From INSERM EMI 0014, Université de Nancy I,
54505 Vandoeuvre, France and
INSERM U-505, Université
de Paris 6, 75006 Paris, France
Received for publication, July 8, 2002, and in revised form, October 14, 2002
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ABSTRACT |
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In the present study, we have determined
the nature and the kinetics of the cellular events triggered by the
exposure of cells to non-fibrillar amyloid- A common feature of Alzheimer's disease (AD),1
the most common form of dementia,
is the accumulation and the aggregation of the amyloid- These results emphasize the necessity to clarify the initial response
of neurons to the non-fibrillar A The aim of the present paper was to identify the primary targets of the
non-fibrillar A In the present study, we have investigated whether the microtubule
network could be an early cellular target for the non-fibrillar A Materials--
A Peptide Solubilization--
To overcome problems of amyloid
peptide solubility at high concentrations, fresh peptide stock
solutions were prepared at 5 mg × ml Neuronal Cell Culture--
Cortical neurons from embryonic day
16-17 Wistar rat fetuses were prepared as described previously (18).
Briefly, dissociated cells were plated at 4.5-5.0 104
cells/cm2 in plastic dishes pre-coated with
poly-L-ornithine (1.5 µg × ml Neuronal Viability--
Experiments were performed on six to
seven DIV neurons. Cell viability was first determined by morphological
observation and cell counting after 5 min of trypan blue staining
(0.4%; Sigma) to evaluate membrane integrity, and the metabolic
activity was assessed by the MTT reduction assay (18, 22). Moreover,
the release of lactate dehydrogenase into the culture medium was
assessed using a cytotoxicity detection kit (Roche Molecular
Biochemicals) according to the recommendations of the manufacturer.
Monitoring of Apoptosis--
Cell nuclei were visualized using
4,6-diamidino-2-phenylindole (DAPI; Sigma). The cells, grown on a glass
coverslips, were washed in PBS, incubated at room temperature for 10 min with DAPI (0.1 µg × ml Measurement of Caspase-like Proteolytic Activities--
The
caspase activities were measured by means of the cleavage of the
substrates DEVD-pNA, YVAD-pNa, LEHD-AMC, and IEPD-AMC (Bachem).
Briefly, at the indicated time points following peptide treatments, the
cells were rinsed three times with ice-cold PBS and incubated for 20 min on ice in a buffer of 25 mM Hepes, pH 7.5, 1% (v/v)
Triton X-100, 5 mM EDTA, 1 mM EGTA, 5 mM MgCl2, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml each of
pepstatin and leupeptin, and 5 µg/ml aprotinin. The lysate was
centrifuged for 15 min at 12,000 rpm and assayed for protein by
Bradford (Bio-Rad). 50 µg of proteins were incubated for 2 h
with 100 µM caspase substrates initially dissolved in
Me2SO. The cleavage of the caspase substrates was
monitored by absorbance measurements at 405 nm for DEVD-pNA and
YVAD-pNa and by fluorescence emission at 460 nm after exciting LEHD-AMC
and IEPD-AMC at 360 nm, using a Fluostar reader plate
(BMG-Labtechnologies).
DCFH-DA Assay--
The measurement of cell oxidation is based on
the oxidation of the non-fluorescent compounds, DCFH-DA, to a
fluorescent derivative, DCF, in a peroxidase-mediated reaction.
Increases in fluorescence emission reflect enhanced cellular oxidative
stress. Briefly, treated cortical neurons were loaded with 100 µM DCFH-DA for 45 min. Before analysis, cells were washed
three times in PBS, and DCF fluorescence was recorded directly on
culture dishes by a Fluostar reader plate (BMG-Labtechnologies),
using 488-nm excitation and 510-nm emission filters.
Electron Microscopy--
Cortical neurons were fixed for 2 h at 4 °C in 2.5% glutaraldehyde and 0.5% tannic acid in 0.1 M cacodylate buffer, pH 7.4. Then the neurons were
postfixed for 2 h at 4 °C in 2% osmic acid in phosphate
buffer. After having been dehydrated in a graded alcohol series, the
samples were embedded in Epon resin (Poly/Bed 812; Polysciences,
Warrington, PA), and ultra thin sections (70-nm) were obtained using a
Reichert Ultracut. Thin sections were counterstained with uranyl
acetate, and lead citrate and examined with a Jeol 100CX microscope.
Immunofluorescence--
For immunofluorescence studies, the
neurons were cultured on glass coverslips that had been coated
overnight with 15 µg/ml poly L-ornithine. Following the
treatments, the neurons were fixed in PBS containing 4%
paraformaldehyde for 30 min at room temperature. The cells were
permeabilized with 0.1% Triton X-100 made up in PBS containing 3%
bovine serum albumin for 30 min and then incubated with a monoclonal
anti- Cell Labeling for Photo Bleaching--
The cells were incubated
for shorter times with A Measurement of the Lateral Fluidity of Cell Membrane--
The
measurement of the lateral diffusion of the molecules in the membrane
by means of FRAP (fluorescence recovery
after photo bleaching) has already been
described (32). A fluorescent probe, here a labeled lipid, was
incorporated into the cell membranes. When a small defined area of the
labeled membrane was photobleached by a high powerful laser pulse, the
intensity of fluorescence was reduced immediately. As the fluorescent
probe present in the vicinity underwent a constant diffusion motion, it
diffused into the bleached area, gradually increasing the fluorescence
intensity after bleaching. Thus, the kinetics of the fluorescent
recovery depends on the diffusion rate of the probe, measured as the
diffusion coefficient, D20,w.
