From the Unité INSERM U-338 de Biologie de la Communication Cellulaire, Centre de Neurochimie, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France
Received for publication, October 24, 2000, and in revised form, December 19, 2000
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ABSTRACT |
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The neurotoxic effects of activated microglia in
neurodegenerative diseases are well established. We recently provided
evidence that chromogranin A (CGA), a multifunctional protein localized in dystrophic neurites and in senile plaques, induces an activated phenotype and secretion of neurotoxins by rat microglia in culture. In
the present study, we focused on the mechanisms underlying neuronal
degeneration triggered by CGA-activated microglia. We found that
neuronal death exhibits apoptotic features, characterized by the
externalization of phosphatidylserine and the fragmentation of DNA.
Microglial neurotoxins markedly stimulate the phosphorylation and
activity of neuronal p38 mitogen-activated protein kinase and provoke
the release of mitochondrial cytochrome c, which precedes apoptosis. Inhibition of p38 kinase with SB 203580 partially protects neurons from death induced by CGA-activated microglia. Furthermore, neurons are also protected by Fas-Fc, which antagonizes the
interactions between the death receptor Fas and its ligand FasL and by
cell-permeable peptides that inhibit caspases 8 and 3. Thus, CGA
triggers the release of microglial neurotoxins that mobilize several
death-signaling pathways in neurons. Our results further support the
idea that CGA, which is up-regulated in many neuropathologies,
represents a potent endogeneous inflammatory factor possibly
responsible for neuronal degeneration.
Destruction of neurons by apoptosis and necrosis is the underlying
mechanism in a variety of neurodegenerative diseases and, thus,
represents an area of intense interest (1, 2). Activated microglial
cells are capable of releasing cytotoxic agents, including cytokines,
complement proteins, proteolytic enzymes, nitric oxide, and reactive
oxygen intermediates, and
N-methyl-D-aspartate-like toxins (3-7).
Therefore, activation of microglia is considered to contribute to
neuronal injury and has important pathogenic implications in
neurodegenerative diseases such as Alzheimer's disease, Parkinson
disease, or immunodeficiency virus-associated dementia (4, 6, 8, 11).
Despite their clinical importance, the brain-derived factors that
control microglial activation and the precise intracellular mechanisms
involved in microglial cytotoxicity remain elusive. The main
constituent of amyloid plaques, Chromogranin A (CGA)1 is a
polypeptide chain of 431-445 amino acids corresponding to a 48-52-kDa
glycoprotein (14) that is widely distributed in endocrine and nervous
tissue. In all tissues examined so far, CGA is proteolytically
processed into peptides, of which some have defined biological activity
(14, 20, 21). For instance, CGA-derived peptides have been shown to
regulate secretion in various endocrine cell types (22-24) and to
modulate adhesion and spreading of fibroblasts (25). CGA and its
proteolytic fragments have been found together with In the present study, we focused on the mechanisms underlying neuronal
death induced by CGA-activated microglia. We found that the death
domain-containing receptors Fas/Apo1/CD95, which belong to tumor
necrosis factor receptor family (29), and the mitogen-activated protein
kinase p38 are possible transductors of the neuronal response to
microglial neurotoxins. The pathways mobilized in neurons also include
release of mitochondrial cytochrome c (28) and activation of
caspases 8 and 3. These results further support the idea that CGA
represents a potent brain endogenous inflammatory factor capable of
inducing the production of microglial neurotoxins responsible for the
mobilization of several apoptotic cascades within neurons.
Purification of Bovine CGA--
CGA was purified from bovine
adrenal medullary chromaffin granules as described by Simon et
al. (30), with an additional purification step on a column of
Ultraspherogel Sec 2000 (7.5 × 30 mm, Beckman). Stock solutions
usually containing 50 µM CGA were separated into aliquots
and stored at
The content of endotoxin in the CGA preparations was determined at the
Institut d'Hygiène et de Médecine Préventive
(Strasbourg, France) with the chromogenic Limulus amoebocyte lysate
test (Coamatic Endotoxin Chromogenix, Biogenic, Maurin, France). At 10 nM, the CGA preparations contained less endotoxin than the
culture medium alone, which itself is unable to trigger the NO
production in microglial cells. CGA was usually dissolved in serum-free
defined medium and applied to cultures by changing the medium at the
indicated concentrations.
Neuronal Cultures--
Neuronal cell cultures were established
after mechanical dispersion of cerebral cortices from Wistar fetal (day
14 of gestation) rat brain as previously described (27). Briefly,
cerebral cortices cleaned from meninges were forced through a nylon
sieve (pore size 48 µm) in nutrient medium consisting of Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% fetal calf
serum. The cell suspension was centrifuged, and cell viability was
determined with trypan blue. Viable cells were plated at a density of
2.5 × 105 cells/well in 24-multiwell Falcon plates
coated with poly-L-ornithine in DMEM supplemented with 10%
fetal calf serum. For immunocytochemistry, each well contained a 12-mm
glass coverslip. Cells were subsequently incubated for 1 h at
37 °C in a 5% CO2-humidified atmosphere. The
serum-containing medium was then replaced by serum-free defined medium
consisting of DMEM supplemented with transferrin (100 µg/ml), insulin
(5 µg/ml), albumin (100 µg/ml), progesterone (6 ng/ml), and sodium
selenite (5.2 ng/ml).
