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Induction of Bcl-2 and Bax was related to hyperphosphorylation of tau and neuronal death induced by okadaic acid in rat brain.

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THE ANATOMICAL RECORD PART A 287A:1236 –1245 (2005)
Induction of Bcl-2 and Bax Was
Related to Hyperphosphorylation of
Tau and Neuronal Death Induced by
Okadaic Acid in Rat Brain
LI-QIN CHEN, JIAN-SHE WEI, ZHI-NIAN LEI, LING-MEI ZHANG, YAN LIU,
AND FENG-YAN SUN*
National Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan
University, Shanghai, China
ABSTRACT
Abnormal hyperphosphorylation of the cytoskeletal protein tau is a characteristic feature
of neurodegeneration in Alzheimer’s disease (AD) brain. Okadaic acid (OA), a protein phosphatase inhibitor, induces neuronal death and hyperphosphorylation of tau. In the present
study using a model of microinjection of OA into rat frontal cortex, we aimed to investigate
if OA-induced hyperphosphorylation of tau and neuronal death are related to the expression
of Bcl-2, an apoptosis inhibitor, or Bax, an apoptosis inducer. Immunohistochemistry and
Western blot analysis showed that OA injection dose- and time-dependently induced the
expression of Bcl-2 and Bax protein in the surrounding of OA injection areas, which were
similar with that of AT8 immunostaining, a marker of hyperphosphorylated tau. However,
the ratios of Bcl-2 over Bax had a negative relationship to the expression of AT8. Furthermore, double fluorescent staining showed that AT8-positive neurons mainly costained with
terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick-end labeling,
a marker of DNA damage, indicating that tau hyperphosphorylation may be associated with
DNA damage in the neurons of rat brain. In the areas more adjacent to the OA injection site,
most neurons with AT8-positive staining showed vulnerability to OA toxicity and could be
triple-stained with Bcl-2 and Bax or double-stained with Bcl-2. However, in the areas further
from the OA injection site, neurons with few AT8-positive staining showed resistance to OA
toxicity and only stained with Bcl-2, but not Bax. The results suggest that the ratios of Bcl-2
over Bax expression may have an effect on tau hyperphosphorylation and neuronal death
following OA injection. © 2005 Wiley-Liss, Inc.
Key words: okadaic acid; apoptosis; tau phosphorylation; Bcl-2; Bax
Alzheimer’s disease (AD) is characterized by neuronal
loss and two pathologic hallmarks, amyloid deposits and
neurofibrillary tangles (NFTs) (Vickers et al., 2000). It has
been shown that the number of NFTs correlates closely
with the degree of dementia (Arriagada et al., 1992). NFTs
are composed of abnormally hyperphosphorylated microtubule-associated protein (MAP) tau (Arriagada et al.,
1992). Tau, mainly residing in neuronal axons, is phosphorylated at very low level in normal adult brain (Iqbal
et al., 2000). Abnormal hyperphosphorylation of tau has
been considered as a key pathologic molecular change
correlated with neurodegeneration in AD brain (Iqbal et
al., 2000). Hyperphosphorylated tau fails to bind microtubules, leading to disassembly of microtubules, disruption
Grant sponsor: National Key Project of Basic Science Research
and Shanghai Metropolitan Fund for Research and Development;
Grant number: G1999054007 and 04DZ00415.
The first 2 authors contributed equally to this paper.
*Correspondence to: Feng-Yan Sun, National Laboratory of
Medical Neurobiology, Shanghai Medical College of Fudan Uni-
versity, 138 Yi Xue Yuan Road, Shanghai 200032, China, Fax:
86-21-64174579. E-mail: fysun@shmu.edu.cn
Received 6 July 2004; Accepted 10 June 2005
DOI 10.1002/ar.a.20241
Published online 1 November 2005 in Wiley InterScience
(www.interscience.wiley.com).
©
2005 WILEY-LISS, INC.
INDUCTION OF Bcl-2 AND Bax
of axonal transport, and ultimately retrograde neurodegeneration (Iqbal et al., 2000).