Analyses were performed at 37 °C with a Zeiss LSM 510 confocal laser
scanning microscope. We used the 488-nm line of a 25-milliwatt argon
laser with a Zeiss C-Apochromat, ×63, numerical aperture 1.2, oil-immersion objective. The pinhole diameter was set to 1 airy unit,
which correspond to a 1.2-µm depth of field, to reduce the
contribution of cytoplasm fluorescence as much as possible. The area of
bleaching was defined as a circle of 3.0-µm diameter, centered on an
apical body cell membrane. The area was photobleached at full laser
power (100% power, 100% transmission) for 230 ms. The extent of the
bleaching typically reached 50 to 80%. Before bleaching, five images
were monitored to define the initial fluorescence. The post-bleached
images were scanned at 0.6% transmission with a delay of 100 ms during
the last 60 s, resulting in a total acquisition of 80 points for
90 s, the time required to complete the maximum recovery of
fluorescence. No photobleaching was observed during recovery. The sets
of scans in which the fraction mobile of fluorophore was less than 65% or more than 110% and bleaching less than 35% were discarded. Cells
presenting debris, heterogeneous labeling, or movements during scanning
were not used for FRAP measurements.
D20,w parameters were calculated
according to the method described by Kubitscheck et al.
(33).
Statistical Analysis--
STAT VIEW computer software was used
for the statistical analysis. Most of the data were from three separate
experiments with three to four determinations each. Values were
expressed as means + S.E. Differences between control and treated
groups were analyzed using Student's t test. Multiple
pairwise comparisons among the groups of data were performed using
ANOVA followed by a Scheffe's post hoc test. Statistical differences
were determined at p < 0.05.
Early Perturbations of the Neurotubule Organization upon A Taxol Prevents Cytoskeleton Disruption and Neurotoxicity Induced by
the Soluble A
In agreement with these morphological and biochemical
observations, we demonstrated that taxol inhibited apoptosis induced by
low concentrations of non-fibrillar A
To further improve our results, we quantified the cytoskeleton
perturbation induced by the non-fibrillar A A A
We reported previously that A Non-fibrillar A
We further investigated the effects of taxol on A In the present paper, we demonstrate for the first time that
(a) the cytoskeleton is an early cellular target for the
non-fibrillar A It has already been suggested that early modifications in the
progression of AD pathology might involve the disorganization of the
cytoskeleton of neurons (37, 38). Here, we show that when exposed to
soluble A Accumulative evidences emphasize the critical role of oxidative stress
in AD and in the neurotoxicity of A Finally, our results demonstrate for the first time that soluble A We have demonstrated recently that the non-fibrillar A Tailing our data, several observations suggest that the deleterious
effects of A The direct relationships between the perturbation of the plasma
membrane properties and cell death induced by the non-fibrillar A Altogether, our data strongly highlight the role of the non-fibrillar
forms of A peptide (A
). When
cortical neurons were treated with low concentrations of soluble A
(1-40), an early reactive oxygen species
(ROS)-dependent cytoskeleton disruption precedes caspase
activation. Indeed, caspase activation and neuronal cell death were
prevented by the microtubule-stabilizing drug taxol. A perturbation of
the microtubule network was noticeable after being exposed to A
for
1 h, as revealed by electron microscopy and immunocytochemistry.
Microtubule disruption and neuronal cell death induced by A
were
inhibited in the presence of antioxidant molecules, such as probucol.
These data highlight the critical role of ROS production in
A
-mediated cytoskeleton disruption and neuronal cell death. Finally,
using FRAP (fluorescence recovery after photo bleaching) analysis, we observed a
time-dependent biphasic modification of plasma membrane
fluidity, as early as microtubule disorganization. Interestingly,
molecules that inhibited neurotubule perturbation and cell death did
not affect the membrane destabilizing properties of A
, suggesting
that the lipid phase of the plasma membrane might represent the
earliest target for A
. Altogether our results convey the idea that
upon interaction with the plasma membrane, the non-fibrillar A
induces a rapid ROS-dependent disorganization of the
cytoskeleton, which results in apoptosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
peptide
(A
), a 39- to 43-amino acid peptide derived from the proteolytic
cleavage of the amyloid precursor protein (1, 2). Although A
represents a key factor in AD (3), the nature of the toxic form of A
early involved in AD pathology remains unclear. Whether it is the
fibrillar or the non-fibrillar peptides that are the more deleterious
remains a controversial issue (4). The amyloid cascade hypothesis
causally links AD clinico-pathological process and neuronal cell death
to the aggregation and deposition of A
(5-7). However, this
hypothesis has been challenged by recent evidences indicating that the
non-fibrillar A
also plays a major role in AD (8, 9). A recent
elegant study has demonstrated that the fibrils from AD brain are
composed of amyloid peptide moieties arranged at right angles to the
backbone of the amyloid P protein wrapped in glycosaminoglycans (10). Thus, the fibrils are not simply made of chains of self-aggregated A
and do not comprise long chains of multimeric A
, similar to those
used to evaluate the neurotoxicity of the fibrillar A
in vitro and in vivo. Moreover, the synaptic loss in AD
brain has been correlated with the soluble pool of A
peptides rather
than the fibrillar one, implying that the non-fibrillar A
may be a crucial pathological factor in AD (11-13). Several studies, based on
the use of transgenic mice, have demonstrated that neurodegeneration and specific spatial learning deficits might occur without amyloid plaque formation (14-17).