Microglial Cultures--
Microglial cells were isolated from
high density glial cell cultures as previously described (26). Briefly,
cells dissociated from cerebral hemispheres of neonatal rat brain
(Wistar strain) were plated at a density of 5 × 104
cells/cm2 in culture medium consisting of DMEM supplemented
with 10% fetal calf serum. Culture medium was changed after 5 days and
then twice a week. After 2 weeks, cultures contained glial cells
including amoeboid microglia, mostly localized on the top of the
cellular layer. The loosely adherent microglial cells were recovered by shaking. After centrifugation (100 × g for 5 min),
cell viability was determined by trypan blue exclusion, and viable
cells were then plated at a final density of 2.5 × 105 cells/well on 24-multiwell Falcon plates in DMEM
supplemented with 10% fetal calf serum. Nonadherent cells were removed
30 min after plating by changing the medium for serum-free defined
medium. Amoeboid microglia isolated from neonatal rat brain cultures
consist of flat, round, or spindle-shaped highly adherent cells. They respond to bacterial endotoxin (32) and express Fc (33) and CR3
complement receptors (26).
Conditioned medium was prepared by incubating microglial cell cultures
for 24 h in serum-free defined medium with or without 10 nM CGA. Medium was subsequently collected and cleared by
centrifugation (1000 × g for 20 min). When necessary,
the medium was stored at Neuron/Microglial Co-cultures--
Co-cultures of neurons and
microglial cells were prepared by plating a suspension of isolated
microglia (2.5 × 105 cells) on neurons maintained 4 days in culture. Cells were incubated for 30 min in DMEM containing
10% fetal calf serum. Serum-free defined medium with or without
additives was then added, and co-cultures were grown for at least 6 days.
Treatment of Cultures--
Neurons were grown for 4 days before
being incubated with conditioned media from CGA-untreated or
CGA-treated microglia for the indicated period of time. Recombinant
human APO-1/Fas-Fc IgG (Fas-Fc) and soluble Fas ligand (sFasL) with
enhancer antibodies (Alexis Corp., Coger, Paris, France) were diluted
in the indicated culture medium. Inhibitors of caspases, Z-DEVD-FMK
(Z-Asp-Glu-Val-Asp-fluoromethyl ketone) and Z-IETD-FMK
(Z-Ile-Glu-Thr-Asp- fluoromethyl ketone) (Calbiochem), diluted in
Me2SO were used at the indicated concentrations. Controls
were performed in the presence of the same concentrations of
Me2SO.
Assay of Viability and Apoptosis--
Neuronal survival in
living cultures was estimated by incubating cells at 37 °C for 10 min with 2.5 µg/ml nuclear fluorochrome propidium iodide (PI, Sigma).
Alternatively, cultures were incubated for 5 min with 0.1% trypan blue
in phosphate-buffered saline (PBS), and stained cells were immediately
counted in three randomly chosen fields.
Nuclei in paraformaldehyde-fixed cells were visualized by staining with
Hoechst 33342 (Sigma) for 10 min at a final concentration of 2.5 µg/ml. Cultures were examined with a microscope equipped with an
epifluorescence system and appropriate filters (Zeiss Axioscope, Carl
Zeiss, Oberkochen, Germany), and those neurons with clearly condensed
and segmented chromatin were counted as apoptotic.
Binding of annexin V was performed by incubating living cells (30 min,
room temperature) with fluorescein-conjugated annexin V (Bioproducts,
France) diluted 1/30 in serum-containing culture medium in the presence
of 0.01% sodium azide. After washing and fixation, cultures were
further processed for immunocytochemistry.
DNA fragmentation was detected in fixed cells using the apoptosis
detection system fluorescein kit (Promega) by incorporating fluorescein-12-dUTP at the 3'-OH ends of the DNA using terminal deoxynucleotidyltransferase according to the manufacturer's instructions.
Immunocytochemical Staining and Confocal Laser-scanning
Microscopy--
Cells grown on coverslips were fixed with 4%
paraformaldehyde for 10 min followed by a 5-min permeabilization with
0.02% Triton X-100 in 4% paraformaldehyde. Cultures were then
incubated at room temperature for 30 min in PBS containing 10% bovine
serum albumin to inhibit nonspecific binding before a 1-h incubation with primary antibodies. Monoclonal antibodies to
microtubule-associated protein 2 (MAP2, SMI 52 Sternberger Monoclonals
Inc.) were used at 1/500 dilution. Monoclonal anti-cytochrome
c antibodies (Pharmingen clone 6H2.B4) were used at a
1/500 dilution. For detection of FasL with rabbit polyclonal antibodies
(N-20 or C-178, Santa Cruz, CA), living cells were incubated with
0.1-0.2 µg/ml of IgG in serum-containing culture medium in the
presence of 0.01% sodium azide at room temperature for 30 min and then
fixed. After washing with PBS (5 changes, 30 min), the coverslips were
incubated for 1 h with either fluorescein- or rhodamine
(tetramethylrhodamine B isothiocyanate)-conjugated affinity-purified
F(ab')2 fragment of goat anti-mouse class G immunoglobulins
used at 1/300 dilution (Fc fragment-specific, Jackson Immunoresearch
Laboratories, West Grove, PA). Coverslips were subsequently washed with
PBS (5 changes, 30 min) and mounted in Mowiol (Calbiochem).
