001.

hippocampus as compared to vehicle-treated group. Melatonin treatment signicantly reduced the neurodegeneration in the DG and CA3 region of hippocampus, showing
a decreased FJB-positive neurons number in the melatonin-treated group as compared to the D-galactosetreatedalone group (Fig. 8C). Next, Nissl staining was used to
observe the extent of neuronal cell death induced by Dgalactose treatment and to examine the neuroprotection
produced through melatonin treatment in the cortex and
hippocampus of D-galactose-treated mice. The number of
survival neurons in the cortex and CA1 and CA3 regions
of hippocampus was reduced in D-galactose-treated mice
compared with vehicle-treated mice. After melatonin treatment, the number of survival neurons signicantly
increased in the cortex and CA1 and CA3 regions of hippocampus in the D-galactose plus melatonin-treated mice
as compared with the D-galactose treatment alone
(Fig. 8D). These Western blot and immunohistochemical
results indicated that melatonin is eective in preventing
apoptosis and neurodegeneration in D-galactose-treated
mice (Figs 7AC and 8AD).

Discussion
Here in, we investigated the ability of melatonin to prevent
the D-galactose-induced memory impairment, synaptic
dysfunction, ROS, oxidative stress, neuroinammation,
and neurodegeneration through RAGE/NF-KB/JNK
pathway. Excessive formation and accumulation of ROS
has been considered a key mediator to induce neuroinammation and neuronal degeneration in various ageassociated neurodegenerative diseases such as AD and
Parkinson disease [3, 4, 47].
Chronic D-galactose administration activates and formation of ROS and has strong anity for free amines of
amino acid in proteins and peptides; and triggers AGEs
accumulation which bind with their receptor RAGE in
vivo, ultimately leading to oxidative stress, resulting neuroinammation, neurodegeneration, and memory impairment [79, 11, 12, 48].
Overexpressed RAGE activates NF-KB, results in the
activation of other inammatory mediators and glial activation, which results in progressive age-related diseases such
as type II diabetes, atherosclerosis, and other chronic neurodegenerative diseases including AD and Parkinson disease
[49, 50]. Elevated NF-KB has been reported in old age [51].
Similarly Terai et al. [52] reported that NF-KB has been
found in neurons, neurobrillary tangles in the brain of
patient with AD after postmortem. Hence, RAGE/NF-KB
signaling pathway is an emerging pathway to study the
progression and treatment of various age-related disease.
Previously it has been established that chronic administration of D-galactose increased ROS level and oxidative

stress, that results in memory and synaptic impairment,

glial activation and reduction in immune responses, ultimately leading to neurodegeneration [53]. In this study,
we examined whether chronic treatment with D-galactose signicantly induced memory impairment consent
with previous evidence [54]. Melatonin treatment
reversed the memory impairment in D-galactose-treated
mice and improved memory via reduced the escape
latency time during trial session, increased the number
of platform crossing and more time spent in the target
quadrant during the probe test, and increased the spontaneous alteration behavior % in the Y-maze task
(Fig. 1AD).
Synaptic decits and loss have been investigated in progressive aging diseases such as AD [55]. The synaptophysin and SNAP-25 levels were decreased in the brain of
patients with AD and animal models of AD [56] and the
postsynaptic proteins PSD95 and p-GluR1 Ser 845 are
involved in the synaptic plasticity and memory function
[57]. Previously, studies have shown that melatonin rescues
the pre- and postsynaptic proteins in various AD models
of neurodegenerative disease [58, 59]. Earlier studies of
Wu et al. [12] shows that D-galactose-induced memory
impairment and decreased associated presynaptic and
postsynaptic protein markers; similarly, our results also
show that presynaptic protein synaptophysin, SNAP25
and postsynaptic protein PSD95 and p-GluR1 (Ser 845)
were reduced in D-galactose-treated mice; these eects
were reversed by melatonin treatment and signicantly
enhanced expression of the protein markers, which might
be associated with memory improvements in the D-galactose-treated mice (Fig. 2).
P-CREB Ser 133 is an important transcription factor of
memory function and synapse formation [60]. Recently,
Yoo et al. [54] showed that melatonin prevented the
depressed p-CREB Ser 133 level and ameliorated memory
impairment in the D-galactose-treated mice. Here, we also
documented that melatonin increased the expression level
of p-CREB Ser 133 and improved memory functions in
the D-galactose-treated mice (Fig. 2).
D-galactose has been used to induce ROS which trigger
oxidative stress and neurodegeneration which accelerates
the normal aging process [7]. We assessed oxidative stress
through an oxidative stress marker, 8-oxoguanin; the
results indicate that melatonin treatment reduced D-galactose-induced oxidative stress via reduction of 8-oxoguanin
(Fig. 3A).
Elevated levels of RAGE have been found in the hippocampal and cortex of the patients with AD [61], suggesting
the involvement of ROS and oxidative stress, and activated by D-galactose in the aging mouse model [13]. It has
been shown that overexpression of RAGE in the D-galactose-treated mice activates gliosis and stimulates both
79

