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Introduction
Neuronal apoptosis sculpts the developing brain.
How-ever, inappropriate apoptosis has been suggested to play a
potentially important role in the pathogenesis of various
neurodegenerative disorders, including Alzheimer's disease,
Parkinson's disease, amyotrophic lateral sclerosis, and
Huntington's disease[1,2]; it has also been implicated in other
types of neurological disorders such as cerebral
ischemia[3]. It is of a great importance to establish therapeutic strategies
to combat apoptosis-induced damage to the central nervous
system and to elucidate the underlying mechanisms.
Thermal preconditioning (TP) can result in the
acquisition of tolerance. It means tissues and cells are subjected to
sub-lethal heat stress to obtain the ability to withstand
subsequent usually lethal stresses. Previous studies have shown
that thermal preconditioning is able to promote neuronal
survival under various types of stress. Although the
underlying mechanisms are usually ascribed to the increased
expression of one or more heat shock proteins
(HSP)[4], some studies have documented that the overexpression of HSP
fails to rescue the neurons from apoptosis induced by heat
or ischemic stress[5]. So the precise mechanisms are still
unclear.
Some evidence suggests that the activation of Akt is
enhanced immediately after heat
stress[6,7]. Akt, a serine/threonine kinase, plays a critical role in regulating neuronal
cell survival responses. Akt is a downstream effector of
phosphatidylinositol 3-kinase (PI3-K), and the
phosphorylated form has been demonstrated to maintain cell survival
by inactivating several apoptosis effectors such as BAD,
forkhead transcription factors, caspase 9 and glycogen
synthase kinase 3b (GSK3b)[8,9]. Moreover, the overexpression
of constitutive Akt by the transfection of active Akt cDNA
into cells can rescue the cells from apoptosis induced by
various types of stress[10]. These findings have led to the
hypothesis that a rise in the activation level of Akt
contributes to the TP-induced neuroprotective effect. However,
how the activation of the PI3-K/Akt pathway is involved in
the TP-induced neuroprotective effect has not been examined.
Cultured cerebellar granule neurons (CGN) from early
postnatal rats represent a highly homogeneous neuron
population. CGN survive and develop characteristics of
mature CGN when maintained in depolarizing concentrations of
potassium (25 mmol/L) medium, but undergo apoptosis
characterized by chromatin condensation and DNA
fragmentation when cultured in physiological low potassium (5
mmol/L) conditions[11]. The low potassium-induced cerebellar
granule neuronal apoptotic paradigm has been proposed as a
suitable in vitro model for studying the mechanisms of
neuronal apoptosis involved in many neurodegenerative
diseases due to its excellent stability.
In this study, we investigated whether the activation of
the PI3-K/Akt pathway was required for thermal
preconditioning to protect CGN against apoptosis induced by low
potassium. Furthermore, we explored the possibility of a link
between the upregulated HSP70 expression and Akt
activation in the acquisition of neuroprotection induced by
thermal preconditioning. We provide a new pathway to
elucidate the mechanisms of the acquisition of tolerance induced
by thermal preconditioning.
Materials and methods
Materials All chemicals, unless otherwise noted, were
purchased from Sigma Chemicals (Sigma-Aldrich, St Louis,
MO, USA). LY294002 was purchased from Calbiochem (San
Diego, CA, USA). Primary antibodies against total Akt,
phospho-Ser473Akt, GSK3b, or phospho-Ser9GSK3b,
secondary antibodies, including horseradish peroxidase
(HRP)-linked anti-rabbit antibody and HRP-linked anti-mouse
antibody and HRP-conjugated anti-biotin antibody were
purchased from Cell Signaling Technology (Beverly, MA, USA).
The primary antibody against HSP70 was from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). The primary
antibody against b-actin was from Neomarkers (Fremont, CA,
USA).