Mounted coverslips were examined with a Zeiss Axioscope microscope
equipped with an epifluorescence system and appropriate filters.
Sequential through-focus images of labeled cells were obtained using a
Zeiss laser-scanning confocal microscope (LSM 410 invert) equipped with
a planapo oil (63×) immersion lens (numerical aperture = 1.4)
and argon 488 nm and a He/Ne 543-nm lasers. The emission signals were
filtered with a 515-565 nm filter (fluorescein) or with a long pass
595-nm filter (TRITC). The images were recorded using identical laser
power, wavelength, and photomultiplier tube voltage. They were
recorded digitally in a 768 × 556-pixel format and saved on a
magneto optical disc.
Preparation of Cell Extracts and Immunoblotting--
Cell
cultures were washed with PBS and lyzed in ice-cold lysis buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 1 mg/ml pepstatin, 1 mM leupeptin, 0.2 mM phenylmethylsulfonyl fluoride, 5 mM sodium
pyrophosphate, and 100 mM sodium fluoride) containing 2%
(v/v) Triton X-100 and 1 µg/ml DNase I. Lysates were cleared by
centrifugation (10,000 × g for 10 min) and both supernatants (Triton X-100-soluble cytosolic fractions) and pellets (Triton X-100-insoluble cytoskeletal fractions) were resuspended in
electrophoresis sample buffer, heated at 100 °C for 10 min, and
stored in aliquots at
Cell extracts were resolved on 10% polyacrylamide-SDS gels and
electroblotted onto nitrocellulose membranes (Sartorius, Goettingen, Germany). Blots were processed using the Western-Light Plus
chemiluminescent detection system (Tropix, Bedford, MA) according to
manufacturer's instructions. The following antibodies to MAP kinases
were used: anti-p38 MAP kinase, anti-phospho-specific p38 MAP kinase
(Thr-180/Tyr-182), anti-c-Jun, anti-phospho-specific c-Jun (Ser-63) II
(New England Biolabs, Beverley, MA), anti-p46 c-Jun N-terminal kinase 1 (JNK1) (C-17) (Santa Cruz, CA), and anti-active JNK pAb (Promega,
Madison WI). Immunoreactive bands were visualized after exposure to
Kodak BioMax Light-1 film (Eastman Kodak Co.). In some cases, blots were stripped in buffer containing 200 mM glycine, 0.1%
SDS, 1.0% Tween 20, pH 2.2, followed by washings before incubating
with blocking buffer and with antibodies to actin.
Immunoprecipitation and Assay of MAP Kinases
Activities--
Cell lysates (400 µg protein) were incubated on a
shaking platform overnight at 4 °C with 5 µl of protein
G-Sepharose conjugated with 1 µg of either anti-p38 MAP kinase or
anti-p46 JNK1 antibodies (StressGen Biotechnologie Corp., Victoria,
Canada). The immunoprecipitates were washed twice with buffer
containing 20 mM HEPES, 10% glycerol, 0.1% Triton X-100,
and 500 mM NaCl followed by two washes with the same buffer
containing 150 mM NaCl. Kinase activity was assayed by
incubating the resuspended pellet at 30 °C in the presence of 10 µCi of [ Presentation of Data--
Experiments were performed on at least
six different cell cultures. In the figures, data are representative of
a typical experiment and are given as the mean of at least three
determinations (± S.E.). The comparison between data obtained under
different experimental conditions was analyzed by Student's
t test. Differences were considered to be statistically
significant when p < 0.01.
Neuronal death in Neuron/Microglial Co-cultures Exposed to
Chromogranin A Exhibits Apoptotic Features--
We previously reported
that exposure of neuron/microglial co-cultures to either bovine or
recombinant human CGA induces a marked reduction in neuronal-specific
[ Chromogranin A-activated Microglia Secrete Diffusable Factors That
Induce Neuronal Apoptosis--
To further analyze the neurotoxic
effect of microglia activated by CGA, we performed experiments on
neurons maintained in pure culture. We previously reported that the
toxic factors secreted by CGA-activated microglia are released into the
culture medium and, as such, can be collected (27). Thus, cortical
neurons were exposed to cell-free conditioned medium (CM) collected
from either resting microglia or microglia treated for 24 h with
10 nM CGA, and the rate of apoptosis and necrosis was
determined. In the early stages of apoptosis, the plasma membrane
remains intact and impermeable to inert dyes such as trypan blue (36). In contrast, necrosis is accompanied by a rapid loss of membrane integrity, and the cell membrane becomes permeable. As shown in Fig.
2, the proportion of necrotic neurons
(stained with trypan blue) in CM from resting microglia does not exceed
10% of the total cell population. Furthermore, the percentage of
apoptotic neurons revealed with the nuclear stain Hoechst 33342 remained lower than 15% over the whole experimental period. In
contrast, in CM from CGA-treated microglia, the proportion of neurons
with apoptotic Hoechst-positive nuclei rapidly increased and reached about 50% of the total neuronal population by 96 h. The number of
neurons permeable to trypan blue also increased but after a lag time of
48 h. These data indicate that the neurotoxic factors secreted by
CGA-activated microglia do not directly affect the integrity of the
neuronal membrane but are more likely to induce an apoptotic process in
neurons.
