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INTRODUCTION |
Apoptosis or programmed cell death is distinguished from lytic or
necrotic cell death by specific biochemical and structural events. The
most characteristic features of apoptosis are the activation of caspase
cascades, nuclear and cytoplasmic condensation, blebbing of cytoplasmic
membranes, and apoptotic body formation. It is generally accepted
that apoptosis is a fundamental cell death pathway contributing to the
regulation of tissue development and homeostasis and that dysregulated
apoptosis may cause a variety of pathologic states such as autoimmune
disease and malignancy. Therefore, apoptosis appears to be highly
controlled by a complex interplay between regulatory proteins such as
Bcl-2 and its family members. However, the biochemical mechanism
involved in the function of these proteins has not been fully elucidated.
Bcl-2 is a 26-kDa integral membrane protein that is found in the
nuclear envelope, the part of the endoplasmic reticulum, and the outer
mitochondrial membrane. Several reports have reported that
overexpression of Bcl-2 can delay or prevent apoptosis by a diverse
number of death-promoting signals in some cell systems (1-8).
Moreover, the study of Bcl-2-knockout mice has revealed that this
protein is involved in regulation of cellular redox in response to
oxidative stress (9). It has also been shown that Bcl-2 inhibits the
activation of caspases by blocking cytochrome c release from
mitochondria (10-13) and prevents the loss of mitochondrial membrane
potential (14, 15). In addition to the anti-apoptosis activity, recent
studies have revealed that Bcl-2 also promotes regeneration of severed
neuronal axons (16), regulates neuronal differentiation (17), and
controls cell proliferation (18-22).
So far, it has been shown that microglia are activated by cytokines to
produce neurotoxic agents such as nitric oxide and reactive oxygen
intermediates (23-26). Therefore, it is considered that activation of
microglia contributes to expansion of neuronal injury and has important
pathogenetic implications in neurodegenerative diseases such as
Alzheimer's disease, multiple sclerosis, and human immunodeficiency
virus-associated dementia (27-30). Despite the clinical importance,
the precise intracellular mechanism for neuronal death triggered by
activated microglia is currently unknown.
To elucidate the biochemical mechanism for activated microglia-induced
neuronal death, control and Bcl-2-transfected neuronal PC12 cells were
treated with activated microglia that were challenged with a
combination of interferon-
/lipoplysaccharide
(LPS)1 in the presence and
absence of the caspase-3-like protease inhibitors.
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EXPERIMENTAL PROCEDURES |
Materials--
Ac-DEVD-CHO, pepstatin A, leupeptin, Ac-YVAD-MCA,
and Ac-DEVD-MCA were purchased from Peptide Institute Inc. (Osaka,
Japan). Calpain inhibitor I was obtained from Bachem (Torrance, CA).
N-Acetyl-L-cysteine (NAC) was obtained from
Nacalai Tesque Inc. (Tokyo, Japan). Recombinant rat IFN-
and LPS
(Escherichia coli serotype 055:B5) were purchased from
Genzyme Corporation (Cambridge, MA) and Sigma, respectively. Nerve
growth factor (NGF) was obtained from Chemicon International Inc.
(Temecula, CA). OX42 and OX6 were purchased from Serotec Ltd
(Bichester, UK). Anti-human Bcl-2 antibody was purchased from Genosys
Biotechnologies Inc. (Cambridge, UK). Papain and DNase were from
Worthington Biochemical (Freehold, NJ). RPMI 1640 medium and
Dulbecco's modified Eagle's medium were from Life Technologies, Inc.
(Grand Island, NY). Calceinacetoxy methyl ester was from Molecular
Probes (Eugene, OR). ApopTag kit was from Oncor (Gaithersburg, MD).
Cell Transfection--
The pheochromocytoma cell line PC12 was
transfected with the plasmid pSTneoB and human Bcl-2/pcDNA/Amp by
the electroporation method (7). The transfected cells were selected by
their resistance to G418 (800 µg/ml) for 2 months in RPMI 1640 medium
supplemented with 10% heat-inactivated horse serum and 5% fetal calf
serum (FCS). The resulting stable neomycin resistant clonal PC12 cell line and Bcl-2 overexpressing clonal cell line were termed Vector N2
and Bcl-2 N2, respectively.