Cell culture and treatment Rat cerebellar granule
neurons were prepared as described by Yan et
al[12]. The cerebella were obtained from Sprague-Dawley rat pups
(postnatal 7_8 d). They were mechanically disrupted, then
trypsinized with 0.025% trypsin (including 0.01% DNase I)
for 15 min at 37 °C. Trypsin digestion was stopped by the
addition of trypsin inhibitor (0.05%). After trituration,
cerebellar granule neurons were plated in poly-lysine-coated
24-well or 35-mm culture plates at a density of
(1.5_1.8)×109 cells/L in basal modified Eagle's (BME) medium containing
10% fetal bovine serum, 25 mmol/L KCl, 2 mmol/L glutamine,
100 kU/L penicillin, and 100 mg/L streptomycin, and
incubated at 37 °C with 5% CO2 in a humidified chamber.
Cytosine arabinoside (10 µmol/L) was added to the medium
24 h after plating to arrest the growth of non-neuronal cells.
D-glucose (5 mmol/L) was added to the cultures on d 6. On
d 8, in vitro CGN were rinsed twice and maintained in 25K
medium (containing 25 mmol/L serum-free KCl) to be used in
the experiments. To induce neuronal apoptosis, CGN were
switched to 5K medium (containing 5 mmol/L serum-free KCl)
for 24 h.
TP CGN were incubated at 43.5 °C with 5%
CO2 in a humidified chamber for 90 min and recovered at 37 °C with
5% CO2 in a humidified chamber for 1 h before low potassium
(5K) treatment[13].
Drug treatment The PI3-K inhibitor, LY294002, was
dissolved in dimethyl sulfoxide (DMSO) as a 1000×50 µmol/L
stock solution and then added to 25K medium; the final
concentration of LY294002 was 5,10, or 20 µmol/L according to
different experiments. The 25K medium with 0.2% DMSO
was used as the control. In the preliminary experiments, we
found that DMSO concentrations up to 0.3% did not affect
neuronal viability of CGN within 120 h of exposure. However,
unless otherwise stated, the final concentration of DMSO
was 0.2%. LY294002 was added to the medium 1 h before
thermal preconditioning.
To examine the role of PI3-K/Akt in the thermal
preconditioning-mediated survival protection of CGN, we designed
an experiment on CGN with a specific PI3-K inhibitor,
LY294002. Thus, CGN were divided into 5 groups as follows:
control (25K group), 5K treatment (5K group), thermal
preconditioning+5K treatment (H+5K group), 20
µmol/L LY294002+thermal preconditioning+5K treatment (LY+H+5K
group), and the 20 μmol/L LY294002+25K (LY+25K group)
groups. The LY+H+5K group was treated with LY294002 for
1 h before thermal preconditioning (at 43.5 °C for 90 min,
then at 37 °C for 1 h) and was then switched to 5K medium.
The LY+25K group was treated with LY294002 for 3.5 h and
was then switched to 25K medium. Cells cultured in 24-well
plates were used to perform MTT assay or fluorescein
diacetate (FDA) staining for evaluating survival
cells. Four wells were used for each group. Cells cultured in 35-mm
plates were used for Hoechst 33258 staining, agarose gel
electro-phoresis, or Western blot analysis. All experimental
procedures were performed according to the Biomedical
Security Rule.
MTT assay and FDA staining for evaluating survival
cells 3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide
(MTT) assay was performed as described
previously[14]. Briefly, the neurons in the 24-well plates were cultured and
treated according to the above methods. MTT was added to
the medium at a final concentration of 1 mg/mL and the cells
were incubated at 37 °C for 4 h. Then the medium was
removed. DMSO was then applied to the wells to dissolve
the formazan crystal. The absorbance of the samples was
measured at a wavelength of 570 nm with 630 nm as
reference wavelength using the Universal Microplate Reader (ELX
800, Bio-Tek, Winooski, Vermont, USA). Values are expressed
as a percentage of control cultures for each experiment. For
FDA staining, as described
previously[12], CGN were incubated with fluorescein diacetate (10 mg/L) at 37 ºC for 5 min
and then examined and randomly photographed by
fluorescence microscope at ×200 magnification. The number of
neurons was counted from the photos by a blind observer.