Effect of Neurotoxic Factors Secreted by Chromogranin A-treated
Microglia on MAP Kinase Phosphorylation and Activity in
Neurons--
To investigate whether the neurotoxic factors secreted by
CGA-treated microglia stimulate MAP kinases in neurons, we examined the
presence and activation of JNK and p38 MAP kinase using antibodies that
recognize all forms or the phosphorylated forms of the kinases. Fig.
3A shows that cultured
cortical neurons express high levels of both JNK and p38 kinase. Both
kinases were essentially present in the cytosol, although a substantial
fraction was also associated with cytoskeletal proteins. The relative
distribution of total and phosphorylated forms of JNK was not
significantly affected by the CM from resting or CGA-stimulated
microglia. As expected, neurons also expressed c-Jun and its
phosphorylated form (results not shown). In contrast, phosphorylated
p38 kinase was not detected in neurons exposed to CM from resting
microglia. However, exposure to CM from CGA-treated microglia triggered
the rapid appearance of the phosphorylated form of p38 kinase,
particularly in the cytosolic fraction (Fig. 3A).
To determine the time course of MAP kinase activation in response to CM
from CGA-treated microglia, we measured kinase activities using
appropriate substrates in immunoprecipitates obtained with specific
antibodies. Fig. 3B shows that JNK is already activated in
neurons, and this activity does not significantly increase further upon
exposure to CM from CGA-activated microglia. In contrast, CGA-induced
microglial toxins provoked a marked and transient stimulation of p38
kinase in neurons. Note that preincubation with 10 µM SB
203580, a treatment reported to inactivate p38 MAP kinase in
vitro (37), abolished the activation of this enzyme in neurons
(Fig. 3B).
To probe the involvement of p38 MAP kinase in the neurotoxic effects
induced by CGA-stimulated microglia, we examined the ability of SB
203580 to prevent apoptosis in neurons. Fig. 3C shows that
pretreatment of neuronal cultures with SB 203580 reduced but did not
completely prevent neuronal death induced by CGA-activated microglia.
At 20 µM, SB 203580 protected ~60% of the total
neuronal population from microglial-induced apoptosis. These results
provide evidence that p38 MAP kinase participates in the apoptotic
cascade induced by CGA-stimulated microglia in neurons, although
additional pathways are likely to be mobilized. Since activation of p38
kinase has been related to altered calcium homeostasis and/or
production of free radicals, which are considered to be potent inducers
of mitochondrial damage (2, 29), we examined whether the release of
cytochrome c from mitochondria may account for the neuronal apoptosis induced by CGA-treated microglia.
Chromogranin A-stimulated Microglia Trigger the Release of
Mitochondrial Cytochrome c in Neurons--
The release of cytochrome
c from mitochondria has recently been described as an early
event that initiates apoptosis in neurons (2) and non-neuronal cells
(29). Once released, cytochrome c interacts with Apaf-1
(apoptotic protease-activating factor 1) and caspase 9 to
proteolytically activate procaspase 3. This activation of caspases is
responsible for the morphological and nuclear changes associated with
apoptosis (10, 38-40). To study whether microglial neurotoxins provoke
the release of cytochrome c from mitochondria in neurons, we
performed a double-staining experiment with anti-cytochrome
c antibody and the nuclear stain propidium iodide (Fig.
4). In neurons exposed to CM from resting microglia (Fig. 4, A and B), anti-cytochrome
c antibody revealed a punctuate staining in cell bodies and
processes, consistent with the mitochondrial location of this enzyme
(40). This pattern of immunostaining was dramatically altered in
neurons exposed to CM from CGA-stimulated microglia (Fig. 4,
C and D). After a 36-h exposure, the punctuate
immunostaining observed with the anti-cytochrome c antibody
had disappeared in most of the neurons, indicating the release of
cytochrome c from mitochondria and its dilution into the
cytoplasm. Time course analysis showed that release of mitochondrial
cytochrome c occurred after 22 h of exposure to
CGA-induced microglial toxins (Fig. 4E). At 36 h, more
than 70% of the neurons in CM from CGA-stimulated microglia had lost their punctuate anti-cytochrome c staining and exhibited
diffuse fluorescence with propidium iodide, indicating that the nuclear chromatin is not yet condensed (Fig. 4, C and D).
Thus, release of mitochondrial cytochrome c appears before
development of nuclear apoptotic changes, suggesting that the leakage
of cytochrome c from the mitochondria might account for the
induction of neuronal apoptosis after treatment with medium from
CGA-stimulated microglia.
Role of the Fas Receptor in Neuronal Death Induced by Chromogranin
A-stimulated Microglia--
The Fas/APO1/CD95 (Fas) transmembrane
receptor including a cytoplasmic death domain and its ligand (FasL)
have been recently described in neurons both in vitro (41,
42) and in the brain (43, 44). Ligation of Fas with soluble or membrane
bound FasL activates death domain, which in turn forms docking sites
for the cytoplasmic adapter protein FAS-associated death domain protein (FADD)/MORT1. FADD binds then to pro-caspase 8, thereby triggering its
autocatalytic activation and the proteolytic cascade leading to cell
death (28, 43, 45). FasL is also expressed on activated microglia and
can be cleaved proteolytically from the cell surface by
membrane-associated metalloproteinases (45-47). Therefore, we examined
the possible participation of Fas/FasL in mediating neuronal apoptosis
in response to CGA-activated microglia.