PC12 Cell Culture--
Stock PC12 cells were cultured in RPMI
1640 medium containing 10% heat-inactivated horse serum, 5% FCS, 50 units/ml penicillin, and 100 µg/ml streptomycin. Neuronally
differentiated PC12 cells were obtained by plating 3-4 × 104 cells onto 24-well collagen-coated dishes in the
presence of 50 ng/ml NGF for a period of 8-10 days in RPMI 1640 medium
supplemented with 10% heat-inactivated horse serum and 5% FCS.
Microglial Primary Culture--
Microglia were isolated from a
primary culture of the rat brain as described previously (7, 31).
Briefly, the cerebral cortex from 3-day-old male Wistar rats was minced
and treated with papain (90 units/ml) and DNase (2000 units/ml) at
37 °C for 15 min. The mechanically dissociated cells were seeded
into plastic flasks at a density of 107 per 300 cm2 in Dulbecco's modified Eagle's medium, 0.3%
NaHCO3, 50 units/ml penicillin, 100 µg/ml streptomycin,
and 10% FCS, and maintained at 37 °C in a 10% CO2,
90% air atmosphere. Subsequent medium replacement was carried out
every 3 days. After 10-14 days in culture, floating cells and weakly
attached cells on the mixed primary cultured cell layer were isolated
by gentle shaking of the flask for 3-5 min. The resulting cell
suspension was transferred to plastic dishes (Falcon 1001, Lincoln
Park, NJ) and allowed to adhere at 37 °C. Unattached cells were
removed after 30 min, microglia were isolated as strongly adhering
cells. About 90% of these attached cells were positive for OX42 or
OX6, makers for macrophage/microglial cell types.
Assay for Cell Survival--
Isolated microglia were directly
plated into 24-well dishes containing neuronal PC12 cells, and then
IFN-
(100 units/ml) and LPS (1 ng/ml) were added in the medium. In
some experiments, isolated microglia were plated into cell culture
inserts (0.4-µm pore size, Falcon 3095) and placed in 24-well dishes
containing neuronal PC12 cells. Cell death was quantified by the
measurement of the cytosolic enzyme lactate dehydrogenase (LDH)
released into the culture medium from degenerating cells. For
determination of LDH activity, the culture medium was collected from
each culture well, and then the well was washed with Dulbecco's
modified Eagle's medium containing HEPES. The medium was centrifuged
at 200 × g for 5 min to sediment nonadherent cells.
The supernatant was transferred to a fresh tube, and pelleted cells
were lysed in 1 ml of 0.1 M potassium phosphate buffer (pH
7.0) containing 0.5% Triton X-100 (lysing buffer). Cells remaining on
the well were lysed in 1 ml of lysis buffer. LDH activities in the
culture medium (supernatant) and in the cell lysates were measured
spectrophotometrically. The percentage of cell death was calculated by
the following formula: cell death = (LDH activity in the culture
medium/total LDH activity) × 100. LDH levels in the culture medium and
the cell lysate from microglia (generally less than 10% of the total
LDH) were determined in the sister cultures and subtracted from the
levels in co-culture experimental systems.
In Situ DNA Nick-end Labeling--
Apoptosis of neuronal PC12
cells after co-culture with activated microglia were detected by
terminal dUTP nick-end labeling (TUNEL) using the ApopTag kit. Neuronal
Vector N2 and Bcl-2 N2 cells plated on the chamber slides at various
days after co-culture with activated microglia were fixed by 4%
paraformaldehyde for 30 min at room temperature. After washes in
phosphate-buffered saline, cells on the slides were stained for TUNEL
following the protocol provided by the manufacturer.
Electrophoresis and Immunoblotting--
Detailed electrophoresis
and immunoblotting procedures were described previously (31, 32).