Regulation of tau phosphorylation by protein kinases
(PKs) and protein phosphatases (PPs) maintains dynamic
balance under normal physiological conditions. Dephosphorylative activity of PP is the prerequisite in vivo for
keeping tau at low phosphorylated level and with biological functions (Kins et al., 2001). PP activity has been
reported to reduce by about 30% in AD brain compared to
control (Gong et al., 1993, 1995). It has also been elucidated that defect of PP activity mimics Alzheimer-like tau
pathology, including aberrant phosphorylation, altered
compartmentalization, and colocalization with ubiquitin
(Kins et al., 2001).
Okadaic acid (OA), an inhibitor of PP, has been a useful
tool to study AD (Bialojan and Takai, 1988). Treatment
neurons with OA in vitro (Arias et al., 1993; Merrick et al.,
1997; Tanaka et al., 1998; Kim et al., 1999) or in vivo
(Arendt et al., 1995, 1998; Nelson and Saper, 1996; Mudher and Perry, 1998) induced aberrant hyperphosphorylation of tau or neuronal death or both. Previous studies
showed that apoptosis was involved in neuronal death
induced by OA or other inhibitors of PP in vitro (Boe et al.,
1991; Nuydens et al., 1998; Fladmark et al., 1999) and in
vivo (Arendt et al., 1998). Bcl-2 and Bax, belonging to
Bcl-2 family, are important apoptotic regulatory molecules. The two, with contrary functions, integrate signals
transducted from extracellular or intracellular insults at
mitochondrial level (Yang et al., 1997; Hengartner, 2000).
Their ratio has been suggested to be able to decide the
cell’s fate under certain conditions. The two molecules
both have widespread neuronal expression in the central
nervous system (CNS) (Krajewski et al., 1994; Merry and
Korsmeyer, 1997). Previous studies have proposed that
Bcl-2 and Bax play important roles in neuronal vulnerability and resistance to degeneration in many CNS diseases, including AD (Krajewski et al., 1995; Martin, 1999;
Vila et al., 2001; Raghupathi et al., 2002). However, it is
not well defined if Bcl-2 and Bax are involved in the
pathphysiological process of Alzheimer-like neuronal
death associated with hyperphosphorylation of tau.
In the present study, we first aimed to demonstrate that
OA could induce neurodegeneration associated with hyperphosphorylation of tau with a model of microinjection
of OA into rat cerebral cortex. Furthermore, with the
model we explored whether the expression of Bcl-2 or Bax
proteins was associated with hyperphosphorylation of tau
induced by OA. Hyperphosphorylation of tau was assayed
by AT8 antibody recognizing the aberrant phosphorylation site at serine 202, threonine 205 of tau.
MATERIALS AND METHODS
Animals
All animal experiments were approved by the Medical
Experimental Animal Administrative Committee of
Shanghai and carried out in accordance with the National
Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize animal
suffering and to reduce the number of animals used. Male
Sprague-Dawley rats from Experimental Animal Center
of Chinese Academy of Science were used for this study.
Animals were anesthetized with chloral hydrate. For immunochemistry and fluorescence immunostaining, animals were transcardially perfused with 0.9% saline solution followed by fixative (4% paraformaldehyde in 0.1 M
1237
phosphate buffer) after anesthetization with chloral hydrate. After perfusion, the brains were removed and postfixed in the same fixative for 8 hr, then immersed in 20%
and 30% sucrose solution in 0.1 M phosphate buffer until
sinking. Coronal serial sections were cut at 30 ␮m thickness on a freezing microtome and stored in a cytoprotectant solution at ⫺20°C. For Western blot analysis, the
cortex tissues were quickly collected on ice, frozen immediately in dry ice, and kept at ⫺70°C.
Application of Okadaic Acid
Saline (vehicle) or saline containing OA (pH 7.4; Sigma)
was injected into the right frontal cortex of rats using
stereotaxic surgery (stereocoordinates: A, 1.20 mm for anterior-posterior; L, 1.90 mm for lateral; H, 2.20 mm for
dorsoventral). Rats were injected with 0.5 ␮L of saline or
saline containing 10, 20, 50, or 100 ng OA and sacrificed at
1 day. For Western blot analysis, injection of 50 ng OA was
omitted. In addition, rats were sacrificed at 3 hr, 6 hr, 12
hr, 1 day, 3 days, or 7 days after 20 ng OA injection. For
Western blot analysis, 6-hr group was not set. The number
of animals used was four in each dose group of doseresponse or each time point group of time-course analysis.