We first asked whether CGA may affect the expression or distribution of
FasL in microglial cells. As illustrated in Fig.
5, immunostaining with polyclonal
anti-FasL antibody revealed that FasL is already present in resting
microglia (Fig. 5A). Treatment with CGA had no pronounced
effect on the overall immunoreactivity but clearly induced the
appearance of a thin slightly irregular staining highlighting the
plasma membrane (Fig. 5B). Similar images were obtained with
two distinct anti-FasL antibodies (results not shown). Thus, CGA seems
to increase the association of FasL with the microglial cell surface.
To test the ability of Fas/FasL in triggering death of cultured
cortical neurons, we used sFasL, corresponding to a recombinant epitope-tagged form of the extracellular domain of human FasL. Incubation of Fas receptor-bearing cells with epitope-tagged sFasL and
anti-tag antibodies clusters sFasL and, thereby, Fas, leading to Fas
activation (29). The addition of sFasL and anti-tag antibodies to
neurons cultured in CM from resting microglia triggered apoptosis in a
dose-dependent manner (Fig. 5C). The induction
of neuronal apoptosis by sFasL was also found in neurons maintained in
unconditioned medium (results not shown). Note however, that sFasL (50 ng/ml) and CM from CGA-stimulated microglia had no additive effect on the percentage of apoptotic neurons, suggesting that the factors released by CGA-activated microglia and sFasL induce apoptosis through
a common mechanism.
To further probe the involvement of Fas signaling in the induction of
neuronal death by neurotoxins secreted by CGA-stimulated microglia, we
assessed the effect of Fas-Fc, which contains the extracellular domain
of recombinant human Fas fused to the Fc domain of IgG1 and antagonizes
the activation of Fas by FasL (28, 42). Fas-Fc inhibited the death of
neurons induced by a 48-h exposure to CM from CGA-stimulated microglia
in a dose-dependent manner (Fig. 5D). The
maximal effect (~75% inhibition) was observed at 5 µg/ml Fas-Fc,
which is close to the dose that prevents the death of cultured
motoneurons (42). Signaling through Fas receptors involves activation
of caspase 8 followed by the downstream caspases, including caspase 3. Caspase 3 is also activated by caspase 9 in response to the release of
mitochondrial cytochrome c (29, 48). To test the involvement
of these caspases in the death process induced by neurotoxins from
CGA-stimulated microglia, we used the cell-permeable peptides IETD and
DEVD, which are reported to inhibit irreversibly and selectively
caspase 8 and caspase 3, respectively (49). The addition of these
inhibitors to cultures of neurons 1 h before incubation in CM from
CGA-stimulated microglia prevented neuronal death (Table
I). After 48 h, IETD and DEVD reduced the apoptotic effect of CGA-activated microglia by 67 and 80%,
respectively, consistent with the requirement for caspases 8 and 3 in
CGA-mediated neuronal apoptosis.
Microglia are the resident macrophages of the central nervous
system. They play an important role in determining neuronal survival
and differentiation and provide the nervous system with a line of
defense against damage and infection by killing invading microorganisms
and removing dying cells. Thus, the activation of microglia represents
a beneficial physiological response in host defense. However, sustained
microglial activity may contribute to the pathogenesis of
neurodegeneration through the extensive release of various neurotoxic
agents such as proteases, proinflammatory cytokines, and reactive
oxygen and nitrogen intermediates (3-7). The specific molecules that
control microglia activity within the brain remain elusive. In numerous
neurodegenerative diseases, CGA is up-regulated and colocalizes with
reactive microglia. CGA is particularly prominent in senile plaques and
constitutes one of the major proteins along with In the present study, we examined the mechanisms by which neurotoxins
released from microglia in response to CGA provoke neuronal death.
Treatment of neuron/microglial co-cultures with CGA triggers two
features characteristic of apoptotic death (1, 36): the translocation
of phosphatidylserine from the inner to the outer face of the plasma
membrane and intranucleosomal cleavage of DNA in neurons. Culture
medium conditioned by CGA-activated microglia induces apoptosis in
neurons after a delay of 22 h, an observation that is consistent
with the release of diffusable neurotoxins from CGA-treated microglia
and the subsequent activation of a death program in neurons. Thus, we
propose that CGA represents an endogenous brain factor that drives
microglia to a neurotoxic phenotype.
Numerous studies have implicated MAP kinase pathways in the induction
of neuronal death, although their precise role is still unclear (1).
JNK activation and phosphorylation of c-Jun have been involved in the
induction of apoptotic neuronal cascades in Alzheimer's disease
(10, 11), and JNK has been associated with apoptosis induced by HIV-1
in human neurons (50). However, c-Jun expression has also been linked
to axonal regeneration and neuronal survival (51). We show here that
exposure of neurons to CGA-induced microglial neurotoxins does not
significantly modify JNK activity but induces a marked and transient
activation of p38 MAP kinase, which preceded the onset of neuronal
apoptosis. Pretreatment of neurons with a selective inhibitor of p38
MAP kinase partially protected neurons, arguing that p38 kinase is a
possible signal transductor for neuronal apoptosis induced by CGA-activated microglial neurotoxins. In cerebellar granular neurons, activation of p38 MAP kinase has been linked with apoptosis induced by
N-methyl-D-aspartate glutamate receptor (52). In
this context, it is interesting to note that Kingham et al.
