Full text of DOI:10.1038/sj.bjp.0704581

British Journal of Pharmacology (2002) 135, 1297 ± 1307
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Kainic acid-induced neuronal cell death in cerebellar granule cells is
not prevented by caspase inhibitors
1
1
1
1Ester Verdaguer, Elvira Garcõa-Jorda, Andres Jimenez, Alessandra Stranges,
2Francesc X. Sureda, Anna M. Canudas, Elena Escubedo, Jordi Camarasa, Merce Pallas &
1
1
1
1
*,1Antoni Camins
1Unitat de Farmacologia i Farmacognosia, Facultat de Farmacia, Universitat de Barcelona, Nucli Universitari de Pedralbes,
E-08028 Barcelona, Spain and Unitat de Farmacologia, Facultat de Medicina i Ciencies de la Salut, Universitat Rovira i
Virgili, C./St. Llorenc 21, E-43201 Reus, Tarragona, Spain
2
1
We examined the role of non-NMDA receptors in kainic acid (KA)-induced apoptosis in cultures
of rat cerebellar granule cells (CGCs). KA (1 ± 500 mM) induced cell death in a concentration-
dependent manner, which was prevented by NBQX and GYKI 52466, non-NMDA receptor
antagonists. Moreover, AMPA blocked KA-induced excitotoxicity, through desensitization of
AMPA receptors.
2
Similarly, KA raised the intracellular calcium concentration of CGCs, which was inhibited by
NBQX and GYKI 52466. Again, AMPA (100 mM) abolished the KA (100 mM)-induced increase in
intracellular calcium concentration.
3
KA-induced cell death in CGCs had apoptotic features, which were determined morphologically,
by DNA fragmentation, and by expression of the prostate apoptosis response-4 protein (Par-4).
KA (500 mM) slightly (18%) increased caspase-3 activity, which was strongly enhanced by
4
colchicine (1 mM), an apoptotic stimulus. However, neither Z-VAD.fmk, a pan-caspase inhibitor, nor
the more speci®c caspase-3 inhibitor, Ac-DEVD-CHO, prevented KA-induced cell death or
apoptosis. In contrast, both drugs inhibited colchicine-induced apoptosis.
5
The calpain inhibitor ALLN had no e€ect on KA or colchicine-induced neurotoxicity.
Our ®ndings indicate that colchicine-induced apoptosis in CGCs is mediated by caspase-3
activation, unlike KA-induced apoptosis.
6
British Journal of Pharmacology (2002) 135, 1297 ± 1307
Keywords: Apoptosis; kainic acid; cerebellar granule cells; Par-4; colchicine; caspases; calpain; Z-VAD.fmk; Ac-DEVD-
CHO; glutamate
Abbreviations: Ac-DEVD-CHO, Ac- Asp- Glu- Val- Asp- aldehyde; ALLN, N-acetyl-Leu-Leu-Nle-CHO; AMPA, a-amino-3-
hydroxy-5-methyl-4-isoxazolepropionic acid; BME, Basal medium with Eagle's salts; BSA, Bovine serum
albumin; CGCs, Cerebellar granule cells; CNS, Central nervous system; Con A, Concanavalin A; CYZ,
Cyclothiazide; FSC, Foetal calf serum; FSC, Forward scatter; KA, Kainic acid; LH-BSA, Locke-HEPES bu€er;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NMDA, N-methyl-D-aspartate; Par-4,
Prostate apoptosis response-4; PBS, Phosphate bu€ered saline solution; PI, Propidium iodide; SSC, Side
scatter; Z-VAD.fmk, Benzyloxycarbonyl-val-ala-asp-(O-methyl)-¯uormethylketone;
Introduction
Glutamate, the main excitatory neurotransmitter in the
central nervous system (CNS), mediates variety of
of neuronal cell death mediated by kainate receptors (Dykens
et al., 1987; Kato et al., 1991). Concerning the nature of cell
death, it has been shown that CGCs exposed to glutamate,
AMPA or KA undergo apoptosis or necrosis depending on
the intensity of exposure (Ankarcrona et al., 1995;
Ankarcrona, 1998; Cebers et al., 1997; Simonian et al.,
1996; Giardina et al., 1998). There is controversy as to which
of the ionotropic glutamate receptors is involved in KA-
induced neuronal cell death in CGCs. Some authors suggest a
process mediated through AMPA receptors (Ambrosio et al.,
2000; Leski et al., 2000) while others point to kainate
receptors (Carroll et al., 1998; Giardina & Beart, 2001).
Whereas necrosis is a passive process of cell death in which
the neuron ultimately lyses its contents into the immediate
surroundings, apoptosis is an active mode of cell death.