Immunohistochemistry
Three sections (Bregma 1.20 mm) from each rat brain
were randomly selected for immunochemistry. Free-floating sections from each rat brain were fixed in 4% paraformaldehyde for 10 min, followed by three rinses in 0.01 M
phosphate-buffered saline (PBS). Endogenous peroxidase
activity was quenched by exposing the sections to 0.3%
H2O2 for 30 min. After blocking with 10% normal goat
serum for 30 min at 37°C, the sections were incubated
with a mouse monoclonal AT8 antibody (1:60; Innogenetics, Belgium), with a rabbit polyclonal antibody against
Bcl-2 (1:60; Oncogene Science), or with a mouse monoclonal antibody against Bax (1:40; Oncogene Science) in 0.01
M PBS containing 1% normal goat serum and 0.3% Triton
X-100 for 2 hr at 37°C and overnight at 4°C. The sections
were then rinsed and incubated for 1 hr at 37°C with
antimouse or biotinalated antirabbit IgG (1:200; ABC kit,
Vector Lab) in 0.01 M PBS containing 1% normal goat
serum and 0.3% Triton X-100. Subsequently, they were
incubated for 1 hr at 37°C with avidin-biotin-horseradish
peroxidase complex (1:200; ABC kit, Vector Lab) following
rinses. Immunostaining was visualized with 0.05% diaminobenzidene (Sigma) as chromogen. Negative control sections received identical treatment except for exposure to
the primary antibodies and showed no specific staining.
Double Staining
Sections (Bregma 1.20 mm) from rats sacrificed at 1 day
after 20 ng OA injection were doubly stained for AT8
and terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick-end labeling (TUNEL). Following incubation with a mouse monoclonal AT8 antibody
as mentioned above, free-floating sections were rinsed
with 0.01 M PBS and incubated with antimouse IgGrhodamine (1:20; Roche) for 1 hr at 37°C and rinsed again.
After that, the sections were incubated with 20 ␮g/ml
protease K in 10 mM Tris-HCl (pH 7.6) for 15 min at 37°C.
After rinsing with 0.01 M PBS, the sections were incubated with 0.1% Triton X-100 in 0.1% sodium citrate for 15
min and rinsed again. Then the sections were incubated
1238
CHEN ET AL.
with TUNEL reaction mixture (in situ cell death detection
kit; Boehringer Mannheim, Germany) composed of 45 ␮l
TUNEL label solution conjugated with fluorescein and 5
␮l TUNEL enzyme for 90 min at 37°C. Negative control
section for TUNEL staining was incubated with 50 ␮l
TUNEL label solution replacing TUNEL reaction mixture.
The fluorescence signals were observed under confocal
laser scanning microscopy (TCS NT; Leica, Germany).
Triple Staining
Sections (Bregma 1.20 mm) from rats sacrificed at 1 day
after 20 ng OA injection were triply stained for AT8, Bcl-2,
and Bax. Free-floating sections were incubated for 2 hr at
37°C and overnight at 4°C in a mixture of two primary
antibodies raised in separate species: a rabbit polyclonal
antibody against Bcl-2 (1:60) and a mouse monoclonal AT8
antibody (1:60). Sections were then rinsed three times
with 0.01 M PBS for 5 min each time and incubated for 1
hr at 37°C with the secondary antibodies, an antirabbit
IgG-rhodamine (1:20; Roche), and an antimouse IgG-fluorescein isothiocyanate (FITC; 1:20; Vector Lab). After
three rinses with 0.01 M PBS, sections were incubated
with a mouse monoclonal antibody against Bax (1:40) for
2 hr at 37°C and overnight at 4°C. Thereafter, sections
were incubated with the secondary antibody, an antimouse IgG-CY5 (1:100; Amersham) for 1 hr at 37°C. The
fluorescence signals were observed under the same microscopy mentioned in double staining. The images were
captured from two kinds of areas. One was the area immediately surrounding the OA injection site and called the
more adjacent area. The other was the region relatively
far from the OA injection site and called the less adjacent
area. The edges of the more adjacent area were 0.42 mm
apart from the injection site. The less adjacent area was
the region of frontal cortex excluding the more adjacent
area.