Neuronal survival was calculated by the following formula:
neuronal survival (%)=(survival cells in the treated
group/survival cells in the control group)×100%.
Hoechst 33258 staining for detecting chromatin
condensation Chromatin condensation was detected by nucleus
staining with Hoechst 33258 as described
previously[12]. CGN grown in the 35-mm dishes were washed with ice-cold
phosphate-buffered saline (PBS) and fixed with 4% formaldehyde
in PBS. Cells were then stained with Hoechst 33258 (5 mg/L)
for 10 min at room temperature. Nuclei were visualized using
a fluorescence microscope at ×1000 magnification. In this
way, apoptotic CGN would be stained into bright blue
because of their chromatin condensation, while normal CGN
were stained to a slight blue colour.
Agarose gel electrophoresis for detecting DNA
fragmentation Genomic DNA was isolated from neurons grown
in poly-lysine-coated 35-mm tissue culture dishes. Cells were
rinsed with BME, scraped, collected in ice-cold PBS, and
centrifuged at 5000×g for 5 min. The pellet was lysed in 600
μL of a buffer consisting of 10 mmol/L Tris-HCl, 10 mmol/L
edetic acid and 0.2% Triton X-100 (pH 7.5). After 15 min on
ice, the lysate was centrifuged at 12 000×g at 4 ºC for 10 min.
The supernatant was extracted first with phenol and then
with phenol-chloroform: isoamylalcohol (24:1). The
aqueous phases were mixed with a 1/10 volume of 3 mol/L sodium
acetate (pH 5.2) and an equal volume of ice-cold isopropanol
for 24 h at -20 °C. The pellet was washed with 70% ethanol,
air-dried and dissolved in 15 μL of Tris-HCl 10 mmol/L plus 1
mmol/L edetic acid (pH 7.5). After digesting RNA with RNase
A (0.6 g/L, at 37 ºC for 30 min), the sample was
electrophoresed in 1.5% agarose gel with TBE buffer. DNA was
visualized by ethidium bromide staining.
Western blot analysis Western blot analysis was
performed as described previously and slightly
modified[15]. In brief, neurons were harvested in a cell lysis buffer (Pierce,
Rockford, IL, USA). After incubation on ice for 15 min and
centrifugation at 18 000×g at 4 °C for 10 min, whole protein
concentrations were determined by BCA assay (Pierce, USA),
using bovine serum albumin as a standard. Cell lysates were
diluted in the SDS sample buffer, and the mixture was boiled
for 5 min. The protein (30 µg) was separated on a 12%
SDS-PAGE. Proteins were transferred to the nitrocellulose
mem-branes. The membranes were incubated for 60 min at room
temperature in Tris-buffered saline containing 0.05%
Tween-20 (TBST) and 5.% nonfat dry milk (NFDM) and then
overnight at 4 ºC with specific antibodies for phospho-Ser473Akt
(1:1000), phospho-Ser9 GSK3β (1:1000), Akt (1:1000),
GSK3β (1:1000), or HSP70 (1:500). The membranes were then washed
in TBST (3×5 min) and incubated for 60 min in
TBST/1%NFDM containing a 1:2000 dilution of a HRP-conjugated
secondary antibody. This was followed by washes (3×15
min) in TBST and subsequently was developed with an
enhanced chemiluminescence system (Pierce, USA). If
necessary, the blots were then incubated in stripping buffer
(67.5 mmol/L Tris, pH 6.8, 2% SDS, and 0.7% b
mercapto-ethanol) at 50 ºC for 30 min and then reprobed with
antibodies against b-actin (1:1000) as loading controls.
Quantification was performed with the Bio-Rad Quantity One software
(Hercules, CA, USA). All data from 3 independent
experiments were expressed as the ratio to optical density values
of the corresponding controls for the statistical analyses.