and to identify the cellular
targets involved in non-fibrillar A
-induced neurotoxicity. Tailing
with these observations, our studies and others rely on the hypothesis
of a close association between neuronal loss and a proapoptotic effect
of soluble forms of A
(18-21). Indeed, it has been established that
the amphiphilic non-aggregated A
may intercalate into the plasma
membrane of neurons, directly altering membrane activities and inducing
neuronal cell death (18, 22-25). Accumulative evidences have laid
emphasis on the critical role of an oxidative stress in AD and in the
neurotoxicity induced by A
(26, 27). However, most of the molecular
mechanisms involved in the neuronal cell death induced by non-fibrillar
forms of A
are yet to be characterized.
and the chronology of the early cellular events
involved in apoptotic neuronal cell death upon A
exposure. Microtubules fulfill a plethora of cellular functions, including axonal
and dendritic growth and stability (28, 29). We have proved recently
that the non-fibrillar A
(1-40) induces apoptosis to rat cortical
neurons (18, 22).
.
We have demonstrated the following sequence: low concentrations of
soluble A
(1-40), or of its shorter (29-40) C-terminal domain, induce apoptotic neuronal cell death by perturbing the fluidity of the
plasma membrane, leading to a disruption of the neurotubule network
depending on the induction of an early oxidative stress.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(1-40), A
(29-40), the caspase
substrate, and inhibitor peptides were purchased from Bachem, and
DCFH-DA was from Molecular Probes. Unless otherwise indicated,
materials used for cell culture were obtained from Invitrogen.
The drug stabilizing cytoskeleton, taxol, and all other chemicals were
of high purity grade from Sigma.
1 in
hexafluoro-2-propanol (Sigma) as described previously (30). For the
incubation of the peptides with the neurons, aliquots of peptide stock
solution were quickly dried under nitrogen and directly solubilized at
the experimental concentrations into the culture medium. Peptide
solutions were then applied onto the cells. Under those conditions, all
the amyloid peptides remained soluble for the determination of their
neurotoxic properties (18).
1;
Sigma). The cells were cultured in a chemically defined
Dulbecco's modified Eagle's medium-F12 medium free of serum
(Invitrogen) and supplemented with insulin (5.10
7
M), putrescine (60 µM), sodium selenite (30 nM), transferrin (100 µM), progesterone
(1.10
7 M), and 0.1% (w/v) ovalbumin.
Cultures were kept at 35 °C in a humidified 6% CO2
atmosphere. After six to seven DIV, cortical population was
determined to be at least 95% neuronal by immunostaining as described
previously (18, 31).
1), washed with PBS,
and examined under a microscope equipped for epifluorescence. To
evaluate the percentage of apoptotic cells, five independent fields of
microscope were counted (around 100 cells) in three separate
experiments, with two determinations each. Under control conditions,
neuronal cells exhibited 12-15% of apoptotic cells at nine DIV. For
experiments in the presence of caspase inhibitors, cells were incubated
2 h with 50 or 100 µM inhibitors before and
throughout A
peptide exposure. Alternatively, DNA fragmentation was
monitored by enzyme-linked immunosorbent assay for the detection of
oligonucleosomes using a kit form purchased from Roche Molecular
Biochemicals. Briefly, cortical neurons were plated in 24-well dishes
(around 200.000 cells per well) and treated at seven DIV for 24 h
with A
. After having been washed, the cells were lysed directly on
wells, and oligonucleosomes were determined according to the
recommendations of the manufacturer.
-tubulin antibody (1:500) (Chemicon) for 1 h under
constant agitation. After several washes in PBS, the cells were
incubated for 1 h with a fluorescein isothiocyanate-conjugated donkey anti-mouse IgG (1: 250) (Santa Cruz Biotechnology), washed with
PBS, labeled with DAPI as described above, and mounted in Fluoprep
(BioMérieux). The microtubules were visualized with a Nikon
microscope using a PlanFluor X40/1.3 objective. For semiquantitative analysis of microtubule organization, at least five microscope fields/condition were imaged using a Nikon DXM1200 digital camera, and
microtubule organization in 100-120 cells/field was classified as
normal, mildly disrupted, or severely disrupted.