Ali et al.
(A)

(B)

(C)

80

Ali et al.
(A)

(B)

78

Ali et al.
(A)

(B)

(C)

(D)

Fig. 8. Melatonin decreases apoptotic markers and neurodegeneration in the D-galactose-treated mice. (A) Western blot analysis of
mouse hippocampus using Cyt.C, caspase-9, activated caspase-3, and cleaved PARP-1 antibodies. The bands were quantied using Sigma
Gel software, and the dierences are represented by a histogram. Anti-b-actin was used as a loading control. The density values are
expressed in arbitrary units (A.U) as the means  SEM for the respective indicated hippocampus proteins (N = 10 mice/group). (B)
Immunoreactive cells of activated caspase-3 antibody were examined in the DG, CA3, and CA1 regions of the hippocampus of the
D-galactose-treated group. Caspase-3-positive cells were increased in the D-galactose-treated mice compared with the control. Treatment
with melatonin signicantly decreased the D-galactose-induced number of caspase-3-positive cells. Scale bar = 200 lm. (C) Representative
images of FJB staining show that neurodegeneration are induced in the D-galactose-treated mice. FJB-stained apoptotic neurons are
shown in the CA3 and DG regions of the hippocampus. The decreased FJB-positive cells indicate that melatonin treatment (10 mg/kg,
i.p. for 30 days) eectively reduced apoptosis in the hippocampus of D-galactose-treated mice. The images are representative of the staining observed in each section (N = 5 animals/group). Magnication 409. Scale bar = 100 lm. FJB-positive cells in the dierent regions of
each section were analyzed using the computer-based Image J program. (D) Representative photomicrograph of Nissl staining in the DG,
CA3, and CA1 regions of the hippocampus and cortex of the D-galactose-treated group. Neuronal cell death increased after injection of
D-galactose in all regions. Melatonin treatment signicantly decreased neuronal cell death. The results were analyzed using the computerbased Image J program Scale bar = 200 lm.* signicantly dierent from the vehicle treated; # signicantly dierent from D-galactose.
Signicance = **P < 0.01, ***P < 0.001, ##P < 0.01, ###P < 0.001.

82

Melatonin prevents D-galactose neurotoxicity

35. LOVELL M, MARKESBERY W. Oxidative DNA damage in mild
cognitive impairment and late-stage Alzheimers disease.
Nucleic Acids Res 2007; 35:74977504.
36. HAMILTON A, HOLSCHER C. The eect of ageing on neurogenesis and oxidative stress in the APPswe/PS1deltaE9 mouse
model of Alzheimers disease. Brain Res 2012; 1449:8393.
37. WU DC, JACKSON-LEWIS V, VILA M et al. Blockade of microglial activation is neuroprotective in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine mouse model of Parkinson
disease. J Neurosci 2002; 22:17631771.
38. GAO HM, LIU B, ZHANG W et al. Novel anti-inammatory
therapy for Parkinsons disease. Trends Pharmacol Sci 2003;
24:395401.
39. BENNER EJ, MOSLEY RL, DESTACHE CJ et al. Therapeutic
immunization protects dopaminergic neurons in a mouse
model of Parkinsons disease. Proc Natl Acad Sci USA 2004;
101:94359440.
40. SALMINEN A, OJALA J, KAUPPINEN A et al. Inammation in
Alzheimers disease: amyloid-[beta] oligomers trigger innate
immunity defence via pattern recognition receptors. Prog
Neurobiol 2009; 87:181194.
41. TOURNIER C, HESS P, YANG DD et al. Requirement of JNK
for stress-induced activation of the cytochrome c-mediated
death pathway. Science 2000; 288:870874.
42. LIU J, LIN A. Role of JNK activation in apoptosis: a doubleedged sword. Cell Res 2005; 15:3642.
43. YEH SL, WU TC, CHAN ST et al. Fructo-oligosaccharide
attenuates the production of pro-inammatory cytokines and
the activation of JNK/Jun pathway in the lungs of D-galactose-treated Balb/cJ mice. Eur J Nutr 2014; 53:449456.
44. EMINEL S, KLETTNER A, ROEMER L et al. JNK 2 translocates
to the mitochondria and mediates cytochrome c release in PC
12 cells in response to 6-hydroxydopamine. J Biol Chem
2004; 279:5538555392.
45. CHAITANYA GV, ALEXANDER JS, BABU PP. PARP-1 cleavage
fragments: signatures of cell-death proteases in neurodegeneration. Cell Commun Signal 2010; 8:31.
46. SCHMUD LC, HOPKINS KJ. Fluro-Jade B: a high anity uorescent marker for the localization of neuronal degeneration.
Brain Res 2000; 874:123130.
47. de IULIIS A, GRIGOLETTO J, RECCHIA A et al. A proteomic
approach in the study of an animal model of Parkinsons disease. Clin Chim Acta 2005; 357:202209.
48. ZHANG Q, LI X, CUI X et al. D-Galactose injured neurogenesis in the hippocampus of adult mice. Neurol Res 2005;
27:552556.
49. SRIKANTH V, MACZUREK A, PHAN T et al. Advanced glycation
endproducts and their receptor RAGE in Alzheimers disease.
Neurobiol Aging 2011; 32:763777.
50. BIERHAUS A, HUMPERT P, MORCOS M et al. Understanding
RAGE, the receptor for advanced glycation end products. J
Mol Med 2005; 83:876886.