Cell Counting
As to each rat brain, three sections (Bregma 1.20 mm)
immunostained were used for cell counting. Bcl-2-positive
cell counts in affected brain areas by OA injection were
measured with the aid of the image processing and analysis system. Counting of AT8- and Bax-positive cells in
affected brain areas by OA injection was done under 400⫻
light microscopy. All values were expressed as mean ⫾
SEM. For AT8, Bcl-2, and Bax immunostaining at each
time point after 20 ng OA injection, data were statistically
analyzed with unpaired Student’s t-test. For AT8, Bcl-2,
and Bax immunostaining at 1 day after serial doses of OA
injection, the amount was evaluated by one-way ANOVA,
followed by unpaired Student’s t-test. Statistical significance was set at P ⬍ 0.05.
Western Blot Analysis
The samples were homogenized in lysis buffer (50 mM
Tris-HCl, pH 7.5, containing 150 mM NaCl, 1% Nonidet
P40, 0.5% sodium deoxycholate, 1 ␮g/ml aprotinin, and
100 ␮g/ml PMSF). Protein concentration was measured by
a Bio-Rad protein assay. Each sample (80 ␮g protein) was
boiled at 100°C in SDS sample buffer for 5 min, electrophoresed on 15% SDS-PAGE gels, and then transferred to
polyvinyldifluoridine (PVDF) membrane (Bio-Rad). The
membrane was incubated sequentially with rabbit polyclonal antibody against Bcl-2 (1:1,000; Santa Cruz Bio-
technology), mouse monoclonal antibody against Bax (1:
1,000; Santa Cruz Biotechnology), and mouse monoclonal
antibody against β–isoform of actin (1:1,000; Sigma) overnight at 4°C. The membrane was washed with TBS containing 0.1% Tween 20, incubated with horseradish peroxidase-conjugated goat antirabbit IgG (1:1,000; Santa
Cruz Biotechnology) and goat antimouse IgG (1:1,000;
Santa Cruz Biotechnology) at room temperature for 60
min and washed three times (15 min each) with TBS/
Tween 20. Western blotting luminal reagent (Santa Cruz
Biotechnology) was used to visualize peroxidase activity.
Controls for nonspecific binding were determined by omission of the primary antibody.
RESULTS
Neuronal damage induced by OA was observed using
cresyl violet staining. Figure 1A–D showed damaged neuronal morphologic changes in ipsilateral cortex after OA
injection. Mechanical neuronal loss and cell debris caused
by injection cannula was obvious after OA or vehicle (saline) injection (Fig. 1A and B). The affected areas by OA
injection were markedly larger than those by vehicle injection (Fig. 1A and B). In the surrounding of vehicle
injection areas, most neurons presented regular morphological characteristics (Fig. 1C). In the surrounding of OA
injection areas, however, many neurons displayed cytoplasmic swelling and inflammatory cells increased (Fig.
1D).
TUNEL staining is a marker for DNA double-strand
damage. Double immunofluorescent labeling of TUNEL
and AT8 was utilized to determine the relationship of tau
hyperphosphorylation and DNA damage caused by OA. It
was shown that TUNEL-positive staining existed in affected areas by OA injection and that most TUNEL staining colocalized with AT8 immunostaining (Fig. 1E).
Immunohistochemical method was used to detect the
dose-response features of AT8, Bcl-2, and Bax immunostaining in affected brain areas by OA injection. Figure 2
illustrated the similar dose-response features of AT8,
Bcl-2, and Bax immunostaining induced by OA. At 1 day
after serial doses of OA injection, neurons in injection
regions displayed AT8, Bcl-2, and Bax immunopositive
staining to different extent. Following vehicle or 10 ng OA
injection, only a few AT8-, Bcl-2-, and Bax-positive cells
existed in injection regions (Fig. 2). In contrast, a lot of
AT8-, Bcl-2-, and Bax-positive cells appeared following 20
ng (Fig. 2), 50 ng (data not shown), or 100 ng (Fig. 2) OA
injection. Moreover, 100 ng OA injection enlarged the
distributional range of AT8-, Bcl-2-, and Bax-positive cells
so that the positive cells presented not only in frontal
cortex but also in parietal cortex (Fig. 2). From microscopic observations, we also noticed that the distributional
range and the number of Bcl-2-positive cells always
seemed to be larger or more than that of the AT8- and
Bax-positive cells at 1 day following 20 ng (Fig. 2) and 50
ng (data not shown) OA injection (Fig. 2). The immunostaining-positive cells in affected brain areas by OA or
vehicle were counted. The results showed that 20, 50, and
100 ng OA injection significantly increased the number of
AT8-, Bcl-2-, and Bax-positive cells in affected brain areas
compared with vehicle and 10 ng OA injection (P ⬍ 0.05,
vs. vehicle and 10 ng OA; Fig. 3). Detailed data on the
dose-response are listed in the Table 1.