(7) describe the release of glutamate from cultured microglia
stimulated with CGA. The activation of p38 kinase has been related to
altered calcium homeostasis and an increase in free radicals and
reactive oxygen species (2), which are considered as potent inducers of
mitochondrial damage (2, 29). Indeed, we found that exposure of neurons
to CM from CGA-activated microglia triggers the release of
mitochondrial cytochrome c into the cytosol before the
development of nuclear condensation. Within the cytosol, cytochrome
c is one of the cofactors that promotes caspase 9 activation, which in turn mobilizes downstream caspases such as caspase
3 (29). Taking into account the observation that cytochrome
c leakage from mitochondria preceded the appearance of
apoptosis together with the neuronal protection obtained with a caspase
3 inhibitor further supports the participation of the p38 MAP
kinase/cytochrome c-activated cascade in the induction of
neuronal apoptosis.
Although few data exist on the involvement of FasL/Fas in
neurodegenerative diseases, FasL and Fas have been recently identified on neurons in vitro and in the brain (41-44). In addition,
Fas was detected in adult postmortem brains of patients with
neurodegenerative disorders, including Alzheimer's disease (53). As an
indication of the role of FasL/Fas signaling in the induction of
neuronal death by microglial neurotoxins, we found that Fas-Fc, a known antagonist of Fas activation by FasL (28, 42), protected neurons exposed to CGA-induced microglial neurotoxins. Microglia express FasL,
and upon exposure to CGA, FasL is more prominent on the plasma
membrane. These findings are in line with previously published data
describing the constitutive expression of FasL by microglia in culture
and in brain and its up-regulation when microglia are activated (47).
Since membrane-associated FasL can be cleaved by metalloproteinase to
generate the active soluble form of the ligand (45, 46), soluble FasL
from CGA-stimulated microglia might contribute to the induction of
apoptosis in neurons. Accordingly, recombinant soluble FasL-triggered
apoptosis in neurons in a dose-dependent fashion and
neuronal death induced by microglial neurotoxins could be prevented by
cell-permeable peptides that inhibit caspase 8 and 3 involved in
FasL/Fas signaling. Thus FasL of microglial origin may well be one of
the neurotoxins secreted by microglia in response to CGA activation.
In conclusion, we provide evidence that neurotoxins released from
microglia activated by CGA trigger apoptotic death of cortical neurons
through pathways involving FasL/Fas receptor and p38 MAP stress kinase
as signal transductors and mitochondrial cytochrome c and
caspases 8 and-3 as apoptotic mediators. The neurotoxicity of activated
microglia can be attributed to a variety of secretory products,
including cytokines, complement proteins, proteolytic enzymes, reactive
oxygen intermediates and nitric oxide,
N-methyl-D-aspartate-like toxins (3-7, 26, 27,
54), and as shown here, membrane-bound or soluble FasL. Thus a
combination of these factors probably mobilizes several death-signaling
pathways in target neurons. The present findings together with the
recent reports describing the toxic cascades induced by CGA in
microglial cells (7, 54) establish CGA as a novel endogenous activator
of microglia able to induce inflammatory processes in the brain.
Inflammatory microglia are characteristic of many neurological
diseases, and the identification of the responsible agents may
therefore be relevant for novel therapeutic strategies. Increased
levels of CGA in cerebrospinal fluids have been found in patients with
Parkinson's disease (55) and has been correlated with early synaptic
degeneration in Alzheimer's disease (56). Our findings point to the
possibility that CGA accumulation is an early marker for the diagnosis
of brain inflammation and that CGA-induced neurotoxicity may be limited
by the use of anti-inflammatory drugs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-amyloid protein, is believed to
play a causal role in the pathogenesis of Alzheimer's disease and
other neuropathological disorders (9-11). Studies with synthetic
peptides show that
-amyloid protein is directly toxic to cultured
neurons (12, 13) and also induces the production of proinflammatory
cytokines in microglia (8-11). However, the initial events that
precede the deposit of the neurotoxic form of
-amyloid protein as
well as the direct contribution of activated microglia to the execution
of neuronal apoptosis are not clear. Understanding these steps may
therefore be relevant for the identification of novel therapeutic
strategies in treatment of neurodegenerative disorders.