51. CALABRESE V, CORNELIUS C, CUZZOCREA S et al. Hormesis,

cellular stress response and vitagenes as critical determinants
in aging and longevity. Mol Aspects Med 2011; 32:279304.
52. TERAI K, MATSUO A, MCGEER PL. Enhancement of immunoreactivity for NF-kappa B in the hippocampal formation and
cerebral cortex of Alzheimers disease. Brain Res 1996;
735:159168.
53. CUI X, ZUO P, ZHANG Q et al. Chronic systemic D-galactose
exposure induces memory Loss, neurodegeneration, and oxidative damage in mice: protective eects of R-a-lipoic acid. J
Neurosci Res 2006; 83:15841590.
54. BARCO A, MARIE H. Genetic approaches to investigate the
role of CREB in neuronal plasticity and memory. Mol Neurobiol 2011; 44:330349.
55. SHANKAR GM, WALSH DM. Alzheimers disease: synaptic dysfunction and Ab. Mol Neurodegener 2009; 4:13264.
56. CANAS PM, PORCIUNCULA LO, CUNHA GM et al. Adenosine
A2A receptor blockade prevents synaptotoxicity and memory
dysfunction caused by b-amyloid peptides via p38 mitogenactivated protein kinase pathway. J Neurosci 2009; 29:14741
14751.
57. KIM MJ, FUTAI K, JO J et al. Synaptic accumulation of PSD95 and synaptic function regulated by phosphorylation of
seine-295 of PSD-95. Neuron 2007; 56:488502.
58. WANG X, WANG ZH, WU YY et al. Melatonin attenuates scopolamine-induced memory/synaptic disorder by rescuing EPACs/miR-124/Egr1 pathway. Mol Neurbiol 2012; 47:373381.
59. ZHU LQ, WANG SH, LING ZQ et al. Eects of inhibiting melatonin biosynthesis on spatial memory retention and tau
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60. YOO DY, KIM W, LEE CH et al. Melatonin improves
D-galactose-induced aging eects on behavior, neurogenesis,
and lipid peroxidation in the mouse dentate gyrus via increasing pCREB expression. J Pineal Res 2012; 52:2128.
61. LUE LF, WALKER DG, BRACHOVA L et al. Involvement of microglial receptor for advanced glycation endproducts (RAGE)
in Alzheimers disease: identication of a cellular activation
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62. HAYDEN MS, GHOSH S. Signaling to NF-kappaB. Genes Dev
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63. GRANIC I, DOLGA AM, NIJHOLT IM et al. Inammation and
NF-kappaB in Alzheimers disease and diabetes. J Alzheimers
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64. MAQBOOL A, LATTKE M, WIRTH T et al. Sustained, neuronspecic IKK/NF-KB activation generates a selective neuroinammatory response promoting local neurodegeneration with
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65. LAN Z, LIU J, CHEN L et al. Danggui-Shaoyao-San ameliorates cognition decits and attenuates oxidative stress-related
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85

Ali et al.
Prof. Myeong Ok Kim for her kind and expert supervision, as she is the corresponding author and holds all the
responsibilities related to this manuscript.