Statistical analysis Data are presented as mean±SD
from independent experiments. For the Western blots, each
experiment was repeated at least 3 times, and in all cases, the
same results were obtained. Statistical analysis of data was
performed by Student's t-test. P<0.05 was considered
significant.
Results
TP protects CGN against apoptosis induced by low
potassium To investigate the effects of TP on apoptosis of
CGN induced by low potassium, at 24 h after 5K treatment,
CGN were stained by Hoechst 33258, a classical way of
identifying apoptotic cells, to test nuclei morphology of neurons.
The results indicated that nuclei of most CGN in the 5K group
were stained bright blue; the nuclei of most CGN in 25K
group and H+5K group showed a slight blue (Figure 1).
Another biochemical feature of apoptosis, DNA fragmentation,
was also tested. We found that CGN exposed to 5K medium
for 24 h showed a typical internucleosomal DNA
fragmenta-tion, while no ladders of oligonucleosomal length DNA were
detected in CGN of the 25K group and the H+5K group
(Figure 2). The results demonstrated that TP protected CGN
against apoptosis induced by low potassium.
LY294002 inhibits the neuroprotective effect on CGN
induced by TP To examine the role of the PI3-K/Akt pathway
in the TP-mediated protection of CGN, we performed an
experiment on CGN with LY294002. Thus, CGN were divided
into 5 groups as described in Materials and methods. At 24
h after the treatment with 5K, cell viability was determined
by MTT assay and FDA staining. Compared with the H+5K
group, cell viability in the LY294002+H+5K group decreased
significantly (P<0.01); however, compared with the 25K
group, cell viability in the LY294002+25K group did not
decrease (P>0.05; Figure 3). The results revealed that LY294002
potently inhibited the neuroprotective effect of TP against
low potassium-induced apoptosis of CGN.
TP induces Akt and GSK3b phosphorylation in a
PI3-K-dependent manner To determine whether the activation of
the PI3-K/Akt pathway was involved in the neuroprotective
activity of TP against low potassium-induced apoptosis of
CGN, after TP following the pretreatment of different
concentrations of LY294002 (5, 10, and 20 µmol/L) for 1 h, cells
were harvested for immuno-blotting. TP resulted in a robust
activation of Akt and an inactivation of GSK3b as indicated
by increased phospho-Ser473-Akt and increased
phospho-Ser9-GSK3b (P<0.05), whereas total Akt and total
GSK3b did not change (P>0.05). Furthermore, LY294002 decreased
TP-induced phosphorylation of Akt and GSK3b
dose-dependently (P<0.05, Figure 4). The results revealed that TP
activated Akt and subsequently inactivated GSK3b via PI3-K.
TP induced Akt and GSK3b phosphorylation in 5K
cultures If the activation of PI3-K/Akt pathway by TP protects
CGN against apoptosis induced by low potassium at 24 h, its
activation may persist for 24 h. So cells of the 5K group, the
H+5K group, and the LY294002+H+5K group were harvested
24 h after the treatment of 5K for immunoblotting. Compared
with the 5K group, TP induced Akt and GSK3b
phosphorylation persistently. Even at the time point of 24 h after
exposure to 5K, the levels of phospho-Ser473 Akt and
phospho-Ser9 GSK3b of CGN in the H+5K group were still much higher
than that of CGN in the 5K group (P<0.05).
Moreover,LY294002 significantly inhibited Akt and
GSK3b phosphorylation induced by TP in 5K cultures
(P<0.05; Figure 5).
Inhibition of the PI3-K/Akt pathway did not affect the
upregulated HSP70 expression induced by TP The above
results show that the activation of the PI3-K/Akt pathway
was required for TP to protect CGN against apoptosis
induced by low-potassium. Our previous report showed that
IP-induced HSP70 expression was also involved in
TP-mediated anti-apoptotic action on
CGN[13]. To explore the possibility of a link between the upregulated HSP70 expression
and the activation of Akt in the acquisition of neuroprotection
induced by TP, we examined the effect of LY294002 (20 µmol/L)
on the upregulated HSP70 expression induced by TP. The
result revealed that TP apparently increased the levels of
HSP70 (P<0.01); however, LY294002 (20 µmol/L) had no
effect on the increased levels of HSP70 induced by TP
(P>0.05; Figure 6).