Briefly, cells in the dishes were washed with phosphate-buffered saline
and mechanically removed. Cells were pelleted by centrifugation and
resuspended in phosphate-buffered saline containing 0.1% Triton X-100.
The soluble cell extracts were prepared by ultrasonication followed by
centrifugation at 105,000 × g for 30 min. The extracts
were subjected to SDS-polyacrylamide gel electrophoresis under reducing
conditions in 7-12% gradient gels after the heat treatment in the
solubilizing buffer at 100 °C for 5 min. For immunoblotting, the
proteins on gels were electrophoretically transferred to nitrocellulose
membranes and then immunostained with anti-human Bcl-2 antibody (10 µg/ml), essentially according to the procedure described previously
(32). As a control, the primary antibody was replaced by preimmune
mouse IgG.
Assays for Caspase Activity--
Assays for caspase-3-like
proteases (caspase-2, -3, and -7) and caspase-1-like proteases
(caspase-1 and -4) were performed at pH 7.4 by using fluorogenic
synthetic tetrapeptide substrates (100 µM), Ac-DEVD-MCA
and Ac-YVAD-MCA, respectively. After the removal of the culture medium
at the appropriate time, the cells were lysed in 500 µl of lysing
buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM dithiothreitol, 5 mM EDTA, 5 mM
EGTA, 1% Triton X-100). After centrifugation, 100 µl of cleared
lysates were subjected to assays for caspase-3-like and caspase-1-like
proteases with the respective substrates at pH 7.4. Fluorescence was
measured with excitation at 360 nm and emission at 460 nm using
CytoFluor II fluorometer (PerSeptive, Eugene, OR). The measurements
were performed in triplicate, and the activity was expressed as change in fluorescence units per hour per 10,000 cells.
Electron Microscopic Analysis--
At 52 h after the
treatment with activated microglia, neuronal Vector N2 or Bcl-2 N2
cells on a glass slide chamber (Falcon) were fixed with 4%
formaldehyde in 0.1 M sodium phosphate buffer (pH 7.4)
containing 1% glutaraldehyde. After washing with 0.1 M
sodium phosphate buffer, the cells were dehydrated in a graded series
of ethanol and then embedded in Epon 812. Serial ultrathin sections
were cut and mounted on nickel grids and stained with 4% uranyl
acetate and lead citrate and then examined with a Hitachi H-7000
electron microscope.
Statistical Analysis--
Data are expressed as means ± S.D. The significance of differences between groups was determined with
two-way analysis of variance (ANOVA), followed by Scheffe's post hoc
test for multiple comparison when F ratios reached significance.
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RESULTS |
Effects of Ac-DEVD-CHO on Activated Microglia-induced Death of
Neuronal PC12 Cells--
The cell viability of neuronal PC12 cells
following co-culture with rat microglia immunostimulated with
IFN-
(100 units/ml)/LPS (1 ng/ml) was examined in the serum-free and
NGF-containing medium. When the neuronal cells were cultured for
72 h in the absence of activated microglia, approximately 20% of
the total cells were dead (Fig.
1A). The neuronal cells, when
treated with IFN-
/LPS in the absence of microglia, exhibited no
significant increase of their death. However, when the neuronal PC12
cells were treated with activated microglia, approximately 60% of the
neuronal cells were dead at 72 h after co-culture. Importantly, no
significant increase of the neuronal death was induced by the treatment
with either unstimulated microglia or activated microglia plated on culture inserts to avoid direct contact with the neuronal cells (data
not shown). The activated microglia-induced neuronal death was
accompanied by the significant increase of the caspase-3-like protease
activity determined with Ac-DEVD-MCA as the substrate (Fig.
1B). When the neuronal cells were pretreated with the
caspase-3-like protease inhibitor Ac-DEVD-CHO (100 µM)
for 24 h, the caspase-3-like protease activity in the cells was
completely inhibited, but the microglia-induced neuronal death was only
partially inhibited, as compared with that of the untreated neuronal
cells. The result indicates that inhibition of the caspase-3-like
protease activity is not sufficient to inhibit activated
microglia-induced neuronal death.

