Download Cannabinoid agonist WIN55,212-2 induces apoptosis in cerebellar

Journal of Neuroscience Research 83:1058–1065 (2006)
Cannabinoid Agonist WIN55,212-2 Induces
Apoptosis in Cerebellar Granule CELLS
via Activation of the CB1 Receptor and
Downregulation of bcl-xL Gene Expression
Giacomo Pozzoli, Giuseppe Tringali, Mauro Vairano, Monia D’Amico,
Pierluigi Navarra, and Maria Martire*
Institute of Pharmacology, Catholic University School of Medicine, Rome, Italy
The endogenous cannabinoid system is involved in the
regulation of a number of physiologic effects in both the
central and peripheral nervous systems. Its role in the
control of neuronal cell proliferation has attracted major
attention because of its potential implications for new
therapeutic strategies. In the present study, we demonstrated that treatment of cultured cerebellar granule cells
with the synthetic cannabinoid WIN55,212-2, causes
cell-body and nuclear shrinkage, which are hallmarks of
neuronal apoptosis, as well as concentration-dependent
decrease in cell viability. Staining with the fluorescent nuclear dye, Hoechst 33258, revealed apoptosis in 27.1%
and 58.5% of cells exposed to 1 and 10 lM of WIN55,
212-2, respectively (P < 0.01 and P < 0.001 vs. control
respectively) after 36 hr. After 24 hr of exposure to
WIN55,212-2, mRNA levels for the anti-apoptotic gene bclxL were reduced to 45.6% of those found in control (P <
0.01). These effects were completely reverted when cells
were exposed to the synthetic cannabinoid in the presence
of the specific CB1-receptor antagonist, SR141716A
(1 lM). Moreover, the pro-apoptotic effect of 10 lM
WIN55,212-2 could be reduced by the addition to the incubation medium of a cell-permeant inhibitor of caspase-1
(50 nM). Finally, WIN55,212-2 significantly increased caspase-1 activity after 24 hr. These findings show that the
activation of CB1 receptors on cerebellar granule cells
induces apoptotic cell death, which is associated with
downregulation of the anti-apoptotic gene, bcl-xL, and at
least in part, activation of caspase-1. VC 2006 Wiley-Liss, Inc.
Key words: cannabinoid; WIN55,212-2; CB1 receptor;
cerebellar granule cells; apoptosis; bcl-xL
Cannabinoids, the active components of Cannabis
sativa, exert a wide spectrum of effects in the central nervous system and at peripheral sites (Pertwee, 2000; Porter
and Felder, 2001). These effects are mediated by binding
to specific plasma-membrane G protein-coupled receptors
(Howlett, 1995). To date, two different cannabinoid
receptors have been characterized and cloned from mammalian tissues: CB1 (Matsuda et al., 1990) and CB2 receptors (Munro et al., 1993). The CB1 receptor is expressed
' 2006 Wiley-Liss, Inc.
mainly in the central nervous system, whereas the CB2 receptor is found primarily in cells of the immune system.
Increasing attention has been focused on cannabinoids
since the discovery of a family of endogenous CB-receptor ligands (Devane et al., 1992; Stella et al., 1997). The
importance of endogenous cannabinoid system is testified
by reports of high-level expression of cannabinoid receptors in the brain (Childers and Breivogel, 1998), by
descriptions of specific mechanisms of synthesis, uptake
and degradation of endocannabinoid (Felder and Glass,
1998) and by the evidence of neuromodulatory actions
exerted by endogenous cannabinoids (Di Marzo et al.,
1998).
Recently, interest has been raised by the possible role
of anandamide (AEA) and other cannabinoids in the regulation of cell growth and differentiation, which might
account for some of the pathophysiologic effects of these
lipids. Cannabinoids can induce growth arrest or apoptosis
in a number of transformed neural and non-neural cells in
culture (Guzmán et al., 2002, Maccarrone and FinazziAgrò, 2003), and they were shown to produce regression
of malignant gliomas in rodents by a mechanism involving
sustained ceramide generation and extracellular signalregulated kinase activation (Galve-Roperh et al., 2000).
Other experimental evidence indicates that cannabinoids
can protect normal neurons from toxic insults, such as glutamatergic overstimulation (Shen and Thayer, 1998), ischemia (Nagayama et al., 1999), or oxidative damage
(Hampson et al., 1998). As for their effects on immune
cells, low doses of cannabinoids may enhance proliferation
Contract grant sponsor: Italian MURST; Contract grant number:
7020863 Catholic University.
*Correspondence to: Maria Martire, Institute of Pharmacology, Catholic
University School of Medicine, Largo F. Vito 1-00168 Rome, Italy.
E-mail: [email protected]
Received 7 September 2005; Revised 6 December 2005; Accepted 22
December 2005
Published online 21 February 2006 in Wiley InterScience (www.
interscience.wiley.com). DOI: 10.1002/jnr.20794
Cannabinoid and Neurotoxicity in CGC
(Derocq et al., 1995) whereas high doses generally induce
growth arrest or apoptosis (Zhu et al., 1998).
To obtain additional evidence of the proapoptotic
capacity of the cannabinoids, we evaluated the in vitro survival of cerebellar granule cells (CGCs) after exposure to the
synthetic cannabinoid, WIN55,212-2. CGC cultures where
chosen because they represent a specific and widely used experimental model for studying the molecular mechanisms of
neurodegeneration (Galli et al., 1995). High expression of
CB1 receptor mRNA has been documented in these cells,
and the receptor seems to be mainly located on parallel
fibers in the molecular layer (Egertová and Elphick, 2000).