(1-40) in Hanks' balanced salt solution
at room temperature and loaded with 4 µM NBD-SM (1 mg/ml
stock solution in chloroform). In the case of longer treatments (up to
24 h), the neurons were incubated with A
(1-40) in normal
culture conditions, then rinsed with Hanks' balanced salt solution and
stained with the fluorescent lipid, and analyzed 10 min thereafter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Treatment--
To investigate the kinetics of microtubule network
disorganization, cortical neurons were exposed to 5 µM
soluble A
(1-40) for short incubation times at the end of which no
morphological feature of apoptosis (e.g. membrane bleeding,
cell shrinkage, and chromatin condensation) was detected (see Figs.
1 and 2). After a 3-h A
(1-40) exposure, we observe a
dramatic perturbation of the neurotubule
network in most of the neurites of the treated neurons (Fig.
1c), as compared with the control cells in which neurotubules elongated within the neurites normally, in a parallel organization (Fig. 1a). Similar results were obtained using
the shorter A
(29-40) which displays also membrane perturbing
properties (data not shown). These observations made through electron
microscopy were confirmed using immunocytochemistry. In the control
cells, a dense and constant microtubule network radiates from the cell bodies to the periphery (Fig. 2, A and B). By
contrast, in the cortical neurons treated with 5 µM
non-fibrillar A
(1-40) for 1 and 3 h (Fig. 2, C and
D, respectively), we observed a peripheral fragmentation and
loss of microtubules without any fragmentation or condensation of
nuclear DNA (Fig. 2C). Despite this severe microtubule
disruption, the treated cells maintained their spreading shape,
implying that the non-fibrillar A
(1-40) did not affect neurofilaments. Higher peptide concentrations or longer incubation times resulted in a more extensive and rapid loss of the microtubule network (not shown).
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Fig. 1.
The non-fibrillar A
(1-40) peptide induces early cytoskeleton perturbations.
a, transmission electron micrographs of untreated rat
cortical neurons, showing a normal parallel organization of the
microtubule network. Cells incubated for 3 h with 100 nM taxol exhibited a normal cytoskeleton morphology
(b and b'), whereas the cortical neurons treated
for 3 h with 5 µM soluble A
(1-40) exhibited
neurotubule disorganization, with very short curly unparalleled
neurotube segments (c). The presence of 100 nM
taxol in the culture medium protects the cortical neurons from soluble
A
(1-40)-induced neurotubule disruption (D). The total
magnification for all pictures was ×16,800.
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Fig. 2.
The non-fibrillar A
(1-40) peptide disrupts neuronal microtubules.
Cortical neurons were incubated in the absence (A and
B) or in the presence of 5 µM soluble A
(1-40) (C and D) for 1 and 3 h,
respectively. The microtubule organization was visualized using
immunofluorescence with an anti
-tubulin monoclonal antibody.
Peptide--
To determine the kinetics of onset of
the early microtubule perturbations induced by the non-fibrillar A
(1-40) and of the apoptotic neuronal cell death, we investigated the
effects of taxol, a microtubule-stabilizing drug, on A
-induced
cytoskeleton disruption and neurotoxicity. Cortical neurons incubated
with 100 nM taxol only displayed a typical microtubule
organization (Fig. 1, b and b') as described
previously (34). Interestingly, the non-fibrillar A
(1-40) was
unable to disrupt the neuronal microtubule network in the cells
preincubated for 2 h with 100 nM taxol before A
(1-40) addition (Fig. 1D). These results have been
confirmed using immunocytochemistry and with taxol being added to the
cortical neurons at the same time as A
(1-40) (not shown). We next
investigated the effects of taxol on non-fibrillar A
-induced
neuronal cell death. As described previously (18), the treatment of
cortical neurons with 5 µM A
(1-40) resulted in a
time-dependent decrease in cell viability monitored by the MTT assay (Fig. 3A). Upon a
6-h exposure to A
(1-40), the MTT reduction level decreased
significantly to 18.3% (p < 0.05) compared with the
control level. Interestingly, whereas taxol alone displayed no effect
on MTT even after 48 h of treatment, its presence protected the
neurons against A
(1-40)-induced neurotoxicity (Fig.
3A). After a 48-h exposure to A
in the presence of 100 nM taxol, the MTT reduction level remained at 82% of
control, whereas the protective effects of taxol diminished after
prolonged incubations. Moreover, the presence of taxol in the culture
medium inhibited the release of lactate dehydrogenase after a 48-h A
(1-40) treatment (Fig. 3B). This suggests that the
stabilization of the microtubule organization by taxol prevents
A
-induced neuronal cell death.
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Fig. 3.
Taxol modulates the neurotoxicity of
non-fibrillar A (1-40). Cortical neurons
were preincubated or not for 2 h with 100 nM taxol and
then treated for the indicated incubation time with 5 µM
non-fibrillar A
(1-40). The neurotoxicity of A
(1-40) was
monitored as a function of time by the MTT assay (A) or by
the measurement of the lactate dehydrogenase release after a 48-h
incubation (B). Data are means (± S.E.) of three different
experiments with four determinations each and are normalized to the
effect of vehicle, designated as 100% (*, p < 0.05;
**, p < 0.01; ***, p < 0.001).
Differences among the subgroups for each condition were performed by
ANOVA followed by a Scheffe's post hoc test. #, p < 0.05 between cells treated with A
alone and cells treated with A
in the presence of taxol. No significant differences were found between
taxol-treated and control cells.