Apoptotic cell death is characterized by several morphologi-
cal and biochemical features, including reduced cell volume,
a
neurological e€ects as a result of ionotropic glutamate
receptor stimulation. Three classes of ionotropic glutamate
receptors are involved in glutamate-induced neuronal cell
death (excitotoxicity): the N-methyl-D-aspartate (NMDA)
receptor, the a-amino-3-hydroxy-5-methyl-4-isoxazolepropio-
nic acid (AMPA) receptor, and the kainate receptor (Bettler
& Mulle, 1995; Bleakman & Lodge, 1998; Meldrum, 2000).
Although the role of NMDA receptor activation in neuronal
cell death has been widely studied, that of AMPA and
kainate receptors is less well known.
Cerebellar granule cells (CGCs), which are particularly
vulnerable to excitotoxins, are used to study the mechanisms
*Author for correspondence; E-mail: camins@farmacia.far.ub.es

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E. Verdaguer et al
Role of caspases in kainic acid-induced cell death
condensation of nuclear chromatin, and DNA fragmentation
(Sastry & Rao, 2000). The most relevant enzymes involved in
neuronal cell death are cysteine proteases. Among them,
calpains participate in both necrotic and apoptotic cell death,
whereas caspases are speci®cally activated only in the
apoptotic process (Wang, 2000). Moreover, several lines of
evidence suggest that caspase-3, a member of the CED-3
subfamily of caspases, controls apoptosis (Marks et al., 1998;
Budd et al., 2000; Mattson, 2000).
Cell culture media and foetal calf serum (FCS) were
obtained from GIBCO (Life Technologies, Paisley, U.K.).
Cell culture salts, enzymes, Mowiol1 4 ± 88 and Triton X-
100 were from Sigma. Other chemical reagents were of
analytical quality and purchased by Panreac Quimica
(Barcelona, Spain).
Cerebellar granule cell cultures
Recently, it has been demonstrated that prostate apoptosis
response-4 (Par-4) has a pro-apoptotic e€ect (Duan et al.,
1999; 2000). This protein belongs to the family of immediate
early gene products, such as c-Myc, c-Fos and c-Jun
(Rangnekar, 1998). Unlike the other immediate early gene
products, Par-4 induction appears to be induced exclusively
by apoptotic stimuli (Camandola & Mattson, 2000). It has
been proposed that Par-4 and caspases are critical mediators
of neuronal apoptosis in neurodegenerative disorders (Liu &
Zhu, 1999). Previous studies have implicated caspase-3 in
apoptosis induced by glutamate (Du et al., 1997a), MPP+
(Du et al., 1997b) and 6-hydroxydopamine in CGCs (Dodel
et al., 1999). In addition, caspase-3 inhibitors protect CGCs
from the neurotoxic e€ect of these compounds. Moreover,
caspase-3 activation contributes to neuronal apoptosis
induced by potassium deprivation (D'Mello et al., 1998;
2000; Marks et al., 1998; Moran et al., 1999). However, in
mice lacking caspase-3, this cysteine protease is not necessary
for neuronal death of CGCs induced by potassium
deprivation, although it is involved in neuronal apoptosis
(D'Mello et al., 2000).
Primary cultures of CGCs were prepared from 7-day-old
Sprague Dawley rat pups following Nicoletti et al. (1986).
Brie¯y, cerebella were quickly removed and cleaned of
meninges, followed by manual slicing with a sterile blade,
dissociation with trypsin and treatment with DNase. Cells
were adjusted to 86105 cells ml71 and plated on both poly-L-
lysine-coated 24-well plates and glass coverslips at a density
of 320,000 cells cm72. Cultures were grown in basal medium
with Eagle's salts (BME) containing 10% FCS, 2 mM L-
glutamine, 0.1 mg ml71 gentamicin and 25 mM KCl, referred
to as a complete medium. Cytosine arabinoside (10 mM) was
added 16 ± 18 h after plating to inhibit the growth of non-
neuronal cells. Cultures prepared by this method were more
than 95% enriched in granule neurons, as assessed by GFAP
immunocytochemistry (data not shown).
Treatment of CGCs and survival assay
CGCs were used after 9 ± 10 days in vitro. KA and colchicine
were dissolved in complete culture medium, and neutralized
with NaOH to pH 7.4 if necessary before addition to the cell
culture (to a maxim volume of 10 ml). To investigate the
e€ect of NBQX, GYKI 52466, (+)MK-801, cyclothiazide,
concanavalin-A, Z-VAD.fmk and Ac-DEVD-CHO were
added to the medium, at the precise concentrations, 30 min
before addition of neurotoxins. Cell death was determined
24 h later by the MTT assay as follows.
Thus, the apoptotic mechanism induced by glutamate is
well known in CGCs, but the pathways involved in KA-
induced apoptosis are less. Although KA may activate
caspase-3 (Nath et al., 1998) and c-jun (Cheung et al.,
1998), the routes involved in KA-induced apoptosis are
unclear.