-amyloid protein
in senile and pre-amyloid plaques (11, 15-17). Moreover, CGA has been
detected in large dystrophic neurites containing the amyloid precursor protein (15, 18, 19), suggesting that it may be one of the endogeneous
factors released from damaged neurons into brain deposits. We
previously determined that CGA is able to induce an activated phenotype
in cultured microglia, characterized by changes in morphology and actin
organization, generation of nitric oxide, secretion of tumor necrosis
factor
, and production of heat-stable diffusable factors that
provoke neuronal degeneration (26, 27). A recombinant N-terminal
fragment of human CGA, vasostatin I, corresponding to residues 1-78,
stimulates secretion of microglial neurotoxins to a similar extent,
suggesting that the active domain is present in the N-terminal region
of the CGA molecule (27).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C. The final CGA preparation was composed of the
native 70-kDa CGA (60% of the total proteins) and smaller processed
components (60 to 43 kDa), together representing 40% of the total
protein, as estimated from scanned monodimensional electrophoretic
profiles. Immunoblotting indicated that all protein bands were
immunoreactive with specific anti-native CGA antibodies (31) and
anti-CGA(124-143) antibodies (30, 31). Sequence analysis
(by automatic Edman degradation on an Applied Biosystems 473A
microsequencer) demonstrated that the final CGA preparation contained
only CGA-derived sequences with 80% of the sequenced material
containing the N-terminal sequence of CGA (LPVNS). The activity of the
CGA preparations was systematically controlled by measuring the
production of NO in microglial cell cultures as described previously
(26, 27).
20 °C and used either nondiluted or
diluted to 80% with serum-free defined medium. In all experiments,
conditioned medium from CGA-untreated and CGA-treated microglia were
collected simultaneously and used in parallel.
20 °C before processing.
-32P]ATP and the appropriate substrate:
myelin basic protein (3 µg) for p38 MAP kinase and glutathione
S-transferase-c-Jun (1 µg) for JNK1. After 20 min, the
reaction was stopped by the addition of electrophoresis sample buffer
and heating at 100 °C for 5 min. The proteins were resolved on 10%
polyacrylamide-SDS gels, and the gels were dried and exposed to
phosphoimaging screens (BioMax Light-1, Kodak).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-3H]aminobutyric acid uptake and leads to neuronal
degeneration (27). To further characterize neuronal death induced by
CGA-activated microglia, we examined the binding of annexin V and the
fragmentation of nuclear DNA in neuron/microglial co-cultures treated
with 10 nM CGA. Binding of annexin V to the cell surface
reveals the translocation of phosphatidylserine from the inner side of
the plasma membrane to the outer layer. When used in conjunction with
propidium iodide exclusion to establish membrane integrity, it
indicates cells undergoing apoptosis (34, 35). In addition, we used the
TUNEL technique to label DNA fragments at the 3'-OH DNA and, thus,
identify nuclear DNA that is cleaved by endonucleases (35). Fig.
1 shows representative confocal images of
neurons in co-culture with microglia for 3 days incubated with or
without 10 nM CGA. In untreated co-cultures (Fig.
1A) annexin V (conjugated with fluorescein) did not bind to
neurons identified with anti-MAP2 antibody. In contrast, most neuronal
bodies and processes in CGA-treated co-cultures were simultaneously
stained for annexin V and MAP2 (Fig. 1B). In both untreated
and CGA-treated co-cultures, nuclei were generally not labeled with PI
when living cells were exposed to PI before fixation, indicating that
the plasma membrane is impermeable to the dye. The percentages of
PI-positive, MAP2-positive neurons in resting and CGA-stimulated
co-cultures were 5.8 ± 1.6 and 9.5 ± 1.9 (mean ± S.E.; n = 3), respectively. Using the TUNEL technique,
labeling of nuclei was observed in only a few MAP2-positive neurons in untreated co-cultures (Fig. 1C). However, almost all nuclei
of MAP2-positive neurons were stained in co-cultures exposed to CGA (Fig. 1D). In addition, CGA-treated co-cultures contained a
substantial proportion of TUNEL-positive, MAP2-negative cells, probably
corresponding to microglia. Taken together, these results suggest that
CGA induces an apoptotic cascade leading to neuronal death in
neuron/microglial co-cultures.
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Fig. 1.
Neuronal death in neuron/microglial
co-cultures exposed to chromogranin A. Neuron/microglial
co-cultures were either untreated (A and C) or
treated for 3 days with 10 nM CGA (B and
D). In A and B living cells were
incubated with propidium iodide (2.5 µg/ml) in culture medium and
then with fluorescein-conjugated annexin V. After fixation, cells were
labeled with anti-MAP2 antibody revealed with rhodamine. In
C and D fixed cultures were processed with TUNEL
technique using dUTP-biotin and streptavidin-fluorescein and with
anti-MAP2 antibody revealed with rhodamine. Confocal images obtained in
the rhodamine and fluorescein channels were recorded simultaneously in
the same optical section by a double-exposure procedure. The
arrow points to a TUNEL-positive MAP2-positive neuron. The
arrowhead indicates several TUNEL-positive, MAP2-negative
cells with smaller nuclei which are presumed to be microglia.
Bar = 25 µm.
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Fig. 2.
Neurotoxic factors released from chromogranin
A-activated microglia induce apoptosis in neurons. Cultured
neurons were grown in cell-free medium collected either from resting
microglia (open symbol) or from microglia stimulated with 10 nM CGA (closed symbol), and necrotic and
apoptotic neurons were counted. Living neurons labeled with trypan blue
(0.1%, 5 min of incubation) were considered as necrotic. The presence
of apoptotic profiles was revealed in neurons after fixation by
staining with Hoechst 33342 dye (2.5 µg/ml, 5 min). Neurons with
segmented and highly condensed nuclear fluorescence were counted as
apoptotic and expressed as the percentage of the total number of cells
present per field. Values are the means of eight determinations ± S.E. and are representative of at least three independent
experiments.
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Fig. 3.