(1-40). The apoptotic nuclei
were visualized and quantified after DAPI staining of cultures treated
with 5 µM A
(1-40) in the absence and
presence of 100 nM taxol. Upon A
exposure, cortical
neurons shared a time-dependent increase in the number of
apoptotic nuclei, which was statistically different from control after
24 h of incubation and reached 58.6 + 3.1% (p < 0.001) after 48 h of incubation (Fig.
4A). The presence of taxol in
the culture medium almost completely inhibited the apoptotic cell death
induced by A
(1-40) after a 24-h incubation, and the effects
persisted for up to 48 h (23.4 + 2.5% of apoptotic nuclei)
(Fig. 4A).
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Fig. 4.
Effects of taxol on soluble
A (1-40)-induced apoptosis and microtubule
disruption. Cortical neurons were preincubated or not for 2 h
with 100 nM taxol and then treated for the indicated
incubation time with 5 µM non-fibrillar A
(1-40).
Apoptotic nuclei were visualized and quantified after DAPI staining
(A and C), and the microtubule perturbations were
quantified using immunofluorescence with an antibody against
-tubulin (B and C). Data are means (± S.E.)
of three different experiments with four determinations each and are
normalized to the effect of vehicle, designated as 100% (*,
p < 0.05; **, p < 0.01; ***,
p < 0.001). Differences among the subgroups for each
condition were performed by ANOVA followed by a Scheffe's post hoc
test. #, p < 0.05 between cells treated with A
alone and cells treated with A
in the presence of taxol. No
significant differences were found between taxol-treated and control
cells.
(1-40) using
immunocytochemistry with an anti-
-tubulin antibody. The A
-induced
microtubule disruption was time-dependent (Fig.
4B). As early as 1 h after the addition of 5 µM soluble A
(1-40) to the cells, we observed a
greater number of neurons exhibiting a mild disruption of microtubules (25.2 + 1.8%, p < 0.01). After a 3-h incubation, the
microtubule network was mildly or severely disrupted in 35.2 + 3.5%
(p < 0.001) of the treated cells, and almost all the
neurons displayed disturbed microtubules after a 24-h incubation (Fig.
4B). Interestingly, the presence of taxol completely
abolished microtubule perturbation induced by the non-fibrillar A
peptide (Fig. 4B). These data strongly emphasize that early
cytoskeleton disruption is required in the neuronal cell death induced
by non-fibrillar A
peptide. Moreover, Fig. 4C showed only
few cells exhibiting both disorganized microtubules and apoptotic
nuclei. Altogether, these results strongly suggest that microtubule
perturbations precede, and might be involved in, a pathway leading to
apoptosis upon soluble A
exposure.
Peptide-induced Cytoskeleton Disruption Precedes Caspase
Activation--
In a previous report, we demonstrated that the caspase
3 inhibitor, DEVD-CHO peptide, reduced neuronal cell death induced by
the non-fibrillar A
(1-40) significantly (18). Here, we clearly
show that the apoptotic cell death induced by non-fibrillar A
(1-40) requires the activation of caspases 3 and 9 by directly measuring caspase-like activity in the lysates of the treated cells
(Fig. 5). The activity of caspases 3 and
9 increased significantly (p < 0.05 as compared with
the control cells) after a 6-h incubation with 5 µM A
(1-40) (Fig. 5, A and D, respectively), whereas
the activation of caspases 1 and 8 was not detected (Fig. 5,
B and C, respectively). To establish a causative
relationship between microtubule disruption and caspase activation, we
performed kinetic experiments of microtubule perturbation in the
absence and presence of caspase inhibitors (Fig.
6). Fig. 6A demonstrates that
both caspase inhibitors markedly reduced apoptosis induced by 5 µM non-fibrillar A
(1-40). However, unlike taxol, the
caspase inhibitors had no effect on the early cytoskeleton
perturbations induced by A
(1-40), even after a prolonged
incubation (Fig. 6B). Moreover, the presence of 100 nM taxol during A
exposure inhibited the activation of
caspases 3 and 9 significantly (not shown). These results suggest that
the microtubule perturbation might occur before caspase activation
under treatment with non-fibrillar A
(1-40).
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Fig. 5.
Neuronal apoptosis induced by non-fibrillar
A (1-40) involves caspase activation.
Cortical neurons were exposed to 5 µM soluble A
(1-40) or A
(29-40) for the indicated incubation times. The
activation of caspase 3 (A), caspase 1 (B),
caspase 8 (C), and caspase 9 (D) was monitored by
measuring the proteolytic cleavage of the caspase-related substrates,
as indicated under "Experimental Procedures." Data are means (± S.E.) of three different experiments with four determinations each. *,
p < 0.05; **, p < 0.01.
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Fig. 6.
Effects of caspase inhibitors on
soluble A (1-40)-induced apoptosis and
microtubule disruption. Cortical neurons were preincubated or not
for 2 h with 100 µM caspase inhibitors and then
treated for the indicated incubation time with 5 µM
non-fibrillar A
(1-40). Apoptotic nuclei were visualized and
quantified after DAPI staining (A), and microtubule
perturbations were quantified by immunofluorescence using an antibody
against
-tubulin (B). Data are means (± S.E.) of three
different experiments with four determinations each and are normalized
to the effect of vehicle, designated as 100% (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
No significant differences were found between caspase inhibitor-treated
and control cells.