Discussion
Neuronal apoptosis resulting from a variety of severe
stresses has been emphasized in the pathogenesis of
numerous neurodegenerative diseases. Mobilizing intrinsic
defensive mechanisms to deal with apoptosis is important for
protecting neurons. TP leads to the phenomenon of thermal
tolerance in which sub-lethal heat stress induces robust
protection against subsequent, potentially lethal heat
stress[16]. Much evidence has shown that TP not only produces
thermal tolerance[17], but also provides the brain and neurons
with a protective response against subsequent various
lethal insults such as ischemia and hypoxia
injury[18,19], glutamate
excitotoxicity[20,21] and toxicity of
1-methyl-4-phenylpyridinium ion[22]
etc. The present study also shows that TP protects CGN against low potassium-induced
apoptosis.
Usually the production of HSP following TP, such as the
HSP70 protein which misfolded or damaged chaperone, is
regarded as one of the critical mechanisms used to bolster
cellular defenses to support neuronal survival in the face of
potentially lethal conditions[23,24]. However, the other
survival-promoting mechanisms besides HSP may also be involved in the TP-mediated neuroprotective effect. The
PI3-K/Akt signaling pathway is considered a classic
pro-survival pathway. PI3-K phosphorylates Akt to give rise to
phosphorylated Akt. The activation of Akt supports cell
survival and counteracts apoptotic signaling, a part of which
relies on the inactivation of GSK3b by converting
phosphorylated GSK3b[25]. In the present study, TP induced the
activation of Akt and the inactivation of GSK3b in a
PI3-K-dependent manner. The inhibition of the PI3-K/Akt pathway by
LY294002 reduced the neuroprotective effect of TP against
low potassium-induced neuronal death; moreover, the
activation of Akt and the inactivation of GSK3b by TP persisted
for 24 h during the treatment of low potassium. So our
findings support the hypothesis that the activation of the
PI3-K/Akt pathway by TP protects CGN against apoptosis induced
by low potassium. Although previously there were reports
of heat stress modulating the activity of the PI3-K/Akt
pathway[7], the present study demonstrates that the activation of
the PI3-K/Akt pathway is required for the TP-induced
neuro-protective effect. Our findings support that TP is a potential
neuroprotective means for neurodege-nerative diseases since
the activation of the PI3-K/Akt pathway is widely involved
in anti-apoptotic action in these diseases.
It has been reported that the upregulated HSP70
expression induced by TP participates in the TP-mediated
neuro-protective effect[13]. Is there a link between the upregulated
HSP70 expression and the activation of PI3-K/Akt pathway
in the acquisition of neuroprotection induced by TP
Considering that neuroprotection by Akt can be mediated
through the downstream induction of HSP70
expression[26], and in renal cell carcinoma (RCC4) cells LY294002 largely
attenuates increased HSP70 expression toward heat
treatment[27], we speculate in this assay that HSP70 might be the
downstream target of Akt and that LY294002 attenuates
TP-induced neuroprotection, probably through inhibiting
increased HSP70 expression. Our data reveals that TP
resulted in the upregulated HSP70 expression, but LY294002
exerted no effects on the TP-induced upregulated HSP70
expression. This means that neuroprotection by the IP-
induced PI3-K/Akt pathway activation was not mediated
through the downstream induction of HSP70 expression in
the present study. Contradictory evidence concerning a
link between Akt activity and HSP70 expression may be due
to cell-specific differences in the signaling pathways that
are recruited. So the possibility of a link between Akt
activity and HSP70 expression in the acquisition of
neuroprotec-tion induced by thermal preconditioning remains to be
clarified.
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