Immunohistochemical assay was also used to examine
the temporal changes of AT8, Bcl-2, and Bax immuno-
Fig. 1. Neuronal damage induced by okadaic acid (OA). A–D: Cresyl
violet staining showing damaged neuronal morphological changes after
OA injection. The arrows in the low-power photomicrographs (A and B)
correspond to those in the high-power photomicrographs (C and D). E:
Double staining for TUNEL (fluorescein) and AT8 (rhodamine) showing
that DNA damage occurred in neurons with AT8-positive immunostaining after OA injection.
1240
CHEN ET AL.
Fig. 2. Photomicrographs showing dose-response features of AT8, Bcl-2, and Bax immunostaining in
affected brain areas at 1 day after OA injection. The images were captured from the areas surrounding the
injection site. The injection site was immediately on the left edge of each picture.
staining in affected brain areas by 20 ng OA injection.
Microscopic observations indicated that AT8, Bcl-2, and
Bax immunostaining induced by OA (20 ng) had parallel
time-courses. In 20 ng OA injection regions, AT8, Bcl-2,
and Bax immunostaining all time-dependently changed.
The number of the three types of positive cells time-dependently increased at 3 hr, 12 hr, and 1 day, then decreased at 3 days (Fig. 4). AT8, Bcl-2, and Bax staining
were all strongest at 1 day and the numbers of all three
types of positive cells were largest at 1 day (Fig. 4). In
addition, we noted that AT8, Bcl-2, and Bax staining had
the intracellular location different from one another. AT8
staining first appeared in distal neurites (at 3 hr), then
gradually developed toward cell bodies along neuronal
processes (at 12 hr), eventually densely accumulated in
soma along single axon-like neurites and displayed NFTlike morphology (at 1 day; Fig. 4). Then AT8 staining
became weak and the positive neurites shortened or disappeared (at 3 days; Fig. 4). Bax staining was mostly
within neuronal soma and proximal neurites (Fig. 4),
1241
INDUCTION OF Bcl-2 AND Bax
Fig. 3. Dose-response features of AT8, Bcl-2,
and Bax immunostaining induced by OA. Rats
were sacrificed at 1 day after the doses indicated
of OA injection. Values were presented as the sum
of positive cells in affected areas per slice and
mean ⫾ SEM. Asterisk, significant difference from
corresponding vehicle and 10 ng OA; number
symbol, significant difference from corresponding
vehicle only (P ⬍ 0.05, one-way ANOVA followed
by Student’s t-test).
TABLE 1. The number of AT8-, Bcl-2- and Bax-positive cells (cells/slice; means ⴞ S.E.M.) appearing in affected
areas by the serial doses of OA injection at 1 d
OA (ng)
AT8
Bcl-2
Bax
0
10
20
50
100
24 ⫾ 3
21 ⫾ 5
23 ⫾ 6
71 ⫾ 8
162 ⫾ 38
44 ⫾ 12
270 ⫾ 48
484 ⫾ 44
175 ⫾ 29
270 ⫾ 70
553 ⫾ 90
173 ⫾ 46
377 ⫾ 62
435 ⫾ 42
249 ⫾ 44
whereas Bcl-2 staining always appeared within neuronal
perinuclear cell bodies (Fig. 4). Cell counting showed the
parallel time-courses of AT8, Bcl-2, and Bax immunostaining in affected areas by 20 ng OA injection. Compared with vehicle injection, 20 ng OA injection significantly increased the number of AT8-, Bcl-2-, and Baxpositive cells at 12 hr, peaked at 1 day, then declined at 3
and 7 days but remained above the basal level at 3 days
(12 hr, 1 day, 3 days; P ⬍ 0.05 vs. vehicle; Fig. 5). Detailed
data on the time-course analysis were presented in
Table 2.