-induced Cytoskeleton Disruption Involves an Oxidative
Stress--
We showed that cell exposure to 5 µM
non-fibrillar A
(1-40) peptide induced a time-dependent
increase ROS formation, as measured by the oxidative stress-sensitive
dye DCFH-DA. A 1- to 3-h treatment with A
caused a significant
increase in ROS production (Fig. 7).
During these short incubation times, the neuronal membrane appeared
intact as determined by trypan blue exclusion (data not shown). The
first clear signs of cell damages, e.g. membrane bleeding and trypan blue staining, appeared only after a 6-h exposure to 5 µM non-fibrillar A
(1-40) (7% of the control cells
were trypan blue-positive versus 16% of the treated
neurons, of 200 cells counted for each condition). In addition, we
tested the effects of several antioxidant molecules on A
-induced
neurotoxicity (Fig. 8). A 2-h
preincubation of the cortical neurons with 10 µM
probucol, 1 µM promethazine, or 5 µM propyl
gallate prior to non-fibrillar A
(1-40) peptide exposure induced a
persistent increase in cell survival monitored by the MTT assay (Fig.
8A) and an inhibition of neuronal apoptosis as determined by
DAPI staining (Fig. 8B). Finally, we clearly demonstrated,
using immunocytochemistry, that antioxidant molecules prevented
A
-induced cytoskeleton disruption (Fig.
9, A, B, and
C). The quantification of the number of cells exhibiting
disorganized microtubules indicates that after a 6-h incubation, the
presence of 10 µM probucol during A
(1-40) treatment significantly reduced the number of cells exhibiting disordered microtubules, even after a 24-h incubation (18.9 + 5.1%,
p < 0.01, versus 40.5 + 4.1% in the
absence of probucol) (Fig. 9D). Moreover, we observed that
the effects of probucol on A
-induced cytoskeleton perturbation was
concentration-dependent (not shown).
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Fig. 7.
The non-fibrillar
A is toxic via oxidative stress. Cortical
neurons were incubated for the indicated incubation time with 5 µM A
(1-40), and the production of reactive oxygen
species was monitored by measuring the fluorescence of DCF as indicated
under "Experimental Procedures." Data are means (± S.E.) of three
different experiments with four determinations each and are normalized
to the effect of vehicle, designated as 100% (**, p < 0.01; ***, p < 0.001).
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Fig. 8.
Antioxidants protect cortical neurons to the
non-fibrillar A -induced cell death.
Before A
exposure, cells were treated for 2 h with 10 µM probucol (PB), 1 µM
promethazine (PM), or 5 µM propylgallate
(PG), and these treatments persisted throughout the 5 µM A
(1-40) exposure. The neurotoxicity of the
soluble A
was monitored by the MTT assay (A) and the
quantification of apoptotic nuclei after DAPI staining (B).
Data are means (± S.E.) of three different experiments with four
determinations each and are normalized to the effect of vehicle,
designated as 100% (**, p < 0.01; ***,
p < 0.001). Differences among the subgroups for each
condition were performed by ANOVA followed by a Scheffe's post hoc
test (#, p < 0.05).
View larger version (62K):
[in a new window]
Fig. 9.
Probucol modulates the disorganization of the
microtubules induced by the non-fibrillar A . Before
A
exposure, the cells were treated for 2 h with the vehicle or
10 µM probucol (PB). The neuronal microtubule
network of control cells (A), cells incubated with PB only
(C), and cells treated with A
(1-40) for 24 h in
the absence (B) and presence of PB (D), was
visualized by immunocytochemistry using an anti-
-tubulin antibody.
E, disorganization of the neuronal microtubule network was
quantified at the indicated incubation times. Data are means (± S.E.)
of three different experiments with four determinations each and are
normalized to the effect of vehicle, designated as 100% (*,
p < 0.05; **, p < 0.01; ***,
p < 0.001). Differences among the subgroups for each
condition were performed by ANOVA followed by a Scheffe's post hoc
test (#, p < 0.05).
(29-40) exhibited membrane fusion
properties (30) and that part of the neurotoxicity of A
(1-40) and
A
(1-42) was triggered by the insertion of their C-terminal ends
into the plasma membrane of the neurons (18). Here, we have
demonstrated that A
(29-40) induced similar kinetics of caspase
activation as A
(1-40) (Fig. 5). Furthermore, the incubation of the
cortical neurons with A
(29-40) resulted in the disorganization of
the microtubule network, prevented by the presence of probucol (Fig.
9). These data highlight the critical role of ROS production in
A
-mediated cytoskeleton disruption and subsequent neuronal cell death.
Induces Primary Plasma Membrane
Disorder--
We next investigated whether the interaction of the
non-fibrillar A
(1-40) with the plasma membrane of cortical neurons
could directly modify the properties of the membrane in living cells. To that end, cortical neurons were incubated with 1 µM
A
peptide, a concentration at which no significant cell death and
ROS production could be observed even after an A
treatment of 3 h. After A
(1-40) treatment during increasing incubation times, the
plasma membrane of the neurons was loaded with 4 µM
NBD-SM for 5 min, and the plasma membrane fluidity was assessed by
measuring the lateral diffusion coefficient
(D20,w) of the phospholipids using FRAP.
