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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Antagonizing -amyloid peptide neurotoxicity of the

anti-aging fungus Ganoderma lucidum

Cora Sau-Wan Lai a , Man-Shan Yu a , Wai-Hung Yuen d , Kwok-Fai So a,b,c ,

Sze-Yong Zee e , Raymond Chuen-Chung Chang a,b,c,
a
Laboratory of Neurodegenerative Disease, Department of Anatomy, The University of Hong Kong, Pokfulam, Hong Kong
b
Research Centre of Heart, Brain, Hormone and Healthy Aging, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong
c
State Key Laboratory of Brain and Cognitive Science, The University of Hong Kong, Pokfulam, Hong Kong
d
Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong
e
Department of Botany, The University of Hong Kong, Pokfulam, Hong Kong

A R T I C LE I N FO AB S T R A C T

Article history: Ganoderma lucidum (Leyss. ex Fr.) Karst. (Lingzhi) is a medicinal fungus used clinically in
Accepted 31 October 2007 many Asian countries to promote health and longevity. Synaptic degeneration is another
Available online 13 November 2007 key mode of neurodegeneration in Alzheimer's disease (AD). Recent studies have shown the
loss of synaptic density proteins in each individual neuron during the progression of AD. It
Keywords: was recently reported that -amyloid (A) could cause synaptic dysfunction and contribute
Ganoderma lucidum to AD pathology. In this study, we reported that aqueous extract of G. lucidum significantly
Synaptotoxicity attenuated A-induced synaptotoxicity by preserving the synaptic density protein,
-amyloid synaptophysin. In addition, G. lucidum aqueous extract antagonized A-triggered DEVD
Synaptophysin cleavage activities in a dose-dependent manner. Further studies elucidated that
Alzheimer's disease phosphorylation of c-Jun N-terminal kinase, c-Jun, and p38 MAP kinase was attenuated by
G. lucidum in A-stressed neurons. Taken together, the results prove a hypothesis that
anti-aging G. lucidum can prevent harmful effects of the exterminating toxin A in AD.
2007 Elsevier B.V. All rights reserved.

1. Introduction 2006). Increasing lines of evidence have shown that G. lucidum

Aging is one of the risk factors for various chronic diseases,
including cancers, cardiovascular disease and neurodegenera-
tive diseases. Recently, herbal medicine has been widely
investigated for developing preventive measure against aging-
related disease. The oriental fungus, Ganoderma lucidum (Leyss.
ex Fr.) Karst. (Lingzhi), has been used for centuries by Asian
people to promote health and increase longevity. G. lucidum has
been shown to prevent chronic diseases in clinical practice
(Kwok et al., 2005; Tang et al., 2005; Chen et al., 2006; He et al.,
has immunoregulatory (Hong et al., 2004; Chen et al., 2006) and
anti-tumor activities (Sliva et al., 2002; Jiang et al., 2004a,b; Sliva,
2006). The G. lucidum extract can promote the healing of acetic
acid-induced ulcers in rats (Gao et al., 2004). Also, it can reduce
plasma cholesterol levels (Berger et al., 2004). While G. lucidum
has long been known to exhibit a wide spectrum of biological
effects, its effect on the nervous system has not been sufficiently
explored and even less is known about its effects in aging-
related neurodegenerative diseases. Although only few studies
have investigated the effect of G. lucidum in the nervous system,

Corresponding author. Department of Anatomy, Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Hong Kong. Fax: +852
2817 0857.
E-mail address: rccchang@hkucc.hku.hk (R.C.-C. Chang).

0006-8993/$ see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2007.10.103
216 BR A I N R ES E A RC H 1 1 9 0 ( 2 00 8 ) 2 1 5 22 4