Here, we study both the ionotropic glutamate receptors
involved in KA-induced apoptosis in CGCs and the role of
caspase-3 and calcium-dependent proteases in KA-induced
apoptosis. We demonstrate that KA-induced apoptosis is
mediated mainly through activation of AMPA receptors and
is not prevented by caspase inhibitors. We suggest that Par-4
Assessment of neuronal injury
MTT was added to the cells to a ®nal concentration of
250 mM and incubated for 1 h to reduce MTT to a dark blue
formazan product (Hansen et al., 1989). The media were
removed and cells were dissolved in dimethylsulphoxide.
Formation of formazan was tested by measuring the amount
of reaction product by absorbance change (595 nm) using a
microplate reader (BioRad Laboratories, CA, U.S.A.).
plays
a key role in KA-induced apoptosis in CGCs.
Therefore, we propose a caspase-independent mechanism of
apoptosis mediated by KA.
Viability results was expressed as a percentage of the
absorbance measured in untreated cells.
Methods
Drugs
Measurement of cytosolic calcium increases
Kainic acid (KA), colchicine, propidium iodide (PI),
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), HEPES and bovine serum albumin (BSA) were
from Sigma Chemical Co. (St. Louis, MO, U.S.A.).
Benzyloxycarbonyl-val-ala-asp-(O-methyl)-¯uormethylketone
(Z-VAD.fmk) and Ac-Asp-Glu-Val-Asp-aldehyde (Ac-
DEVD-CHO) were from Bachem AG (Bubendorf, Switzer-
land). N-acetyl-Leu-Leu-Nle-CHO (ALLN) was from Cal-
biochem. AMPA, (+)MK-801, GYKI 52466 and NBQX
were purchased from TOCRIS (Ballwin, MO, U.S.A.). Fura-
2 AM was from Molecular Probes (Eugene, OR, U.S.A.).
The increase in intracellular free Ca2+ was determined in
CGCs grown in glass cover slips (Corning Costar Corp.,
Acton, MA, U.S.A.) using a Mg2+-free, Locke-HEPES bu€er
(LH-BSA), which consisted of (in mM): NaCl 154, KCl 5.6,
NaHCO3 3.6, CaCl2 1.3, D-glucose 5.6, HEPES 10 and 0.1%
(w v71) BSA (pH=7.35). After 9 ± 10 days in culture, one
cover slip was carefully transferred to a Petri dish containing
3 ml of LH-BSA bu€er and 2 mM Fura-2 AM, and incubated
at 378C for 1 h on a cell incubator. For ¯uorescence
recording, the cover slip was carefully rinsed in LH-BSA
bu€er, mounted on a speci®c holder (coverslip accessory
British Journal of Pharmacology vol 135 (5)

E. Verdaguer et al
Role of caspases in kainic acid-induced cell death
1299
L2250008, PerkinElmer, Inc., Wellesley, MA, U.S.A.) and
placed in a quartz cuvette containing 1.3 ml LH-BSA bu€er.
Measurements were made at 378C under continuous mild
stirring in a LS50B PerkinElmer ¯uorescence spectrometer,
equipped with a fast-®lter accessory for Fura-2 ¯uorescence
ratio measurements. Emission data (510 nm) were collected
with alternate excitation at 340 and 380 nm, and the ratio
expressed as percentages of the absorbance measured in
vehicle-treated cells.
Western blot analysis
Aliquots of cells homogenate, containing 30 mg of protein per
sample, were placed in sample bu€er (0.5 M Tris-HCl pH 6.8,
10% (w v71) glycerol, 2% (w v71) SDS, 5% (v v71) 2-b-
mercaptoethanol and, 0.05% bromophenol blue) and dena-
tured by boiling at 95 ± 1008C for 5 min.
F340/F380 was calculated in real time, using proprietary
software (FL WinLab 2.0)
Analysis of apoptosis rate by flow cytometry
Samples were separated by electrophoresis on 10% (w v71
)
acrylamide gels. Proteins were then transferred to polyviny-
lidene ¯uoride (PVDF) sheets (ImmobilonTM-P, Millipore
Corp., Bedford, MA, U.S.A.) using a transblot apparatus
(BioRad). Membranes were blocked overnight with 5%
(w v71) nonfat milk dissolved in TBS-T bu€er (Tris 50 mM;
NaCl 1.5% (w v71); Tween 20 0.05% (v v71), pH 7.5).
Membranes were then incubated with a primary rabbit
polyclonal antibody against Par-4 (1 : 1000, Santa Cruz
Biotechnology, Santa Cruz, CA, U.S.A.). After 90 min, blots
were washed thoroughly in TBS-T bu€er and incubated for
1 h with a peroxidase-conjugated anti rabbit IgG antibody
(Amersham Corp., Arlington Heights, IL, U.S.A.). Immu-
noreactive protein was visualized using a chemiluminiscence-
based detection kit following the manufacturer guidelines
(ECL kit; Amersham Corp.).