The intensity of the fluorescent signal enabled us to discriminate the
apical plasma membrane from the inside of the cell using confocal
microscopy. All experiments were therefore performed at 37 °C before
the accumulation of the fluorescent probe in the inner membranes. After
a 2-h incubation with 1 µM A
(1-40), the cortical
neurons displayed a slight increase of the lateral diffusion of NBD-SM,
i.e. 0.280 + 0.052 µm2/s (n = 27) and 0.402 + 0.044 µm2/s (n = 26)
(p < 0.05) in control cells and in cells treated for 2 h with A
, respectively (Fig.
10). Prolonged incubation times up to
24 h, with the non-fibrillar A
resulted in a continuous decrease of the D20,w (after 24 h
of incubation, D20,w = 0.109 + 0.064, p < 0.05), as compared with the control cells (Fig.
10). Interestingly, the alteration of the lateral diffusion of NBD-SM
induced by 1 µM non-fibrillar A
(1-40) in primary
neurons was similar in the human neuroblastoma HN-SY5Y cell line. After 3 h of incubation, we observed an apparent increase of the
D20,w, i.e. 0.306 + 0.046 µm2/s (n = 21) and 0.432 + 0.053 µm2/s (n = 20), for control and
A
-treated cells, respectively, followed by a dramatically decrease
of the D20,w (not shown). Finally, we
demonstrated that, under the same experimental conditions, the
C-terminal fragment of A
, e.g. the A
(29-40) peptide,
exhibited similar effects on membrane fluidity as A
(1-40) (data
not shown).
View larger version (37K):
[in a new window]
Fig. 10.
Non-fibrillar A
(1-40) induces a biphasic change in the plasma membrane fluidity
of cortical neurons. The membrane fluidity was monitored using
FRAP analysis of the lateral diffusion of incorporated
NBD-sphingomyelin in the plasma membrane of the living neurons, as
described under "Experimental Procedures." The lateral diffusion
coefficient (D (µm2/s)) was
calculated for increasing incubation times of cells with 1 µM non-fibrillar A
(1-40). *, p < 0.05.
peptide-induced
modifications of the membrane fluidity. In separate experiments than
those described in Fig. 10, cortical neurons pre-treated for 2 h
with 100 nM taxol exhibited similar values of the
D20,w as control cells
(D20,w = 0.269 + 0.036 µm2/s for control cells (n = 23),
D20,w = 0.287 + 0.052 µm2/s for taxol-treated cells (n = 23)).
A 2-h pre-incubation of the cortical neurons with 100 nM
taxol did not prevent the modification of
D20,w after a 2-h treatment with 1 µM A
(1-40) (D20,w = 0.421 + 0.035 µm2/s for cells treated with A
only
(n = 30), and D20,w = 0.392 + 0.051 µm2/s for A
-treated cells in the
presence of taxol (n = 32)). We moreover observed a
similar decrease of D20,w after a 24-h
treatment with 1 µM A
in the absence or presence of
taxol (not shown). Finally, we demonstrated that the presence of
antioxidant molecules did not counteract the disturbing effects of the
non-fibrillar A
on the membrane (not shown). Altogether, these data
suggest that A
-induced membrane perturbation might represent a
primary event of A
-induced neuronal cell death.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and that the disruption of the microtubule network
is required for A
-induced neuronal cell death, (b) the
non-fibrillar A
induces a rapid oxidative stress that precedes and
induces the microtubule disorganization, and (c) the
earliest detectable action of soluble A
on the neurons is a rapid
and biphasic modification of the plasma membrane fluidity preceding
latter cellular events leading the cells into an apoptotic pathway.
This process involves the activation of caspases, the neo-synthesis of
proteins, and DNA fragmentation (18, 22). Cell death occurs in a time-
and dose-dependent manner via a pathway involving the
activation of several classes of caspases, including caspases 3 and 9. However, this pathway did not involve the activation of caspase 8, as
demonstrated through the measurement of the caspase 8-like activity and
the absence of effect of caspase 8 inhibitors on soluble A
-induced
cell death. Our results contrast with previous studies demonstrating
that cell death induced by fibrillar A
consisted partly in a rapid activation of caspase 8 (35, 36). This corroborates our previous hypothesis (18, 22), which was based on the idea that the cellular
targets and the molecular mechanisms involved in cell death induced by
A
strongly depended on peptide conformation.