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Fig. 1.
Effects of Ac-DEVD-CHO on the action of
activated microglia on neuronal PC12 cells. A,
induction of neuronal cell death during co-culture with activated
microglia. Both Ac-DEVD-CHO-treated and control neuronal PC12 cells
were co-cultured with the activated microglia for 72 h. Then cell
viability was measured by the LDH assay. Each column and
bar represents the mean ± S.D. of three to five
individual experiments. ***, p < 0.001 as compared
with the IFN- /LPS-treated neuronal PC12 cells in the absence of
microglia. , p < 0.05 as compared with the activated
microglia-treated neuronal cells in the absence of Ac-DEVD-CHO (two-way
ANOVA). B, activation of caspase-3-like proteases in the
activated microglia-treated neuronal PC12 cells. After treatment with
the activated microglia for 72 h, both control and
Ac-DEVD-CHO-treated neuronal PC12 cells were lysed, and the
caspase-3-like protease activity in their cell extracts were measured
using the fluorogenic substrate. Each column and
bar represents the mean ± S.D. of three to five
individual experiments. ***, p < 0.001 as compared
with the IFN- /LPS-treated neuronal cells in the absence of
microglia.   , p < 0.001 as compared with the
activated microglia-treated neuronal cells in the absence of
Ac-DEVD-CHO (two-way ANOVA).
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Morphological Characteristics of Activated Microglia-induced Death
of Neuronal PC12 Cells--
At 72 h after treatment with
activated microglia, the majority of neuronal cells became
TUNEL-positive and contained various sizes of multiple apoptotic
bodies (Fig. 2B), indicating
the occurrence of apoptotic cell death. When Ac-DEVD-CHO (100 µM) was added to the culture medium at 24 h before
and during treatment with activated microglia, the majority of neuronal
cells were not stained with TUNEL (Fig. 2C). However, damage
of neuronal cells was clearly evident by morphological alterations such
as their retracted neurites. Neither TUNEL-positive staining nor
neuronal damage was observed in the neuronal cells treated with
IFN-
/LPS in the absence of activated microglia (Fig.
2A).

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Fig. 2.
TUNEL staining in neuronal PC12 cells
following co-culture with activated microglia in the presence and
absence of Ac-DEVD-CHO. A, neuronal PC12 cells at
48 h after the treatment of IFN- /LPS in the absence of
microglia. Scale bar, 20 µm. B, neuronal PC12
cells at 48 h after treatment with the activated microglia.
TUNEL-positive neuronal cells contained multiple apoptotic bodies.
C, neuronal PC12 cells pretreated with Ac-DEVD-CHO at
48 h after the treatment with activated microglia. Most of the
neuronal cells were not stained with TUNEL but showed morphological
changes such as retracted neurites, indicating their damage.
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Effects of Overexpression of Human Bcl-2 on Activated
Microglia-induced Cell Death of Neuronal PC12 Cells--
We then
examined the effect of high levels of Bcl-2 protein in the neuronal
cells on the activated microglia-induced neuronal death. The stable
neomycin-resistant clonal PC12 cells without (Vector N2) or with human
Bcl-2 protein (Bcl-2 N2) were differentiated to neurons by NGF
treatment. Then both serum and NGF were deprived from cultures of the
respective cell types. As shown in Fig.
3A, neuronal Vector N2 cells
were markedly induced to neuronal death by the trophic factor
deprivation in a time-dependent manner, whereas neuronal
Bcl-2 N2 cells were more resistant to death induced by deprivation of
trophic factors (Fig. 3A). These data indicate that Bcl-2 is
expressed highly enough in neuronal Bcl-2 N2 cells to suppress cell
death induced by deprivation of trophic factors. In contrast, no
significant difference with regard to the extent of the activated
microglia-induced neuronal death was observed between Vector N2 and
Bcl-2 N2 cells, indicating that high levels of Bcl-2 cannot protect the
neuronal cells from the activated microglia-induced cell death
(Fig. 3B).