Apoptosis was measured 24 h after KA or colchicine
addition. In brief, PI (10 mg ml71) and 0.1% (v v71) Triton
X-100 were added to culture media 30 min before cyto-
¯uorometry analysis. Cells were collected from culture plates
by pipetting. Flow cytometer experiments were carried out
using an Epics XL ¯ow cytometer (Coulter Corp. Hialeah,
FL, U.S.A.). The instrument was set up with the standard
con®guration: excitation of the sample was performed using
as a standard 488 nm air-cooled argon-ion laser at 15 mW
power. Forward scatter (FSC), side scatter (SSC) and red
(620 nm) ¯uorescence for PI were acquired. Optical align-
ment was based on optimized signal from 10 nm ¯uorescent
beads (Immunocheck, Epics Division). Time was used as a
control of the stability of the instrument. Red ¯uorescence
was projected on a 1024 monoparametrical histogram.
Immunocytochemistry against Par-4
Detection of apoptotic nuclei by propidium iodide staining
CGCs were grown on sterile glass slides. After stimuli, cells
were washed twice in PBS and ®xed in 4% (w v71
)
PI staining was used to detect morphological evidence of
apoptosis (Atabay et al., 1996). CGCs were grown on glass
coverslips and after treatment with KA (500 mM) or
colchicine (1 mM), they were ®xed in 4% (w v71) paraformal-
dehyde/phosphate bu€ered saline solution (PBS), pH 7.4 for
1 h at room temperature. After washing with PBS, they were
paraformaldehyde/PBS, pH 7.4 for 1 h at room temperature
and then permeabilized in 0.3% (v v71) Triton X-100 for
10 min. After blocking with goat serum (1 h at room
temperature), cells were incubated with rabbit anti-Par-4
(1 : 400, Santa Cruz Biotechnology) overnight at 48C. They
were then washed extensively and incubated with secondary
antibody (anti-rabbit-FITC conjugated (1 : 100, Sigma) for
1 h at room temperature. Coverslips were thoroughly washed
and mounted in Mowiol1 4 ± 88 and immunosignal analysis
incubated for 3 min with
a solution of PI in PBS
(10 mg ml71). Coverslips were mounted in Mowiol1 4 ± 88.
Stained cells were visualized under u.v. illumination using the
206objective (Leica DMRB ¯uo microscope, Leica Micro-
systems AG, Germany) and their digitized images were
captured.
was performed using a confocal microscope at 606
magni®cation (Leica, TCS, 4D).
Apoptotic cells showed shrunken, brightly ¯uorescent,
apoptotic nuclei showing with high ¯uorescence and con-
densed chromatin, compared with nonapoptotic cells.
Apoptotic cells were scored by counting at least 500 cells of
six ®elds for each sample in three experiments.
Statistics
The data are presented as the mean+s.e.mean. For statistical
comparisons, one-way analysis of variance followed by
Tukey's test was used for multiple comparison and Student's
test for pairs of data. Probability values lower than 0.05 was
accepted as statistically signi®cant.
Assay of caspase-3 enzymatic activity
We used the colorimetric substrate Ac-DEVD-p-nitroaniline
(Oncogen) for the determination of caspase-3 activity
described below. Twelve, 18 or 24 h after treatment with
500 mM KA and 24 h after treatment with 1 mM colchicine,
CGCs were collected in lysis bu€er (HEPES 50 mM, NaCl
100 mM, 0.1% (w v71) CHAPS and EDTA 0.1 mM, pH 7.4).
0.5 mg ml71 of protein was incubated with 200 mM Ac-DEVD-
p-nitroaniline in assay bu€er (HEPES 50 mM, NaCl 100 mM,
0.1% (w v71) CHAPS, 10 mM dithiothreitol and, 0.1 mM
EDTA, pH 7.4) in 96-well plates at 378C for 24 h.
Absorbance of the cleaved product was measured at
405 nm in a microplate reader (BioRad). Results were
Results
Characterization of kainic acid-induced toxicity in
cultured rat CGCs
Exposure of cultured CGCs to various concentrations of KA
(1 ± 500 mM) for 24 h was neurotoxic in a concentration-
dependent manner, as revealed by the MTT assay. Maximal
toxicity was obtained at a concentration of 500 mM, which
decreased viability to 27+5.7% (n=6) (Figure 1). Nonlinear
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E. Verdaguer et al
Role of caspases in kainic acid-induced cell death
Figure 1 E€ect of exposure to KA (1 ± 500 mM) for 24 h on CGCs
viability, based on MTT assay. Each point is the mean+s.e.mean of
5 ± 6 cultures, carried out in quadruplicate.
analysis of viability values yielded an EC50 of 68+8.9 mM
(n=6). The AMPA/kainate receptors antagonists NBQX and
GYKI 52466 signi®cantly prevented KA-induced (100 mM)
cell death (Figure 2A,B). However, (+)MK-801 (10 mM), a
selective antagonist of NMDA receptors, did not prevent
KA-induced cell death (Figure 2A).