, most of the neurons displayed a disrupt microtubule
architecture, even after 3 h of incubation and before the
morphological and biochemical alterations typical of apoptotic cell
death. Indeed, the perturbations of the microtubules precede caspase
activation and nuclear DNA fragmentation and condensation. Moreover,
the presence of a microtubule-stabilizing drug, e.g. taxol,
inhibited neuronal cell death together with non-fibrillar A
-induced
perturbation of the cytoskeleton. It remains to establish the
specificity of A
-induced microtubule perturbations. Using immunoblotting, we observed that the treatment of the cortical neurons
with non-fibrillar A
did not modify the tubulin pools (data not
shown). It could be hypothesized that the microtubule perturbations
might occur via post-translational modifications and/or proteolytic
cleavage of microtubule-associated proteins (MAP). Modifications of the
phosphorylation level of tau and MAP2 in AD brains
have been described (39-41). In agreement with these observations, it
has been demonstrated that the products of lipid peroxidation disrupted
the microtubules of cortical neurons directly (42).
(26, 27). Our results highlight
the critical role of ROS production in the cytoskeleton disruption and
the subsequent neuronal cell death induced by soluble A
. The
incubation of cortical neurons with low concentrations of soluble A
(1-40) results in an early time-dependent increase in the
ROS production, preceding both microtubule perturbation and caspase
activation. Indeed, the presence of anti-oxidant molecules prevents all
of the intracellular events triggered by non-fibrillar A
.
(1-40) peptide induces a rapid modification of the membrane fluidity
in living cells. Using FRAP analysis, we show that low concentrations
of soluble A
(1-40) (1 µM) induce a biphasic
modification of the fluidity of the neuron plasma membrane as monitored
by a global determination of the lateral diffusion coefficient of lipids. Upon short incubation times of cortical neurons with soluble A
, we observed a reproducible increase in the membrane fluidity. In
agreement with previous works (42-44), longer incubation times result
in a significant decrease of the membrane fluidity, which could be
attributed to lipid peroxidation associated with A
-induced oxidative
stress described in the present paper.
(1-40)
displays fusogenic properties because of the membrane perturbing activity of its C-terminal domain, e.g. 29-40 (30, 45).
Here, we demonstrate that A
(29-40) induces effects on the membrane fluidity and subsequent cell death similar to those induced by A
(1-40). These results strongly support the idea that the effects of
A
on the membrane fluidity are partly mediated by the fusogenic properties of its C-terminal fragment and correlate the physicochemical properties to the neurotoxicity of non-fibrillar A
.
on neuron viability might involve the interaction of
A
with the lipid phase of the plasma membrane. A recent study by
Cotman and co-workers (46) reports that the effects of A
on neuronal
viability were not mediated by specific interactions with a receptor
but more likely by changes in the structure and dynamics of the lipid
constituents of the membrane (46). Most studies describe the action of
aggregated forms of A
on model membranes or synaptosomes,
demonstrating that the fibrils of A
displayed a high affinity with
the membrane and decreased the fluidity of the lipid phase (47, 48).
Furthermore, aggregated A
(1-40) has strong electrostatic
interactions with the surface of model membranes that appear to mediate
its neurotoxicity (49). However, whereas both soluble and aggregated
A
interact differently with rat synaptic plasma membranes, both
transiently increase their membrane fluidity (50, 51). Our results
emphasize the importance of the early interactions of non-fibrillar
A
with lipids in A
-mediated neuronal cell death.
are presently under investigation. The transient increase of the plasma
membrane fluidity induced by low concentrations of soluble A
may
explain the reported effects of A
on the permeability of the neurons
to extracellular calcium and the increase in KCl-induced neuronal
calcium in brain neurons and lymphocytes (52, 53). Indeed, the presence
of inhibitors of calcium influx reduced markedly neuronal cell death
induced by soluble A
(1-40) and
(29-40).2 The interactions
between soluble A
and the lipids might modify the function of
ion channels or/and directly create ion pore, as described recently
(25, 54).
in the progression of AD. Accordingly, recent findings
have demonstrated that only in a non-aggregated conformation, A
activates the phosphoinositide signaling pathway (55) and mediates
vasoconstriction activity both in vitro and in
vivo (56, 57). Uncovering the neuronal toxicity of non-fibrillar
A
gives new insights into the development of AD therapy, which might
take into account both amyloid deposit prevention and an efficient clearance of non-fibrillar A
peptides.
![]() |
FOOTNOTES |
---|
* This work was supported in part by INSERM and by a grant from the Aventis French Network on Molecular Mechanism in Alzheimer's disease.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.
§ Contributed equally to this work.
¶ Supported by a postdoctoral fellowship from Aventis Pharma (Vitry-Sur-Seine, France).
** To whom correspondence should be addressed: INSERM EMI 0014, Université de Nancy I, 9 Avenue de la Forêt de Haye, BP 184, 54505 Vandoeuvre, France. Tel.: 33-3-83-68-32-74; Fax: 33-3-83-68-32-79; E-mail: Thierry.Pillot@bcmn.facmed.u-nancy.fr.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M206745200
2 I. Sponne, A. Fifre, and T. Pillot, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
AD, Alzheimer's
disease;
A, amyloid-
;
DCFH-DA, 2',7'-dichlorofluorescein
diacetate;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide;
DAPI, 4,6-diamidino-2-phenylindole;
PBS, phosphate-buffered
saline;
NBD-SM, 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-sphingomyelin;
ANOVA, analysis of variance;
DIV, day in vitro, AMC,
7-amido-4-methylcoumarin;
FMK, fluoromethyketone;
DCF, 2',7'-dichlorofluorescein.
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