results from these studies suggested that G. lucidum has sedative 2.3. Effects of GLA against A neurotoxicity
effects in vivo (Chu et al., 2007), neuroprotective effects against
oxidative stress in vitro (Zhao et al., 2004, 2005) and has the effect From the results in Fig. 1, it can be concluded that GLA pre-
to induce neuronal differentiation (Cheung et al., 2000). There- serves synapses by attenuating the down-regulation of
fore, G. lucidum may have beneficial effects on age-associated synaptophysin. In order to explore antagonizing effects of
diseases. In view of the fact that neurodegeneration is the key GLA against A peptide, primary cultured neurons were
problem in AD, we aim to investigate if G. lucidum attenuates A exposed to 25 M of A2535 peptide. DEVD-cleavage activity
neurotoxicity. was used to assess caspase-3-like activity. Colorimetric
In addition to neuronal apoptosis, synaptic degeneration
has been recognized as an important mode of neurodegenera-
tion. Synaptic dysfunction and loss of synapse in AD are well
documented (Scheff and Price, 2003; Coleman and Yao, 2003;
Gylys et al., 2004; Reddy et al., 2005). Several synaptic density
proteins including synaptophysin (Masliah et al., 1989; Shi-
mohama et al., 1997; Reddy et al., 2005), synaptotagmin (Yoo
et al., 2001; Reddy et al., 2005) and PSD-95 (Gylys et al., 2004) are
reduced in the AD brain. As a result, synaptic strength for
normal neurotransmission is markedly weakened. Base on
these neurodegenerative mechanisms involved in AD and the
anti-aging characteristics of G. lucidum, we hypothesized that
extract from G. lucidum could preserve synaptic protein as well
as antagonize A peptides neurotoxicity.

2. Results

2.1. Effect of GLA on preserving synapse in A

peptide-treated neurons

Loss of synaptic connection (synaptic degeneration) is one of the

key modes of neurodegeneration in AD. In order to allow the
establishment of an extensive neurite network in primary
cortical neuronal culture and to maintain fasciculation of
neurons, neurons on day 14 in vitro were used for the experiment.
A2535 at 10 M was used to induce synaptic degeneration
without triggering neuronal apoptosis. Immunofluorescent
staining of synaptophysin was examined by multiphoton
confocal microscopy. Figs. 1a and d showed that the synapto-
physin immunoreactivity was evenly distributed in the control
and 500 g/ml GLA-treated group. However, the immunoreactiv-
ity of synaptophysin was markedly decreased along the neuritis
in A2535-treated neurons (Fig. 1b). In addition, the staining of
synaptophysin was not evenly distributed. Instead, patches of
aggregated staining of synaptophysin were observed along the
neurites. Pre-treatment of GLA inhibited down-regulation of
synaptophysin immunoreactivity induced by A2535 (Fig. 1c).

2.2. Effects of GLA against neuritis damage by A peptide
The morphology and neurite network of the A2535-treated
group was significantly different when compared to the primary
cortical neurons in control group. As shown in Fig. 2b, the
neurite network was destroyed at 25 M A2535-treated
group as highlighted by a white circle. Shrinkage of the cell
body could also be observed in A2535-treated group. Pre-
treatment of GLA could preserve the neurite network at high
dose (100 g/ml and above in Figs. 2h, j and l). As shown in
Fig. 2, the morphology of neurons exposed to GLA was well
preserved corresponding to the antagonizing effects in dose-
dependent manner.
Fig. 1 GLA inhibits A-induced down-regulation of
synaptophysin immunoreactivity. Cultured cortical neurons
(14 DIV) were treated with various concentrations of GLA for
1 h, and then subjected to A2535 (10 M) for 24 h (ad).
Micrographs are multiphoton confocal laser scanning images
stained with synaptophysin. (e) Quantitative analysis of
mean intensity of synaptophysin immunofluorescence
staining (mean pixel intensity). p < 0.001 vs. control; **p < 0.05
treated with A peptide per se. Significant difference between
different groups were compared by one-way ANOVA for
multiple comparison and StudentNewmanKeuls test as
post hoc test. Pictures are representative of three independent
experiments. Scale bar = 20 m.
 BR A I N R ES E A RC H 1 1 9 0 ( 2 00 8 ) 2 1 5 2 24 217

caspase-3-like activity assay was applied to evaluate the effect

of GLA against A2535 neurotoxicity. A peptide induced
DEVD-cleavage activity to 3.2 0.1-fold of control (Fig. 3). Pre-
treatment of GLA at 250 g/ml significantly (p < 0.001) attenu-
ated the A2535-induced DEVD-cleavage activity to 1.6 0.1-
fold of control. Furthermore, GLA reduced A2535 toxicity in a
dose-dependent manner. 500 g/ml of GLA showed similar

Fig. 3 Antagonizing effect of GLA on A-induced cytotoxicity.