Western blots were applied to analyze the changes in
protein levels of Bcl-2 and Bax after OA injection. Figure
6 showed that the protein levels of Bcl-2 and Bax dose- and
time-dependently changed. The protein expression of
Bcl-2 and Bax increased at 1 day after the serial doses of
OA injection (Fig. 6A). The protein levels of Bcl-2 and Bax
gradually increased and then decreased after injection of
20 ng OA (Fig. 6B).
Using triple immunofluoresent staining combined with
confocal laser scanning microscopic analysis, brain slices
from rats sacrificed at 1 day after 20 ng OA injection were
employed to illustrate the topographic relationship between Bcl-2, Bax, and AT8 immunostaining stimulated by
OA. In brain areas more adjacent to OA injection site,
where neurons showed vulnerability to OA toxicity, AT8
immunostaining was costained with Bcl-2 and Bax immunostaining or only with Bcl-2 immunostaining (Fig. 7). In
brain areas less adjacent to OA injection site, where neurons showed resistance to OA toxicity, OA only induced
positive staining of Bcl-2 but not that of Bax and AT8
(Fig. 7).
DISCUSSION
Our results demonstrated that OA could induce neurodegeneration associated with hyperphosphorylation of tau
in vivo. Cresyl violet staining showed that OA caused
neurodegenreation including neuronal damage and loss.
The colocalization of TUNEL staining and AT8 immunostaining illustrated that OA could induce neuronal DNA
damage, which was associated with hyperphosphorylation
of tau. The observations of neuronal death and hyperphosphorylation of tau induced by OA were consistent with
previous studies (Boe et al., 1991; Arendt et al., 1998;
Nuydens et al., 1998; Fladmark et al., 1999). The colocalization relationship between neuronal DNA damage and
hyperphosphorylation of tau supported a previous report
that cytoskeletal pathology including hyperphosphorylation of tau had a major role in OA-induced neuronal death
(Tanaka et al., 1998).
We examined the effects of OA on protein expression of
Bcl-2 and Bax and further compared their features of
time-course and dose-response with that of hyperphosphorylation of tau. In the present study, immunohistochemical observations illustrated that quite a few neurons basally expressed low level of Bcl-2 and Bax proteins in rat
frontal cortex. The results of both immunohistochemistry
and Western blot analysis showed that protein expression
of Bcl-2 and Bax was highly induced in rat frontal cortex
after OA injection and the induction had features of doseresponse and time-course. Moreover, immunohistochemical results indicated induction of Bcl-2 and Bax expression
changed dose- and time-dependently in parallel with hyperphosphorylation of tau stimulated by OA, suggestive of
1242
CHEN ET AL.
Fig. 4. Photomicrographs showing temporal changes of AT8, Bcl-2, and Bax immunostaining in affected
brain areas after OA (20 ng) injection.
INDUCTION OF Bcl-2 AND Bax
1243
Fig. 5. Time-courses of AT8, Bcl-2, and Bax
immunostaining induced by OA (20 ng). A–C respectively reflect time-courses of AT8, Bcl-2, and
Bax immunostainging. Data were expressed as the
sum of positive cells in affected areas per slice and
represent mean ⫾ SEM. Asterisk, double asterisk,
and number symbol: significant difference from
corresponding vehicle (asterisk, P ⬍ 0.01; double
asterisk, P ⬍ 0.001; number symbol, P ⬍ 0.05,
Student’s t-test).
certain association between hyperphosphorylation of tau
and the expression of Bcl-2 and Bax. In respect to doseresponse, these three types of immunostaining all remarkably increased at 1 day after 20, 50, and 100 ng OA
injection. As to time-course, all of them markedly in-
creased at 12 hr, peaked at 1 day, and reduced at 3 day but
remained above the basal level after 20 ng OA injection.
These results were significant in light of the possibility
that integration of apoptotic signals mediated by Bcl-2
and Bax was involved in neurodegenerative process
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CHEN ET AL.