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Melatonin prevents D-galactose neurotoxicity

35. LOVELL M, MARKESBERY W. Oxidative DNA damage in mild
cognitive impairment and late-stage Alzheimers disease.
Nucleic Acids Res 2007; 35:74977504.
36. HAMILTON A, HOLSCHER C. The eect of ageing on neurogenesis and oxidative stress in the APPswe/PS1deltaE9 mouse
model of Alzheimers disease. Brain Res 2012; 1449:8393.
37. WU DC, JACKSON-LEWIS V, VILA M et al. Blockade of microglial activation is neuroprotective in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine mouse model of Parkinson
disease. J Neurosci 2002; 22:17631771.
38. GAO HM, LIU B, ZHANG W et al. Novel anti-inammatory
therapy for Parkinsons disease. Trends Pharmacol Sci 2003;
24:395401.
39. BENNER EJ, MOSLEY RL, DESTACHE CJ et al. Therapeutic
immunization protects dopaminergic neurons in a mouse
model of Parkinsons disease. Proc Natl Acad Sci USA 2004;
101:94359440.
40. SALMINEN A, OJALA J, KAUPPINEN A et al. Inammation in
Alzheimers disease: amyloid-[beta] oligomers trigger innate
immunity defence via pattern recognition receptors. Prog
Neurobiol 2009; 87:181194.
41. TOURNIER C, HESS P, YANG DD et al. Requirement of JNK
for stress-induced activation of the cytochrome c-mediated
death pathway. Science 2000; 288:870874.
42. LIU J, LIN A. Role of JNK activation in apoptosis: a doubleedged sword. Cell Res 2005; 15:3642.
43. YEH SL, WU TC, CHAN ST et al. Fructo-oligosaccharide
attenuates the production of pro-inammatory cytokines and
the activation of JNK/Jun pathway in the lungs of D-galactose-treated Balb/cJ mice. Eur J Nutr 2014; 53:449456.
44. EMINEL S, KLETTNER A, ROEMER L et al. JNK 2 translocates
to the mitochondria and mediates cytochrome c release in PC
12 cells in response to 6-hydroxydopamine. J Biol Chem
2004; 279:5538555392.
45. CHAITANYA GV, ALEXANDER JS, BABU PP. PARP-1 cleavage
fragments: signatures of cell-death proteases in neurodegeneration. Cell Commun Signal 2010; 8:31.
46. SCHMUD LC, HOPKINS KJ. Fluro-Jade B: a high anity uorescent marker for the localization of neuronal degeneration.
Brain Res 2000; 874:123130.
47. de IULIIS A, GRIGOLETTO J, RECCHIA A et al. A proteomic
approach in the study of an animal model of Parkinsons disease. Clin Chim Acta 2005; 357:202209.
48. ZHANG Q, LI X, CUI X et al. D-Galactose injured neurogenesis in the hippocampus of adult mice. Neurol Res 2005;
27:552556.
49. SRIKANTH V, MACZUREK A, PHAN T et al. Advanced glycation
endproducts and their receptor RAGE in Alzheimers disease.
Neurobiol Aging 2011; 32:763777.
50. BIERHAUS A, HUMPERT P, MORCOS M et al. Understanding
RAGE, the receptor for advanced glycation end products. J
Mol Med 2005; 83:876886.

51. CALABRESE V, CORNELIUS C, CUZZOCREA S et al. Hormesis,

cellular stress response and vitagenes as critical determinants
in aging and longevity. Mol Aspects Med 2011; 32:279304.
52. TERAI K, MATSUO A, MCGEER PL. Enhancement of immunoreactivity for NF-kappa B in the hippocampal formation and
cerebral cortex of Alzheimers disease. Brain Res 1996;
735:159168.
53. CUI X, ZUO P, ZHANG Q et al. Chronic systemic D-galactose
exposure induces memory Loss, neurodegeneration, and oxidative damage in mice: protective eects of R-a-lipoic acid. J
Neurosci Res 2006; 83:15841590.
54. BARCO A, MARIE H. Genetic approaches to investigate the
role of CREB in neuronal plasticity and memory. Mol Neurobiol 2011; 44:330349.
55. SHANKAR GM, WALSH DM. Alzheimers disease: synaptic dysfunction and Ab. Mol Neurodegener 2009; 4:13264.
56. CANAS PM, PORCIUNCULA LO, CUNHA GM et al. Adenosine
A2A receptor blockade prevents synaptotoxicity and memory
dysfunction caused by b-amyloid peptides via p38 mitogenactivated protein kinase pathway. J Neurosci 2009; 29:14741
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