We studied the implication of kainate receptors in the
neurotoxic e€ects of KA using concanavalin A (Con A,
ranging from 1 mg ml71 to 250 mg ml71), a lectin that inhibits
the desensitization of kainate receptors. Con A neither
decreased nor enhanced KA toxicity in CGCs (Figure 3A).
Exposure of CGCs to AMPA (100 mM) slightly decreased
cell viability. Cyclothiazide (CYZ, 50 mM), a speci®c inhibitor
of AMPA receptor desensitization, potentiated AMPA-
induced cell death (Figure 3B). However, CYZ alone had
no e€ect on cell survival. To further demonstrate that KA
neurotoxicity is mediated by interaction with AMPA
receptors, KA (100 mM) was incubated in the presence of
increasing concentrations of AMPA (10 ± 100 mM). Viability
assays showed that KA toxicity was signi®cantly inhibit by
100 mM AMPA (P50.05) (Figure 4).
Figure 2 (A) Protective e€ect of AMPA/kainate receptor antagonists
(10 mM) on KA-induced toxicity. CGCs were pre-treated with drugs
30 min before KA addition (100 mM). The data represent the mean+
s.e.mean of four independent experiments carried out in quadruplicate,
and are expressed as the percentage change of control cells, arbitrarily
set at 100%. When necessary, the statistical analysis was carried out
with the one-way ANOVA followed by Tukey's test *P50.05,
***P50.001 vs KA. (B) Concentration-response curves of NBQX
and GYKI 52466 vs KA-induced toxicity (100 mM) in CGCs.
We studied e€ect of CYZ on KA toxicity. Again, CYZ
(50 mM) slightly promoted the neurotoxic e€ects of KA.
Although the di€erence was not signi®cant, viability
decreased from 54+8.4 (n=3) to 43+3.1 (n=6) in the
presence of CYZ (Figure 4).
or absence of AMPA (100 mM). Added 5 min before KA
(100 mM), AMPA completely blocked the increase in cytosolic
calcium concentration. (Figure 5B,C). These results further
support the hypothesis that KA-induced responses are mainly
due to the activation of AMPA receptors (Leski et al., 2000).
Kainic acid-induced effects on intracellular Ca2+
concentration in CGCs
Kainic acid activates caspase-3 in CGCs
Exposure of CGCs to KA (500 mM) for 24 h induced a slight,
but signi®cant increase (18+8.7%; n=6) in caspase-3 activity
as soon as 12 h after KA addition to cultures (Figure 6). KA
(100 mM) did not induce caspase-3 activity. Colchicine (1 mM),
used as a positive control of caspase-3 activation, strongly
enhanced (150+40.5%, n=6) caspase-3 activation.
We also evaluated the e€ect of KA on cytoplasmic calcium in
CGCs using the speci®c probe Fura-2. KA (10 ± 100 mM)
strongly increased intracellular calcium concentration. The
nonlinear regression analysis yielded an EC50 of
128.8+3.3 mM (n=3) (Figure 5A). The e€ect of various
concentrations of GYKI 52466 and NBQX (0.01 ± 10 mM)
were tested on the KA (100 mM)-induced increase in calcium
concentration, yielding IC50 values of 9.1+0.87 mM and
2.8+0.9 mM, respectively (n=3). To assess the implication
of AMPA receptors in KA e€ects in CGCs, the e€ect of KA
on intracellular calcium increases was assessed in the presence
Caspase inhibitors do not prevent kainic acid-induced
toxicity
To investigate the involvement of the apoptotic process in
excitotoxic neuronal death mediated by KA, we used two
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E. Verdaguer et al
Role of caspases in kainic acid-induced cell death
1301
Figure 3 (A) E€ect of various concentrations of concanavalin A on
KA-induced toxicity in CGCs. (B) Cyclothiazide potentiated the e€ect
of AMPA on CGCs viability. Data was obtained from 3 ± 4
experiments and are the mean+s.e.mean of the percentage change
of control cells. The statistical analysis was carried out using the one-
way ANOVA followed by Tukey's test *P50.05, ***P50.001 vs KA.
Figure 5 E€ect of KA on intracellular calcium concentration in
cultured CGCs. Recordings of the ratio of the ¯uorescent emission of
Fura-2, under the excitation at 340 and 380 nm were measured as
described in the Methods section. The ratio F340/F380 is indicative of
changes in cytosolic free calcium. (A) Concentration-response curve
of the increase in intracellular calcium concentration induced by KA.
(B) E€ect of KA on intracellular calcium concentration in the
absence or presence of AMPA 100 mM. Tracings are a representative
experiment using
a sample ®rst preincubated with AMPA and
exposed to increasing concentrations of KA (grey tracing). After
recording, cells were washed twice and the experiment was repeated
in the absence of AMPA (black tracing). (C) Bar graph showing the
data from three experiments using the same procedure as in (B). Each
value is the mean+s.e.mean. The statistical analysis was carried out
using Student's test *P50.05 vs KA.