TABLE 2. The number of AT8-, Bcl-2- and Bax-positive cells (cells/slice; means ⴞ S.E.M.) appearing in affected
areas by 20 ng OA injection at 12 h, 1 d and 3 d
12 h
AT8
Bcl-2
Bax
1d
3d
vehicle
OA
vehicle
OA
vehicle
OA
19 ⫾ 2
34 ⫾ 8
27 ⫾ 5
102 ⫾ 6
328 ⫾ 20
69 ⫾ 9
24 ⫾ 3
21 ⫾ 5
23 ⫾ 6
270 ⫾ 48
484 ⫾ 44
175 ⫾ 29
30 ⫾ 5
20 ⫾ 5
21 ⫾ 5
92 ⫾ 22
165 ⫾ 16
77 ⫾ 10
Fig. 6. Changes in protein levels of Bcl-2 and Bax after OA injection. Western blots showed that the protein levels of Bcl-2 and Bax altered
depending on dose (A) and time-course (B). The protein levels of Bcl-2 and Bax increased at 1 day after the doses indicated of OA injection (A). The
protein levels of Bcl-2 and Bax gradually increased and then decreased after injection of 20 ng OA (B).
caused by cytoskeletal pathology following PP inhibition
with OA. For the first time, it was clarified that apoptotic
regulatory molecules of Bcl-2 and Bax were highly induced
by PP inhibition. But the causes of induction of Bcl-2 and
Bax expression by OA were unclear now.
Sequentially, the topographic relationship between hyperphosphorylation of tau and inductive expression of
Bcl-2 and Bax was determined. Triple immunofluorescent
staining delineated that hyperphosphorylation of tau colocalized with the inductive expression of Bcl-2 and Bax or
only with the inductive expression of Bcl-2 in areas more
adjacent to OA injection site, where neurons were more
vulnerable to OA toxicity. However, only Bcl-2 immunostaining was seen, but neither AT8 nor Bax immunostaining in areas less adjacent to OA injection site, where
neurons were more resistant to OA toxicity. It has been
reported that Bax promotes neuronal death in many neurodegenerative processes and Bcl-2 supports neuronal
survival (Krajewski et al., 1995; Martin, 1999; Vila et al.,
2001; Raghupathi et al., 2002). Bax only appeared in areas
where neurons were more vulnerable to OA toxicity, suggesting that high induction of Bax by OA possibly participated in neuronal death associated with hyperphosphorylation of tau. We noted that there were more cells that
express Bcl-2 than those that express Bax. There was
substantial Bcl-2 expression even in cells that express
Bax. The phenomena were consistent with immunostaining observations that the number of Bcl-2-positive cells
was always more than that of Bax-positive cells after
injection of the same-dose OA at the same time point. It
has been proposed that the ratio of Bcl-2 and Bax is able
to decide a cell’s fate under certain condition. Whereas in
the areas less adjacent to OA injection site, neurons with
few AT8-positive staining showed resistance to OA toxicity and only stained with Bcl-2, but not Bax. It seemed an
attempt at cell survival existed after OA treatment. The
colocalization of highly expressed Bcl-2 and Bax possibly
reflected an active antagonism between the two proteins.
A previous report showed that Bcl-2 overexpression could
Fig. 7. Photographs showing relationship of AT8, Bcl-2, and Bax
immunostaining induced by OA (20 ng). The sections are triple-labeled
with anti-AT8 (fluorescein), anti-Bcl-2 (rhodamine), and anti-Bax (CY5).
INDUCTION OF Bcl-2 AND Bax
completely inhibit the death of NGF-differentiated PC12
cells induced by OA by a mechanism that Bcl-2 increased
stability of microtubules (Nuydens et al., 2000). But the
things may be more complicated. PP has been found to
localize at the mitochondrial membrane and to dephosphorylate Bcl-2 (Klumpp et al., 2002). OA, a potent inhibitor of PP, would increase Bcl-2 phosphorylation. Increased phosphorylation of Bcl-2 has been suggested to
reduce its function of promoting cell survival in case that
kainic acid induced neuronal excitotoxicitory damage
(Korhonen et al., 2003).
In conclusion, the present study proposes that the induction of Bcl-2 and Bax was both related to hyperphosphorylation of tau and neuronal death induced by OA in
rat brain. It is the first to clarify that Bcl-2 and Bax
expressions was induced by PP inhibitor.
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acid, death, induction, induced, rat, okadaic, brain, bcl, tau, bax, neuronal, hyperphosphorylation, related
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