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Fig. 3.
Effects of Bcl-2 overexpression in neuronal
PC12 cells on their death induced by deprivation of trophic factors
(A) and treatment with activated microglia
(B). A, time-course of changes in cell
viability after deprivation of both NGF and serum in the neuronal
Vector N2 (filled circle) and Bcl-2 N2 (open
circle) cells. Cell viability was measured by the LDH assay. More
than 50% of the control Vector N2 cells died at 72-h postdepletion,
whereas about 75% of the Bcl-2 N2 cells were apparently viable at the
same time point. Each point and bar represents
the mean ± S.D. of five experiments, respectively. ***,
p < 0.001 as compared with Vector N2 cells (two-way
ANOVA). B, time course of changes in cell viability after
treatment with activated microglia in neuronal Vector N2 (filled
circle) and Bcl-2 N2 (open circle) cells. No
significant difference in the extent and profile of cell death was
observed between Vector N2 and Bcl-2 N2 neuronal cells. Each
point and bar represents the mean ± S.D. of
five to ten individual experiments, respectively.
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To examine the possibility that the lack of protective effect of Bcl-2
expression on the activated microglia-mediated neurotoxicity may be
because of the changes of its cellular levels or molecular forms during
incubation, the cellular levels of Bcl-2 protein were analyzed by
immunoblotting (Fig. 4). When the same
amounts of proteins from the cell extracts of neuronal Bcl-2 N2 before and after treatment with activated microglia for 72 h were
subjected to SDS-polyacrylamide gel electrophoresis followed by
immunoblot analysis, single immunoreactive bands with an apparent
molecular mass of 26 kDa were observed with both samples at the same
intensity, and their electrophoretic mobilities were also
indistinguishable. Therefore, it is unlikely that the activated
microglia-induced neuronal death of Bcl-2 N2 cells is caused by either
the reduction of cellular levels of Bcl-2 or its molecular change.

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Fig. 4.
Bcl-2 protein expression in neuronal Bcl-2 N2
cells during co-culture with activated microglia. Western blot
analysis was performed on the neuronal Bcl-2 N2 cells before and after
treatment with activated microglia for 72 h to ensure the
unchanged expression of Bcl-2 protein during the process of the
activated microglia-induced neuronal death. The same amount of protein
(30 µg) of each cell extract was subjected to SDS-polyacrylamide gel
electrophoresis and analyzed with anti-human Bcl-2 antibody.
Right lane, undifferentiated Bcl-2 N2 cells; middle
lane, neuronal Bcl-2 N2 cells; left lane, neuronal PC12
N2 cells after treatment with activated microglia for 72 h.
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In the neuronal Bcl-2 N2 cells, the proteolytic activities of
caspase-1-like and caspase-3-like proteases were determined after
treatment with activated microglia by use of the fluorogenic substrates
Ac-YVAD-MCA and Ac-DEVD-MCA, respectively (Fig.
5). The hydrolytic activity of
Ac-YVAD-MCA was barely detectable in the extracts of both neuronal
Vector N2 and Bcl-2 N2 cells during the whole period of co-culture with
activated microglia. No significant activity of Ac-DEVD-MCA was
detected in the extract of neuronal Bcl-2 N2 cells during the
co-culture, although only a slight increase in the activity was
observed around 42 h after the co-culture. In contrast, the
extract of neuronal Vector N2 cells showed a gradual increase in the
hydrolytic activity of Ac-DEVD-MCA to attain the peak value at around
42 h after the treatment with activated microglia. This activity
profile appeared to be correlated with their death as measured by LDH
activity in the culture medium per total LDH activity. No hydrolytic
activity of either Ac-YVAD-MCA or Ac-DEVD-MCA was detected in cytosolic
extracts of microglia. Moreover, various protease inhibitors, other
than Ac-DEVD-CHO, such as the aspartic proteinase inhibitor pepstatin A
(30 µM for 48 h), the cysteine proteinase inhibitor
leupeptin (100 µM for 24 h), and calpain inhibitor I
(30 µM, for 24 h) had no significant inhibitory
effect on either the Ac-DEVD-MCA hydrolytic activity or the death of
neuronal Bcl-2 N2 cells following co-culture with activated microglia
(data not shown).