Cultured cortical neurons were treated with different
concentrations of GLA for 1 h, followed by a 24-h exposure to
A2535 (25 M). Afterwards, colorimetric DEVD-cleavage
activity assays were determined by the absorbance (at 405 nm)
of the yellow product (pNA) cleaved from the substrate. Results
were expressed as mean SE from at least three independent
experiments. *p< 0.001 vs. the group treated with A peptide
per se. Significant difference between various groups was
compared by one-way ANOVA for multiple comparison and
StudentNewmanKeuls as post hoc test.

neuroprotective effect (1.7 0.1-fold of control) without any

significant toxicity in this high dosage.

2.4. Effect of GLA on the number of apoptotic bodies in

neurons exposed to A1 42 peptide

In order to maximize the neuroprotective effect of GLA, 500 g/ml

of GLA was used in the following experiments to further confirm
whether GLA could antagonize A peptide toxicity. We used
another method, TUNEL staining, to evaluate the number of
apoptotic bodies. In addition, we used full-length A142 to con-
firm results obtained from A2535. Numbers of TUNEL-positive
neurons were counted under a fluorescent microscope. Five
microscopic fields were counted. Total counts of each treatment
were at least 300 neurons. Representative pictures of the TUNEL
staining are shown in Figs. 4a to d. As shown in Fig. 4e, the
quantitative results of the apoptotic neurons counts, A142
peptide increased the percentage of apoptotic neurons to 1.51
0.09-fold of control. Exposure to GLA at 500 g/ml significantly

Fig. 2 Morphology of neurons after treatment with GLA and

A. Cultured cortical neurons were pre-incubated with GLA for
1 h, followed by a 24-h exposure to A2535: (a) control, (b) A2535
(25 M), (c) GLA (10 g/ml), (d) GLA (10 g/ml)+A2535 (25 M),
(e) GLA (50 g/ml), (f) GLA (50 g/ml)+A2535 (25 M), (g) GLA
(100 g/ml), (h) GLA (100 g/ml)+A2535 (25 M), (i) GLA
(250 g/ml), (j) GLA (250 g/ml)+A2535 (25 M), (k) GLA
(500 g/ml), (l) GLA (500 g/ml)+A2535 (25 M). The circled
areas indicate the destroyed neurite network. Pictures are
representative of three independent experiments.
218 BR A I N R ES E A RC H 1 1 9 0 ( 2 00 8 ) 2 1 5 22 4