Figure 4 E€ect of AMPA receptor modulators on KA-induced
neurotoxicity. Each point is the mean+s.e.mean of four wells of
three cultures, expressed as the percentage change of control cells,
arbitrarily set at 100%. The statistical analysis was carried out using
the one-way ANOVA followed by Tukey's test *P50.05 vs KA.
compounds, the pan-speci®c caspase family inhibitor Z-
VAD.fmk and the speci®c caspase-3 inhibitor Ac-DEVD-
CHO. Neither Z-VAD.fmk (0.1 mM) nor Ac-DEVD-CHO
(100 mM) prevented the excitotoxic e€ects induced by KA
(500 mM, 24 h). However, Z-VAD.fmk (but not Ac-DEVD-
CHO) inhibited the decrease in viability induced by colchicine
(1 mM). We also studied the involvement of calpain-1 in KA
neurotoxicity. Treatment of CGCs with the calpain-I
inhibitor ALLN (1 mM) did not protect the cells from KA-
induced neuronal damage (Figure 7).
Kainic acid-induced apoptosis in CGCs is not prevented by
caspase inhibitors
KA-induced apoptosis in CGCs was evaluated by two
methods: DNA fragmentation by ¯ow cytometry and
counting the fraction of cells with nuclear condensation.
When NBQX (10 mM) or GYKI 52466 (10 mM) were co-
incubated with KA (500 mM, 24 h), they markedly reduced
the percentage of apoptotic cells (Figure 8). On the other
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1302
E. Verdaguer et al
Role of caspases in kainic acid-induced cell death
signal expressed after various apoptotic stimuli in CGCs,
immunohistochemical and Western blot analyses were
performed. Immunoblot analysis revealed a marked increase
in the level of Par-4 protein expression after KA (500 mM) or
colchicine (1 mM) treatment (Figure 10). This was con®rmed
by immunocytochemistry, since KA-treated CGCs showed a
marked increase in Par-4 expression. NBQX (10 mM)
prevented the increase in KA-induced Par-4 expression.
Discussion
Our results show that KA-induced apoptosis in cerebellar
granule neurons is mediated through AMPA receptors and is
not prevented by caspase inhibitors. The type of receptor
responsible for KA e€ects on neuronal damage and/or
apoptosis in CGCs is a matter controversy. Some authors
suggest that kainate receptors are involved in KA-induced
neuronal damage in several cell types (Carroll et al., 1998),
while others implicate AMPA receptors (Ohno et al., 1997;
1998; Leski et al., 2000). Recently, Gasull et al. (2001) have
demonstrated that AMPA receptors, but not kainate
receptors, are responsible for KA-induced neuronal damage
in rat cortical neurons. Likewise, other investigators have
found similar results on hippocampal neurons (Ohno et al.,
1997; Ambrosio et al., 2000) or retina (Ferreira et al., 1998).
Previous studies have detected speci®c kainate receptors in
CGCs by electrophysiological methods (Pemberton et al.,
1998; Savidge et al., 1999) or by measuring changes in
intracellular calcium concentration (Savidge et al., 1997;
Savidge & Bristow, 1998). Moreover, KA, through kainate
receptors, enhances the activation of the AP-1 transcription
factor, and this e€ect is increased by concanavalin A, whereas
cyclothiazide has no e€ect (Kovacs et al., 2000).
However, few reports examine which of the receptors that
are activated by KA are responsible for neuronal death in
CGCs. Our study demonstrates that KA-induced toxicity is
mediated by AMPA receptors, as supported by several
®ndings. First, addition of AMPA to the culture medium
protects cells from KA neurotoxicity and prevents the
increase in cytosolic calcium concentration induced by KA.
Since this agonist quickly evokes AMPA receptor desensi-
tization, the inhibition of KA-induced neuronal damage in
CGCs demonstrates that the induction of desensitization at
this receptor impedes KA e€ects. Moreover, the selective
inhibitor of the desensitization of the AMPA receptor
cyclothiazide promoted KA-induced neurotoxicity, although
the e€ect was not signi®cant. Probably, KA does not totally
desensitize the AMPA receptor, and when desensitization is
blocked, its e€ects are boosted. However, when desensitiza-
tion is by AMPA incubation, KA-induced neurodegenera-
tion is prevented. Conversely, the speci®c inhibitor of
kainate receptor desensitization concanavalin A did not
a€ect KA neurotoxicity. Finally, GYKI 52466, the selective
AMPA antagonist, prevented KA-induced neuronal death.
These results strongly suggest that KA or AMPA-induced
neurotoxicity in CGCs is due to the activation of AMPA
receptors.
Figure 6 Bar chart showing the percentage of caspase-3-like activity
in CGCs exposed to KA (500 mM) and colchicine (1 mM). Results are
the mean+s.e.mean of four cultures. The statistical analysis was
carried out using Student's test *P50.05.