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Fig. 5.
Time-course of changes in the caspase-1-like
(A) and caspase-3-like proteolytic activities
(B) after treatment with activated microglia in the
cell extracts of control and Bcl-2-overexpressing neuronal PC12
cells. A, time course of caspase-1-like protease
activity. The activity was determined with the fluorogenic substrate
Ac-YVAD-MCA. B, time course of caspase-3-like protease
activity that was determined with Ac-DEVD-MCA.
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Inhibition of DNA Fragmentation in Neuronal PC12 Cells
Overexpressing Bcl-2 during the Process of Degeneration Induced by
Activated Microglia--
At 48 h after treatment with activated
microglia, the majority of neuronal Vector N2 cells became
TUNEL-positive and contained various sizes of multiple apoptotic bodies
(Fig. 6B). A number of
TUNEL-positive apoptotic bodies were attached to neighboring microglia
and were engulfed by the microglia (Fig. 6, D and
E). Some of the TUNEL-positive apoptotic bodies appeared to
be extensively degraded in microglia (Fig. 6F).
Approximately 40% of the total TUNEL-positive apoptotic bodies were
engulfed by microglia. On the other hand, the majority of neuronal
Bcl-2 N2 cells were not stained by TUNEL even at 48 h after
treatment with activated microglia but were nevertheless clearly
damaged by the treatment as evidenced by their retracted neurites and
accumulated vacuoles in their perikarya (Fig. 6C). No
staining was observed in neuronal Vector N2 cells co-cultured with
unstimulated microglia (Fig. 6A).

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Fig. 6.
TUNEL stainings on control and
Bcl-2-overexpressing neuronal PC12 cells after treatment with activated
microglia for 48 h. A, the neuronal Vector N2
(control) cells that were treated with unstimulated microglia.
B and D-F, the control cells after treatment
with activated microglia. TUNEL-positive apoptotic bodies were
frequently attached to microglia (D) and engulfed by
microglia (E and F) and degraded (F).
C, the neuronal PC12 cells overexpressing Bcl-2 (Bcl-2 N2)
after treatment with activated microglia. The cells were scarcely
stained by TUNEL but had retracted neurites and accumulated vacuoles in
the perikarya. The nuclei of microglia were indicated by m
in panels A-F. Scale bars in panels A
and D = 10 µm.
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Ultrastuctural Analyses of Neuronal Vector and Bcl-2 N2 Cells
Treated with Activated Microglia--
After treatment with
IFN-
/LPS, most of the microglia showed characteristic ruffled
membrane structures on their cell surface and had a large number of
phagosome-like dense bodies containing phagocytosed materials (Fig.
7A). Fig. 7B shows
an apoptotic body of neuronal Vector N2 cells containing distinct
chromatin condensation in the vicinity of activated microglia (an
arrowhead indicates the ruffled cell membrane of microglia).
Apoptotic bodies of neuronal Vector N2 cells were found to be
frequently phagocytosed by activated microglia. Nuclei of Vector N2
cells were also characterized by chromatin uniformly aggregated into
large discrete masses abutting on the nuclear membrane (Fig.
7C). In contrast, in the activated microglia-treated
neuronal Bcl-2 N2 cells, their chromatin was slightly condensed to form
irregular shaped masses (Fig. 7D), but their nuclear
membranes were still maintained and formed some boundaries between
nuclear and cytoplasmic matrixes. Importantly, these degenerating Bcl-2
N2 cells frequently showed severely disintegrated perikarya, dilated
rough endoplasmic reticulum, and swollen mitochondria, suggesting that
the degenerating process is similar to the necrotic death pathway
rather than the apoptotic death pathway.

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Fig. 7.