Fig. 4 GLA inhibits A142-triggered apoptosis. Cultured cortical neurons were treated with 500 g/ml of GLA for 1 h, and then
subjected to A142 (20 M) for 24 h. TUNEL staining for DNA breaks was performed to distinguish normal and apoptotic cells
under fluorescent microscopy. Panels a to d show representative pictures of the TUNEL staining. Panel e show quantitative
analysis of the percentage of apoptotic neurons. Results were expressed as mean SE from at least three independent
experiments. p < 0.001 vs. control; **p < 0.05 vs. the group treated with A peptide only by one-way ANOVA for multiple
comparison and StudentNewmanKeuls as post hoc test.
 BR A I N R ES E A RC H 1 1 9 0 ( 2 00 8 ) 2 1 5 2 24
Fig. 5 Western blot analysis of phospho-JNK,
phospho-c-Jun I, phospho-c-Jun II, phospho-ERK and
phospho-p38 MAPK in neurons treated with GLA. Cultured
cortical neurons were pretreated with GLA (10, 100 or 500
g/ml) for 1 h, followed by exposure to A2535 peptide
(25 M) for 2 h. Proteins extracted were subjected to Western
blot analysis to detect the level of phospho-JNK,
phospho-c-Jun I (Ser73), phospho-c-Jun II (Ser63), phospho-p38
MAPK, total JNK, total c-Jun, total p38 MAPK and -actin. -
Actin was used as the internal control. Results were obtained
from at least three independent experiments.
(p < 0.05) reduced the number of apoptotic bodies triggered by
A142 peptide to 1.19 0.05-fold of control.
2.5. Effect of GLA on activation of stress kinases triggered
by A peptide
To investigate whether GLA antagonized A peptide by mod-
ulating stress kinases, Western blot analysis of their phosphor-
ylation state was performed. Similar to our findings and other
findings, neurons exposed to A2535 peptide for 2 h exhibited a
significant increase in phosphorylation of JNK I, c-Jun, and p38
MAPK (Fig. 5). Pre-treatment of GLA markedly attenuated the
levels of phosphorylated JNK, phosphorylated c-Jun, and
phosphorylated p38 MAPK in a dose-dependent manner. The
GLA alone showed a dose-dependent decrease in phospho-p38.
This phenomenon implied that the low dose (10 g/ml) and high
dose (500 g/ml) of GLA had different effects on signaling
pathways.
3. Discussion
In the present study, we have provided evidence by showing
that GLA antagonizes A peptide neurotoxicity including
synaptotoxicity as well as neuronal apoptosis. We further
demonstrate that antagonizing effects of GLA are mediated by
modulating the activation of stress kinases (JNK and p38 MAP
kinase) triggered by A peptide.
219
Recent reports suggest that polysaccharide isolated from
G. lucidum is responsible for its anti-tumor properties (Sliva,
2006). G. lucidum has been shown to inhibit proliferation and
induce apoptosis of prostate, colon, liver and breast cancer
cells (Sliva et al., 2002; Hsu et al., 2002; Lin et al., 2003; Hong
et al., 2004; Jiang et al., 2004a,b). It has been shown that
G. lucidum down-regulates phosphorylation of Akt and is
followed by the inhibition of NF-B activity in breast cancer
cells (Jiang et al., 2004a), suggesting its modulation on survival
signaling pathways. In addition, G. lucidum can activate JNK
and p38 in hepatoma cells and dendritic cells (Lin et al., 2003;
Lin, 2005). G. lucidum seems to exhibit different effects on
different cell types. Hsu's laboratory has shown that poly-
saccharide extracted from G. lucidum inhibits spontaneous
and Fas-mediated apoptosis in human neutrophils via activa-
tion of the phosphatidylinositol 3 kinase/Akt signaling path-
way (Hsu et al., 2002, 2003). All the above reports suggest that
G. lucidum exhibits differential effects to benefit our body and
the effects depends on cell type and tissue. Apart from the
anti-tumor effects, a report has shown that G. lucidum poly-
saccharide induces neuronal differentiation (Cheung et al.,
2000). A recent animal study shows that aqueous extracts
from G. lucidum exert sedative effects by decreasing sleep
latency and increases sleep time in pentobarbital-treated rats
via a GABAergic mechanism (Chu et al., 2007). In addition,
Lin's group has reported that G. lucidum polysaccharide re-
duces hypoxia/reoxygenation-induced oxidative stress on
cortical neurons (Zhao et al., 2004, 2005). A did not exhibit
its toxicity by oxidative stress in our model. Our previous
study showed that A could not significantly induce ER stress
(Yu et al., 2006a,b), which implies that oxidative stress may not
mainly contribute to the toxicity. Therefore, the antagonizing
effects of GLA against A peptide should not mediate via anti-
oxidation.