Figure 7 E€ect of caspase (Z-VAD.fmk, 0.1 mM, and Ac-DEVD-
CHO, 100 mM) and calpain (ALLN, 10 mM) inhibitors on KA or
colchicine-induced toxicity. CGCs were pre-treated with drugs 30 min
before KA (500 mM) or colchicine (1 mM) addition. The data represent
the mean+s.e.mean of three/four independent experiments carried
out in quadruplicate, and are expressed as the percentage change of
control cells, arbitrarily set at 100%. The statistical analysis was
carried out using the one-way ANOVA followed by Tukey's test
**P50.05 vs colchicine.
hand, Z-VAD.fmk (0.1 mM) and Ac-DEVD-CHO (100 mM)
did not modify the percentage of the hypodiploid population.
However, co-incubation of colchicine (1 mM) with Z-
VAD.fmk or Ac-DEV-CHO decreased the percentage of
apoptotic cells from 41+2.1 to 15+3.3 and 18+2.8,
respectively (data are the mean+s.e.mean of 4 ± 8 experi-
ments performed in duplicate).
Apoptotic features were also characterized by changes in the
morphology of the nuclei, after staining with PI observed under
¯uorescence. The number of cells with chromatin condensation
increased after treatment with KA (500 mM, 24 h). NBQX
prevented KA e€ects on nuclear morphology. However, neither
Z-VAD.fmk (0.1 mM) nor Ac-DEVD-CHO (100 mM) blocked
KA-induced nuclear condensation (Figure 9).
Kainic acid induces the expression of the prostate
apoptosis response-4 (Par-4) protein
In vivo studies support the hypothesis that kainate
receptors are involved in the excitotoxic process because
they enhance the release of glutamate (Malva et al., 1998). To
rule out that the e€ect of KA depended on NMDA receptor
In previous studies, a correlation has been shown between the
induction of Par-4 expression and neuronal apoptosis (Duan
et al., 1999; 2000). To assess whether Par-4 is a common
British Journal of Pharmacology vol 135 (5)

E. Verdaguer et al
Role of caspases in kainic acid-induced cell death
1303
Figure 8 Flow cytometry analysis of KA-induced apoptosis in permeabilized CGCs shown by propidium iodide ¯uorescence
histograms. Bar chart shows the percentage of apoptotic cells in the conditions tested. The statistical analysis was carried out using
the one-way ANOVA followed by Tukey's test ***P50.001 vs control.
activation, we used the selective NMDA antagonist (+)MK-
801. In agreement with other authors, this compound did not
prevent KA-induced cell death in cultures of CGCs
(Pemberton et al., 1998; Savidge et al., 1999).
Thus, our data reveal the key role of AMPA receptors in
excitotoxicity and suggest that whereas NMDA and AMPA
receptors are essential to glutamate-mediated excitotoxicity,
the function of kainate receptors in excitotoxicity remains to
be determined.
Caspase-3 has been identi®ed as a central executioner of
programmed cell death in neurons from Alzheimer's disease
patients (Stadelmann et al., 1999; Shimohama et al., 1999),
British Journal of Pharmacology vol 135 (5)

1304
E. Verdaguer et al
Role of caspases in kainic acid-induced cell death
inhibitor of caspases) and Ac-DEVD-CHO (caspase-3 speci®c
inhibitor) to prevent KA-induced apoptosis. KA may exert
its neurotoxic e€ects in CGCs via a caspase-independent
pathway. KA excitotoxicity may be associated with damage
of the plasma membrane due to cell swelling (Kiedrowski,
1998; Rago et al., 2001), whereas glutamate excitotoxicity is
associated with a prolonged alteration of the mitochondrial
membrane potential. The authors also suggest that the
intracellular sodium increase through the stimulation of
AMPA/kainate receptors is essential to KA-induced excito-
toxicity in CGCs. Conversely, NMDA receptor-induced
excitotoxicity, involves a rise in cytoplasmic calcium con-
centration, and the intracellular calcium concentration is
altered in CGCs exposed to glutamate. The di€erences in
caspase-3 activation between glutamate and KA may be due
to the distinct ion permeability of NMDA and AMPA
receptors (Savidge et al., 1997; 1999; Savidge & Bristow,
1997; 1998; Rago et al., 2001).
Nevertheless, a caspase-independent apoptotic mechanism
has also been reported in other apoptotic processes, since
caspase inhibitors do not protect from neuronal cell death
induced by b-amyloid peptide (Saez-Valero et al., 2000) in
mice lacking caspase-3 (apoptosis is prevented but not
neuronal cell death) (D'Mello et al., 2000). Moreover, the
mechanism of neuronal cell death induced by methylmercury
in CGCs (Castoldi et al., 2000; Dare et al., 2000), serum
deprivation in cortical neurons (Hamabe et al., 2000) and
lack of Bax in mice (Miller et al., 1997) seems to be caspase-
independent. Calpain I is activated during apoptosis (Chan &
Mattson, 1999). ALLN, a calpain I inhibitor, did not prevent
KA-induced neurotoxity. Thus, we conclude that this protein
is not involved in this apoptotic/necrotic process.