Electron microscopic graphs of neuronal
Vector N2 and Bcl-2 N2 cells after treatment with activated
microglia. A, typical activated microglia having
ruffled membrane structures on the cell surface. B, an
apoptotic body with uniformly condensed chromatin of Vector N2 cells
close to the ruffled cell membrane of microglia (an
arrowhead). C, apoptotic bodies of neuronal
Vector N2 cells engulfed by microglia (its nuclei was indicated by
m). D, a degenerating neuronal Bcl-2 N2 cell
after treatment with activated microglia, where its nuclear membrane
was still maintained to form boundaries between the nuclear and the
cytoplasmic matrix. Scale bars, 2 µm (A and
B) and 4 µm (C and D).
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Effects of N-Acetyl-L-cysteine on Activated
Microglia-induced Death--
Several lines of evidence have revealed a
role of reactive oxygen species (ROS) as a mediator of cell death.
Indeed, generation of ROS has been commonly observed after treatment
with agents such as tumor necrosis factor-
(33), LPS (34), and
ceramide (35). Trophic factor deprivation can also stimulate the ROS production (36). In addition, ROS scavengers, such as NAC, are known to
inhibit cell death induced by various death stimuli (9, 37-39).
Therefore, to elucidate the involvement of ROS in the activated microglia-induced death of neuronal PC12 cells, we examined the effect
of NAC (60 mM). Both neuronal Vector N2 and Bcl-2 N2 cells, when treated with NAC (60 mM) for 1 h before and
during the treatment with activated microglia, exhibited significant
resistance to the microglia-induced neuronal death (Fig.
8). It was also noted that NAC-treated
neuronal Bcl-2 N2 cells were more resistant to killing by activated
microglia than NAC-treated neuronal Vector N2 cells.

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Fig. 8.
Inhibition of activated microglia-induced
death by NAC in neuronal Vector N2 and Bcl-2 N2 cells. Both
control (Vector N2) and Bcl-2-overexpressing neuronal PC12 cells (Bcl-2
N2) were pretreated with 60 mM for 1 h, and then the
cells were treated with the activated microglia for 72 h in the
presence of NAC (60 mM). Cell viability was measured by the
LDH assay. Each column and bar represents the
mean ± S.D. of four to eight individual experiments. *,
p < 0.05; ***, p < 0.001 as compared
with the respective nontreated neuronal cells; , p < 0.05 as compared with the NAC-treated Vector N2 cells (two-way
ANOVA).
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DISCUSSION |
The present study has clearly demonstrated that activated
microglia can effectively induce the apoptotic cell death of neuronal PC12 cells. This apoptosis was accompanied by caspase-3-like protease activation and morphological changes characteristic of apoptosis, such
as nuclear and cytoplasmic condensation, DNA fragmentation, and the
formation of apoptotic bodies that were finally phagocytosed by
neighboring microglia. This demonstrates the predominance of the
caspase-3-executed apoptotic cell death of neuronal cells by activated
microglia. When the neuronal cells were pretreated with Ac-DEVD-CHO, no
activation of caspase-3-like proteases was observed. Nevertheless,
activated microglia-induced neuronal death occurred, suggesting that
the activation of caspase-3-like proteases is not essential for the
execution of activated microglia-induced neuronal death. However, since
the morphological hallmarks of apoptosis, such as chromatin
condensation, DNA fragmentation, and apoptotic body formation, were not
observed in this process, the microglia-induced neuronal death executed
in the presence of inhibitor seems to be non-apoptotic. There is
increasing evidence that cells can die without chromatin condensation
and DNA fragmentation after apoptotic stimuli in the presence of the
inhibitor for caspase-3-like proteases (40-42). Recent evidence also
indicates that caspase-3 is not required for apoptosis in the PC12
cells (43) and that thymocytes isolated from caspase-3 knockout mice
exhibit normal Fas-induced apoptosis (44). Taken together, the present
data indicate that although caspase-3-like proteases are essential for
chromatin condensation and DNA fragmentation, they are not required for
other apoptotic events in the cytoplasm or the cell death itself. It
should also be noted that this inhibitor rescued the same cells from
death induced by trophic factor deprivation.