G. lucidum has been used clinically in many Asian countries
to prevent and even treat of different aging-related diseases. A
randomized, double-blind, placebo-controlled study in the
Chinese population has shown that G. lucidum can effectively
reduce the clinical score of fatigue (Tang et al., 2005). Another
clinical study done in Hong Kong has shown that there was no
significant changes in routine coagulation screen, fibrinogen
concentration, von Willebrand ristocetin cofactor activity,
platelet function and thrombelastography activity in healthy
volunteers receiving G. lucidum orally for 4 weeks (Kwok et al.,
2005). Therefore, this study suggests that G. lucidum has no
adverse effect on healthy people and that it is safe for clinical
trial. In terms of its anti-cancer effects, an open label study in
Asian countries has shown that G. lucidum can increase
mitogenic response for lymphocytes but decrease production
of cytokines suggesting that G. lucidum is a promising adjunct
approach for cancer treatment (Chen et al., 2006). The results
presented in this study together with the clinical trials on
other diseases suggests that animal model of G. lucidum
should be done in order to investigate the neuroprotective
effects of G. lucidum against A peptide toxicity. One of the
concerns on animal model of Chinese medicine in the nervous
system is whether the herbal extracts could pass through the
blood brain barrier to exert its effect. The recent animal study
shows the sedative effects of G. lucidum aqueous at a dose of
120 mg/kg. Although the report did not investigate whether
220 BR A I N R ES E A RC H 1 1 9 0 ( 2 00 8 ) 2 1 5 22 4
G. lucidum could pass through the bloodbrain barrier, the
results suggested that aqueous G. lucidum extracts could exert
its effect on the nervous system (Chu et al., 2007). Indeed,
polysaccharides from other medicinal herbs have been shown
to affect the CNS (Chang and So, in press). In the present study,
a maximum dosage of 500 g/ml GLA was used to prove that
there is no toxicity even in such high dosage and it that is still
preserved the neuroprotective effect of GLA. Indeed, both
250 g/ml and 500 g/ml dosages of GLA showed similar
prominent effects against A toxicity, which suggested that
lower dosage could be used with the preservation of its
neuroprotective effect on nervous system.
Synaptic degeneration has been recognized as an impor-
tant mode of neurodegeneration. Synaptic dysfunction and
loss of synapse in AD are well documented (Scheff and Price,
2003; Coleman and Yao, 2003; Gylys et al., 2004; Reddy et al.,
2005). Several synaptic proteins including synaptophysin
(Masliah et al., 1989; Shimohama et al., 1997; Reddy et al.,
2005), synaptotagmin (Yoo et al., 2001; Reddy et al., 2005) and
PSD-95 (Gylys et al., 2004) have been reported to be decreased
in AD brain. Synaptophysin is one of the most abundant pre-
synaptic vesicle proteins (Phelan and Gordon-Weeks, 1992),
which can be served as a synaptic marker to evaluate the
synaptotoxicity of A in the present study. Our results demon-
strate for the first time that G. lucidum attenuated A-induced
synaptotoxicity by preserving a synaptic density protein,
synaptophysin, in cultured neurons. In the present study,
synaptophysin is not only a marker for functional synapse; but
also a marker for the axonal vesicle transport. A not only
decreases the number of synaptophysin immunoreactivity
along neurites, but also induces aggregation and accumula-
tion of synaptophysin. The aggregation and accumulation of
synaptophysin may account for the blockage of axonal trans-
port of synaptic vesicles. A recent report shows that the stress
signaling pathway of JNK is involved in axonal transport. This
report found that nitric oxide inhibits the axonal transport of the
synaptic vesicle precursor containing synaptophysin, while
inhibition of JNK phosphorylation can resume the axonal trans-
port of synaptophysin in hippocampal neuron (Stagi et al., 2005).
We report here that GLA can significantly inhibit the phosphor-
ylation of JNK in A-stressed neuron. Inhibiting the phosphor-
ylation of JNK by GLA may also explain the attenuation of A-
induced blockage of synaptophysin transport.
It has been shown that A peptide, triggers activation of
caspase-3 resulting in neuronal apoptosis (Harada and Sugi-
moto, 1999; Chang et al., 2002a,b; Suen et al., 2003; Lin et al.,
2004). From the biochemical assay of caspase-3-like activity
and TUNEL staining to access apoptosis, GLA significantly
antagonizes A-induced neuronal apoptosis. The results sug-