The mechanism by which KA induces apoptosis is not well
understood, but it is known that KA induces the expression
of some apoptotic signals such as c-Jun, the cell-cycle
regulatory proteins p21 and p53 (Uberti et al., 1998; Chan
& Mattson, 1999; Grilli & Memo, 1999) and cyclin D
activation (Giardina et al., 1998). To further characterize the
apoptotic process induced by KA in CGCs, we studied the
expression of Par-4, an immediate early gene involved in the
neuronal apoptotic biochemical cascade. Our results indicate
that both KA and colchicine-incubation evoke the expression
of the pro-apoptotic protein Par-4. Moreover, this increase in
Par-4 expression depends on AMPA/kainate receptor activa-
tion, since it is prevented by NBQX. Par-4 is a protein
identi®ed in prostate tumour cells undergoing apoptosis (Sells
et al., 1997). It plays a key role in the apoptotic process
induced by several toxins and excitatory amino acids (Duan
et al., 1999; Camandola & Mattson, 2000). Moreover, both in
Alzheimer's disease and in amyotrophic lateral sclerosis, Par-
4 levels increase (Guo et al., 1998; Pedersen et al., 2000).
However, the mechanism by which Par-4 evokes neuronal
apoptosis in unclear, but it is characterized by a mitochon-
drial alteration (Duan et al., 1999). We would like to
highlight that mitochondrial dysfunction releases cyt-c and
AIF (apoptotic induction factor), which activate the
apoptosis executioner machinery.
Figure 9 Chromatin condensation in permeabilized CGCs exposed
to KA (500 mM) for 24 h. After exposure to KA, the CGCs were
®xed, stained with propidium iodide and photographed under the
¯uorescence microscope, calibration bar, 10 mM. The nuclei were
counted under the ¯uorescence microscope, distinguishing the normal
from the condensed nuclei with the criteria stated in Methods. The
statistical analysis was carried out using one-way ANOVA followed
by Tukey's test **P50.01, ***P50.001 vs Control; ###P50.001 vs
KA 500 mM. Colchicine, 1 mM.
Parkinson's disease patients (Tatton, 2000) and after
traumatic brain injury (Yakovlev et al., 1997). Moreover,
caspase-3 is activated in CGCs by glutamate (Du et al.,
1997a), dopamine (Dodel et al., 1999), MPP+ (Du et al.,
1997b), colchicine (Bonfoco et al., 1995) and serum/
potassium deprivation (Marks et al., 1998). There is evidence
that NMDA increases caspase-3 activity in CGCs but this
increase is much lower than that observed in low potassium-
treated cells (Nath et al., 1998). The same authors showed
that KA-induced caspase activation by detecting an a-
spectrin breakdown product and NMDA clearly increases
caspase activity, whereas KA-treated cells show only a slight
increase. On the other hand, in cultures of cortical neurons,
NMDA receptor activation dramatically increases caspase-3
activity and also induces apoptosis. Stimulation of AMPA/
kainate receptors slightly modi®es these parameters (Hira-
shima et al., 1999).
Here, we demonstrate that KA induces a modest increase
in caspase-3 activity compared with colchicine. These results
suggest that caspase activation does not play a pivotal role in
KA-induced neuronal cell death in CGCs. This hypothesis is
supported by the inability of Z-VAD.fmk (broad acting
In conclusion, caspase-3 activation is not a crucial event in
KA-induced apoptosis in CGCs, since caspase inhibitors do
not prevent apoptotic features like nuclear condensation.
Moreover, the expression of Par-4 may play a key role in
CGCs apoptosis.
British Journal of Pharmacology vol 135 (5)

E. Verdaguer et al
Role of caspases in kainic acid-induced cell death
1305
Figure 10 Evidence that Par-4 mediates KA-induced apoptosis in CGCs. Upper panels show confocal images of Par-4
immunoreactivity in CGCs, calibration bar, 30 mM. Lower panel shows levels of Par-4 assessed by Western blot. Control (CT),
kainate (KA, 500 mM), NBQX (10 mM) colchicine (CX, 1 mM).
We thank the Language Advice Service of the University of
Barcelona for revising the manuscript. We also thank Dr Jaume
Comas and Ms Rosario Gonzalez, from the Scienti®c-Technical
Services of the University of Barcelona, for their technical
assistance in ¯ow cytometry. This study was supported by the
CICYT Grant PM98-0195 and Fundacio La Marato de TV3.
E. Verdaguer is the recipient of a fellowship from the University of
Barcelona.
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(Received November 26, 2001
Revised January 3, 2002
Accepted January 3, 2002)
British Journal of Pharmacology vol 135 (5)