Then we examined whether high levels of Bcl-2 protein in neuronal PC12
cells inhibited the activated microglia-induced neuronal apoptosis. The
results indicated that Bcl-2 overexpression effectively protected the
neuronal cells from apoptosis induced by deprivation of both serum and
NGF, whereas it did not abrogate the activated microglia-induced
neuronal death. In either case, the caspase-3-like protease activity
was effectively inhibited in neuronal PC12 cells overexpressing Bcl-2
protein, but the trophic factor deprivation-induced cell death only was
prevented. Importantly, the Bcl-2-overexpressing cells after treatment
with activated microglia showed morphological changes different from
apoptosis. These results suggest that apoptotic signaling pathways
dependent on the activation of caspase-3-like proteases would be
changed to non-apoptotic signaling pathways independent of the
activation of caspase-3-like proteases.
It has recently been shown that, in addition to the caspase-3-like
proteases, some other intracellular proteolytic systems are identified
as executioners to activate cell death. Cathepsin D, a typical
lysosomal aspartic proteinase, has been shown to mediate
cytokine-induced apoptosis of some cell types (45, 46). Calpain, a
calcium-dependent cysteine proteinase, has also been shown
to mediate cell death in some cell systems (47-51). However, it is
unlikely that the intracellular proteolytic machinery mediated by
either cathepsin D or calpain is responsible for the activated microglia-induced neuronal death of Bcl-2 overexpressing PC12 cells in
the present study because pepstatin A, leupeptin, or calpain inhibitor
I failed to protect the cells from death.
Recently, several lines of evidence support a role for ROS as a
mediator of both apoptosis and necrosis. Hydrogen peroxide and
superoxide caused apoptosis in a variety of cell lines (9, 52, 53). In
addition, antioxidants such as NAC can inhibit apoptosis of neuronal
PC12 cells and sympathetic neurons induced by trophic factor
deprivation (54, 55). ROS and resulting cellular redox changes are
known to be part of the signal transduction pathway during apoptosis.
On the other hand, ROS toxicity has been also shown to result in
necrosis of some cell types (56, 57). More recently, Vercammen et
al. (58, 59) have reported that enhanced production of ROS is
responsible for TNF-induced necrosis of L929 cells in the presence of
caspase inhibitors. Accordingly, we attempted to determine whether ROS
was important in the activated microglia-induced death of neuronal
Vector N2 and Bcl-2 N2 cells. The results indicated that NAC
significantly but partially decreased the activated microglia-induced
death of both cell types, suggesting that ROS production is partially involved in death pathways of both neuronal PC12 cells. It should also
be noted that NAC showed more effective protection in the neuronal
Bcl-2 N2 cells than the control Vector N2 cells against the activated
microglia-induced cell death. This suggests that the activated
microglia-induced death of neuronal Bcl-2 cells is accompanied by the
enhanced production of ROS. Although the enhanced production of ROS may
be one of the mediators for non-apoptotic death of neuronal Bcl-2 N2
cells, the precise mechanism is to be elucidated in future studies.
In conclusion, it must be pointed out that activated microglia
predominantly induce caspase-3-like protease-executed apoptosis of
neuronal PC12 cells but alternatively trigger non-apoptotic cell death
when the caspase-3-like protease cascade is inhibited by protease
inhibitors and Bcl-2 overexpression. Thus, it is more likely that
whether activated microglia induce apoptosis or non-apoptosis, the
neuronal cells depend on the prominence of caspase-3-like protease
activation in the death process as well as other factors such as the
ROS production. These findings also suggest that adaptation to one type
of death pathway may render cells more susceptible to alternative death
pathways and that the hypersensitive response of the neuronal cells may
be caused by the process of adaptation to the microglia-induced
apoptosis. This could be an effective backup mechanism to execute the
activated microglia-induced neuronal death. To our knowledge, the data
presented here provide the first evidence for a possible mechanism for
the regulation of neuronal death by activated microglia.