gest that GLA can indeed provide beneficial effects on neurons
against both synaptotoxicity as well as neuronal apoptosis
elicited by A peptide.
Activation of stress kinases such as JNK, ERK and p38 MAP
kinase has been demonstrated to be associated with AD in
both in vitro and in postmortem AD brain (Anderson et al.,
1994; Hensley et al., 1999; Zhu et al., 2000, 2001; Atzori et al.,
2001; Medina et al., 2005; Chong et al., 2006). Upon A peptide
stimulation, JNK are activated by phosphorylation (Troy et al.,
2001; Morishima et al., 2001; Chang et al., 2002a,b; Yu et al.,
2005; Lai et al., 2006). It has been demonstrated that activation
of JNK was found in AD autopsied brain samples (Zhu et al.,
2001; Savage et al., 2002). Activation of ERK has also been
found in vitro and postmortem AD brain tissue (Dineley et al.,
2001; Medina et al., 2005; Chong et al., 2006). Inhibition of ERK
kinase, MEK by U0126 can reduce A peptide-mediated neu-
ronal cell death (Medina et al., 2005; Chong et al., 2006). p38
MAP kinase is another major MAP kinase responsive to
cellular and environmental stresses. Activation of p38 has
also been described in AD (Hensley et al., 1999; Zhu et al., 2000;
Atzori et al., 2001). While activation of these signaling path-
ways are involved in survival and/or differentiation (Encinas
et al., 1999; Cheung et al., 2000; Lambeng et al., 2003; Canon
et al., 2004; Almeida et al., 2005), these signaling pathways can
also be considered pro-apoptotic (Anderson et al., 1994;
Hensley et al., 1999; Zhu et al., 2000, 2001; Atzori et al., 2001;
Medina et al., 2005; Ito et al., 2006; Chong et al., 2006; Amadoro
et al., 2006). From the Western blot analysis (Fig. 5). GLA at
500 g/ml significantly inhibited A-induced activation of JNK,
and p38 MAP kinase. Furthermore, c-Jun as a substrate of JNK
also has implications in neurodegeneration of AD (Anderson
et al., 1994). GLA significantly attenuated the phosphorylation
levels of c-Jun at both serine 67 and serine 73 phosphorylation
sites in A-stressed neurons (Fig. 5). These results demon-
strate that GLA suppresses A toxicity by inhibiting the
apoptotic signaling including JNK-c-Jun, p38 MAP kinase
signaling pathways. It is important to note that the effects of
G. lucidum vary in different cells. Nevertheless, inhibition of
pro-apoptotic signaling pathways in neurons by G. lucidum is
somewhat similar to what have been found in human
neutrophils (Hsu et al., 2002, 2003).
With the advance in medical treatment, the average life
expectancy is continuously increasing and the aged popula-
tion dramatically increases in accordance. To provide good
quality of life to the elderly is now one of the biggest challenges
the pharmaceutical industry faces. Many herbal medicines
have been widely used for their well-known anti-aging effects.
The search for anti-aging herbal medicine for preventive
measures or even disease-modifying agent may open a new
avenue for interventions in neurodegenerative disease. Pre-
viously, our group has demonstrated that another anti-aging
herb, Lycium barbarum, also exhibits neuroprotective properties
in vivo (Chan et al., 2007) and in vitro (Yu et al., 2006a,b, 2007a,b;
Ho et al., 2007). The findings regarding the neuroprotective
effects of G. lucidum makes it yet another candidate in the
development of anti-aging drugs from herbal medicine.
Taken together, our results suggest that GLA exhibits bene-
ficial effects on neurons by antagonizing A peptide neuro-
toxicity. GLA inhibits JNK, ERK and p38 MAP kinase signaling
pathways. It also markedly attenuates the down-regulation
of synaptophysin immunoreactivity in A-stressed neurons.
G. lucidum may antagonize A synaptotoxicity prior to the
activation of apoptosis. The results indicate that anti-aging
herbal medicine can preserve neurons in AD. G. lucidum has
been widely used as plant drug for many years, it has few
undesirable side effects and is considered to be a functional
food in many Asian countries (Kwok et al., 2005). These results
further demonstrate that anti-aging herbal medicine around
the world contains a large pool of potential disease-modifying
agent for the treatment of aging-related neurodegenerative
diseases.
 BR A I N R ES E A RC H 1 1 9 0 ( 2 00 8 ) 2 1 5 2 24
4. Experimental procedures
4.1. Materials
The fruiting bodies of G. lucidum (Fr.) Karst were cultivated and
collected in the Shanxi province of the People Republic of
China. The fungus was purchased in a local Shanghai market