Effect of conditioned medium from resting or
chromogranin A-stimulated microglia on JNK and p38 MAPK in
neurons. A, Western blotting showing the protein level
of the total and phosphorylated forms of JNK and p38 MAP kinases in
neurons incubated for 15 min with medium from microglia cultured
without ( CGA) or with 10 nM CGA (+CGA). Triton
X-100-soluble cytosolic (lane 1) and Triton X-100-insoluble
cytoskeletal (lane 2) protein fractions from neurons were
resolved by electrophoresis and transferred onto nitrocellulose, and
blots were probed with antibodies against p46 JNK1 (JNK),
phosphorylated JNK (pJNK), p38 MAP kinase (p38),
and phosphorylated p38 MAP kinase (pp38). Immunodetection of
actin (arrowhead) is shown as an internal standard for
protein loading. The results shown are representative of four
independent experiments. B, the activity of JNK or p38 MAPK
was measured in immunoprecipitates from neurons exposed for the
indicated period of time to CM from resting (
CGA) or CGA-stimulated
(+CGA) microglia. To inhibit p38 MAPK, neurons were incubated for
1 h with 30 µM SB 203580 before the addition of CM.
The radioactivity incorporated into the substrates was estimated after
electrophoresis by optical scanning densitometry on the corresponding
autoradiogram. Values are expressed relative to the kinase activities
detected in neurons incubated for 15 min with CM from resting
microglia. Results are representative of three independent experiments.
Student's t test was used for estimating significance: **,
p < 0.001. C, neurons were incubated for
48 h with CM from resting (
CGA) or CGA-stimulated (+CGA)
microglia in the presence of the indicated concentrations of SB 203580 added 1 h before treatment with CM. The number of apoptotic nuclei
was assessed by Hoechst 33342 staining. Data are the mean percentage of
apoptotic cells ± S.E. and are representative of three
independent experiments. Student's t test results: *
p < 0.01.
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Fig. 4.
Neurotoxic factors secreted by chromogranin
A-activated microglia induce the release of mitochondrial cytochrome
c in neurons. Representative photographs of fixed
cultured neurons double-labeled with anti-cytochrome c
antibody (A and C) and the nuclear stain
propidium iodide (B and D). Neurons were exposed
for 36 h to CM collected from resting microglia (A and
B) or to CM from CGA-activated microglia (C and
D). Confocal images were taken under identical set-up
conditions. Bar = 12 µm. The arrow points
to a neuron with reduced immunoreactivity for cytochrome c
(C) and having a nucleus showing noncondensed chromatin
(D). E, time course of the loss of
anti-cytochrome c immunostaining in neurons exposed to CM
from CGA-stimulated microglia. Values are the means of eight
determinations performed with the three cultures ± S.E.
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Fig. 5.
FasL/Fas signaling in neuronal apoptosis
triggered by CGA-activated microglia. Double-staining with
anti-FasL antibody and propidium iodide (2.5 µg/ml) of microglial
cells untreated (A) and treated with 10 nM CGA
(B). Note the appearance of FasL immunoreactivity on the
plasma membrane of microglia exposed to CGA (arrows).
Bar = 12 µm. C, effect of sFasL on
neuronal apoptosis. Tagged sFasL at the indicated concentrations
together with 1 µg/ml anti-tag antibody were added to neurons
cultured with CM from resting ( CGA) or CGA-stimulated microglia
(+CGA). Apoptotic nuclei were assessed by Hoechst 33342 staining after
24 h. Student's t test results: * p > 0.01. D, effect of Fas-Fc, an inhibitor of Fas activation by
FasL, on neuronal apoptosis induced by CGA-induced microglial toxins.
Neurons were preincubated for 30 min with the indicated concentrations
of Fas-Fc and enhancer antibody (1 µg/ml) before the addition of CM
from resting (
CGA) or CGA-stimulated (+CGA) microglia. Apoptotic
cells were revealed after 48 h by Hoechst staining. Histograms are
representative of three independent experiments. Data are the mean
percentage of apoptotic cells ± S.E.
Death of cortical neurons triggered by neurotoxic factors from
CGA-activated microglia involves caspases 8 and 3
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-amyloid protein in
extracellular deposits associated with Alzheimer's disease (11,
15-17). The presence of CGA in brain lesions led us to examine whether
CGA could contribute to neuronal degeneration. We found that CGA can induce an activated phenotype in rodent microglial cells (26) and
triggers the release of microglial factors that cause injury and
degeneration of brain cortical neurons (27).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. N. J. Grant and Dr. K. Langley for suggestions and for revising the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: CNRS UPR-2356, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France. Tel.: 33 88 45 67 15; Fax: 33 88 60 16 64; E-mail: ciesielski-treska@
neurochem.u-strasbg.fr.
§ Present address: CNRS UPR-2356 Neurotransmission et Sécrétion Neuroendocrine, Centre de Neurochimie, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M009711200
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ABBREVIATIONS |
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The abbreviations used are: CGA, chromogranin A; DMEM, Dulbecco's modified Eagle's medium; sFasL, soluble Fas ligand; PI, propidium iodide; PBS, phosphate-buffered saline; MAP2, microtubule-associated protein 2; MAP kinase, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; TUNEL, terminal dUTP nick-end labeling; CM, conditioned medium.
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