and identified by Professor X.-L. Huang by comparison with a
voucher specimen (No. 317), previously deposited in the her-
barium of the Phytochemistry Department of the Shanghai
Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai, People's Republic of China.
Culture medium for neuronal cells was purchased from
Gibco-BRL (Grand Island NY, USA). A peptides (both 142 and
2535 fragments) were obtained from Sigma-Aldrich (Saint
Louis, USA). Caspase-3 substrate (Ac-DEVD-pNA), protease in-
hibitor cocktail, phosphatase inhibitor cocktail and stauros-
porine were obtained from Calbiochem (La Jolla, CA, USA). In
situ cell death detection kit was purchased from Roche (Roche
Diagnostics, Mannheim, Germany). Rabbit polyclonal anti-JNK,
rabbit polyclonal anti-phosphorylated JNK (threonine-183 and
tyrosine-185), rabbit polyclonal anti-phosphorylated c-Jun
(serine 73), rabbit polyclonal anti-phosphorylated c-Jun (Serine
63) II, rabbit anti-c-Jun, rabbit polyclonal anti-phosphorylated
p44/42 MAPK (threonine-202 and tyrosine-204), were obtained
from Cell Signaling Technology (Beverly, MA, USA). Mouse
anti-synaptophysin antibody was obtained from Calbiochem.
Mouse anti--actin monoclonal antibody was purchased from
Sigma-Aldrich. Horseradish peroxidase-conjugated goat anti-
rabbit and goat anti-mouse antibodies were purchased from
DAKO (Glostrup, Denmark). Goat anti-mouse Alexa Fluor-568
antibody was obtained from Invitrogen (Molecular Probes,
Eugene, OR, USA). PVDF membrane was purchased from Bio-
Rad (Richmond, CA, USA). Biomax X-ray film was from Kodak
(Tokyo, Japan). Enhanced chemiluminescence (ECL) detection
kit was from Amersham-Biosciences (Buckinghamshire, UK).
4.2. Isolation of GLA from G. lucidum
The G. lucidum aqueous extract GLA was isolated according to
the previous procedure (Bao et al., 2002). Briefly, the air-dried
G. lucidum fruit bodies (1 kg) were defatted with 95% alcohol
and extracted with 20 l of boiling water for 6 h. The aqueous
extract was filtered and treated with trichloroacetic acid at
4 C to remove proteins. The resulting aqueous fraction was
dialyzed against running water for 2 days (molecular weight
cutoff: 30005000 Da). The retentate was concentrated to a
volume of 300 ml under reduced pressure and treated with
four volumes of 95% ethanol. The resulting precipitate was
isolated by centrifugation and then washed sequentially with
ethanol and acetone. The product was vacuum-dried at 40 C
to yield a brownish powder [GLA, yield: 3.4% (w/w)]. The
chemical analysis revealed that GLA contained 68.1% carbo-
hydrate and 20.9% protein (Fang et al., 2005a,b). Furthermore,
the glycosyl composition analysis indicated that galactose
and glucose (1.0:8.3) were the major carbohydrate found in
GLA. Triterpenes were not detected in GLA by HPLC or
visualization by UV. GLA has also been examined by endotoxin
test (E-toxate, Sigma) and was found to be free of endotoxin.
221
4.3. Primary cortical culture
Primary culture for cortical neurons were prepared from day 17
embryonic Sprague-Dawley rats (Laboratory Animal Unit, The
University of Hong Kong, Hong Kong, SAR) as described
elsewhere (Chang et al., 2002a,b; Suen et al., 2003; Lin et al.,
2004; Yu et al., 2004, 2005, 2006a,b, 2007a,b; Lai et al., 2006; Ho
et al., 2007). Briefly, the meninges were removed and the cortices
were mechanically dissociated in PBS supplemented with
glucose (18 mM). Neurons were then plated onto poly-L-lysine
(25 g/ml)-coated 6-well plate (2 106 cells per well) or 12-well
plate with coverslip (3 105 cells per well), respectively. Neurons
were cultured with Eagle's minimum essential medium (GIBCO-
BRL, Rockville, MO, USA) supplemented with 5% heat-inacti-
vated fetal bovine serum, glucose (18 mM), L-glutamine (2 mM),
insulin (5 g/ml), progesterone (0.02 M), putrescine (100 M),
selenium (30 pM), penicillin/streptomycin (100 units and 100 g/
ml) at 37 C in a humidified 5% CO2 atmosphere. Neurons were
cultured for 7 or 14 days prior to treatment.
4.4. Preparation of fribillar A peptides
Both A2535 and A142 were prepared by dissolving in water at
concentrations of 2 mg/ml and 0.2 mg/ml, respectively. A
peptide solution was incubated at 37 C for 7 days to allow fibril-
lation. After incubation, peptides were stored at 20 C before use.
4.5. Treatment protocol
To investigate GLA neuroprotective effects against A toxicity,
primary culture for cortical neurons at 7 days in vitro were
treated with GLA at various concentrations for 1 h and fol-
lowed by exposure to A peptides for 24 h. After treatment, cell
lysates were collected for caspase activity assay or Western
blot analysis. DAPI staining was performed after fixation of
treated neurons. For immunostaining, cortical neurons at
14 days in vitro were treated with GLA at 500 g/ml for 1 h,
followed by exposure to A (10 M) for 24 h. Neurons were
seeded onto 6-well plate for caspase-like activity assay and
Western blot analysis, and 12-well plate for DAPI staining and
immunocytochemical analysis.
4.6. Assay for caspase-like activity
After treatments, neurons were washed with ice-cold Tris-
buffered saline (pH 7.4), and then harvested according to our
published methods (Chang et al., 2002a,b; Suen et al., 2003; Lin
et al., 2004; Yu et al., 2004, 2005, 2006a,b, 2007a,b; Fang et al.,
2005a; Lai et al., 2006; Ho et al., 2007). Briefly, 60 g of cellular
proteins were used for determining catalytic activity of
caspases. Substrate for caspase-3 (Ac-DEVD-pNA) was incu-
bated with protein extract for 2 h at 37 C to yield p-nitroanilide
in yellow color, of which absorbance was measured by
spectrophotometric reader at 405 nm. We have tested that
the extract per se did not affect the assay.
4.7. TUNEL staining
After treatment, cultured neurons were fixed in methanol at
20 C for 20 min and permeabilized in 0.1% Trition X-100 at
222 BR A I N R ES E A RC H 1 1 9 0 ( 2 00 8 ) 2 1 5 22 4
room temperature for 5 min. Neurons were blocked in 10% BSA
for 1 h. In situ cell death detection kit was used for the TUNEL
staining following the protocol from manufacturer. Pictures
were captured under fluorescence microscope. Data were
obtained from three independent experiments and expressed
as the fold of control of percentage of apoptotic bodies. Five
microscopic fields were counted. Total counts of each treat-
ment were at least 300 neurons.
4.8. Immunofluorescence staining
Neurons were rinsed with PBS, fixed in methanol at 20 C for
20 min and permeabilized in 0.1% Triton X-100 at room tempe-
rature for 5 min. Neurons were blocked in 10% BSA and incubated
with anti-synaptophysin (1:500) for 2 h. After washing, neurons
were incubated with Alexa 568-conjugated goat-anti mouse
(1:500) for 1 h. Neurons were mounted with Antifade-mounting
medium (Molecular Probes). Optical section images and z-stacks
of images were obtained with a multiphoton confocal laser
scanning microscope (LSM-Meta510; Zeiss, Jena, Germany)
using a plan apochromat 63/1.4 numerical aperture oil lens.
4.9. Western blot analysis
After treatment, cellular proteins form culture of cortical
neurons were extracted as reported elsewhere (Chang et al.,
2002a,b; Suen et al., 2003; Lin et al., 2004; Yu et al., 2004, 2005,
2006a,b, 2007a,b; Fang et al., 2005b; Lai et al., 2006; Ho et al., 2007).
Briefly, extracted proteins were separated by SDSpolyacryla-
mide gel electrophoresis and were then transferred onto PVDF
membrane (Bio-Rad). The membrane was blocked with 5%
nonfat milk in Tris-buffered saline (pH 7.4) containing Tween-20
(0.1%) for 1 h. Afterwards, the membrane was incubated with
the rabbit anti-JNK (1:1000), rabbit anti-phosphorylated JNK
(threonine-183 and tyrosine-185) (1:1000), rabbit anti-phos-
phorylated c-Jun (serine 73) I (1:1000), rabbit anti-phosphory-
lated c-Jun (Serine 63) II (1:1000), rabbit anti-c-Jun (1:1000), rabbit
anti-phosphorylated p38 MAPK (threonine-180 and tyrosine-
182) (1:1000), rabbit anti-p38 MAPK, or mouse anti--actin
(1:5000) for 2 h at room temperature and subsequently with
horseradish peroxidase-conjugated secondary antibody (1:2000)
for 1 h at room temperature. Bands were developed and
visualized on Biomax X-ray film using ECL plus kit.
4.10. Statistical analysis
Data for multiple variable comparisons were analyzed by one-
way analysis of variance (ANOVA). For the comparison of
significance, StudentNewmanKeuls test were used as post
hoc comparison according to a statistical program SigmaStat
(Jandel Scientific, Chicago, IL, USA). Results are expressed as
the mean standard error (S.E.) from at least three indepen-
dent experiments.
Acknowledgments
The authors would like to thank for the technical support from
Ms. Dorothy Chan. The project is supported by HKU Strategic
Research Theme on Healthy Aging, HKU Seed funding for Basic
Research (200611159206), earmarked Competitive Research
Grant from Research Grant Council of Hong Kong (HKU 7699/
05M, 7552/06M). CSWL is grateful to the support of studentship
from the Graduate School and MSY is supported by the
postdoctoral fellowship from The University of Hong Kong.
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