Cerebellar CB1 receptors seem to be involved in some of
the motor effects associated with acute administration of
D9-tetrahydrocannabinol (D9-THC), e.g., hypolocomotion,
ataxia, and catalepsy (Chaperon and Thiebot, 1999).
We found that treatment of CGCs with WIN55,
212-2 produces DNA fragmentation and diminishes bclxL mRNA levels. Furthermore, the apoptosis produced
by the drug was blocked by the selective CB1 receptor
antagonist, SR141716A and reduced by the pretreatment
with the cell-permeable inhibitor of caspase-1, YVAD-CHO.
MATERIALS AND METHODS
Preparation of Cell Cultures
Primary cultures of CGCs were prepared from 7-dayold rats as described previously (Cambray-deakin, 1995). In
brief, cerebella were sliced and the tissue was dissociated
through trypsinization in 0.025% trypsin solution (15 min at
378C) and trituration in the presence of DNase (0.01%) and
trypsin inhibitor (0.05%). Dissociated cells were collected after
centrifugation by passing through a 4% BSA gradient and
resuspended in basal medium Eagle (BME; Biochrom, Berlin,
Germany) supplemented with 10% fetal calf serum, 2 mM
glutamine, 25 mM KCl and 100 IU penicillin/100lg/ml
streptomycin. Cells were plated onto dishes coated with 10
lg/ml poly-D-lysine at a density of 7 3 106 cells per 60 mm
dish, and maintained in a humidified atmosphere of 5% CO2
95% O2 at 378C. 10 lM cytosine arabinofuranoside was
added after 18 hr of culture to inhibit the growth of non-neuronal cells and did not affect the survival of cultured neurons
as assessed through morphologic and biochemical analyses
(data not shown). Cultures set up this way contain >98%
granule cells and a small amount of glial and endothelial cells
as contaminants.
Treatments were carried out after 7 days in vitro (DIV).
Cells were washed twice in serum-free, 2 mM glutamine,
25 mM KCl, penicillin/streptomycin BME medium and
then maintained in this medium. To study the effects of
WIN55,212-2, this was added directly to the 25 mM KCl
medium at appropriate concentrations. In experiments with
antagonists, SR141716A and YVAD-CHO were added to the
incubation medium 20 min before and then during the incubation in the presence of WIN55,212-2.
Microscopic Analysis of Apoptotic Neuronal Death
The morphologic features of apoptotic degeneration
were analyzed through the use of fluorescence microscopy
Journal of Neuroscience Research DOI 10.1002/jnr
1059
with the nuclear dye Hoechst 33258 (Forloni et al., 1993).
Cells were plated at a density of 2.5 3 106 cells per 35-mm
dish containing glass coverslips coated with 1 mg/ml poly-D-lysine. After exposure to experimental treatments, cells were
washed with PBS and then fixed in 4% paraformaldehyde in
PBS for 30 min at room temperature. Cells were then washed
three times with PBS and incubated for 10 min in a humidified
atmosphere of 5% CO2, 95% O2 at 378C with the DNA-binding dye Hoechst 33258 (0.5 lg/ml in PBS). After being washed
with water, coverslips were mounted onto glass slides and photographed on a fluorescence microscope (Zeiss Axiovert 25)
with excitation at 360 nm, at a magnification of 3400.
Cells with bright, condensed nuclei were counted as
positive for apoptosis, whereas cells were considered viable if
their chromatin was diffuse and evenly distributed throughout
the nucleus. Each condition was represented in three dishes
per experiment. Normal and apoptotic neurons were counted
from three fields per dish in a fixed pattern.
RNA Extraction
Total RNA was extracted by the guanidine thiocyanate
lysis method of Chomczynski and Sacchi (1987). The average
yield of RNA was 40–45 lg/7 3 106 cells.
RNase Protection Assay
To measure mRNA expression of a number of apoptotic-related genes, the RiboQuantTM multi-probe template set
rAPO-1 (Pharmingen, La Jolla, CA) containing cDNA templates for rat FAS, bcl-xL, bcl-xS, FASL, caspase-1, caspase-2
(L) and (S), caspase-3, bax, and bcl-2, was used. Templates for
the analysis of rat mitochondrial ribosomal protein L32 and rat
glyceraldeide-3-phosphate dehydrogenase (GAPDH) housekeeping genes are also included in this set to allow assessments
of total RNA levels for normalizing sampling. Nucleotide
antisense riboprobes for the above mentioned genes were synthesized by using T7 RNA polymerase in the presence of
[a32-P] UTP (800 Ci/mM).
RNase protection analyses were carried out (Pozzoli
et al., 1996) by hybridizing 20 lg total RNA in 24 ll deionized formamide plus 6 ll hybridization buffer containing
1.0 3 105 cpm of each riboprobe. After heating at 808C, the
samples were hybridized at 568C for 15 hr and subsequently
digested by RNase (40 lg/ml RNase A and 350 U/ml
RNase T1) at room temperature for 60 min. The samples
were resolved on 5% polyacrylamide-8M urea gels. Quantitative analysis was carried out using the ImageMaster1 VDS
and the Imagesystem software package (Amersham-Pharmacia
Biotech, San Francisco, CA). The intensity of the protected
apoptosis-related genes fragments were normalized to the intensity of the protected L32 and GAPDH fragments of the
same sample, and results reported as corrected arbitrary units.
Measurement of Caspase-1 Activity
Caspase-1 activity was measured by using the Carboxyfluorescein FLICA caspase-1 assay kit from B-Bridge International Inc. (Sunnyvale, CA) following manufacture’s instructions. CGC were plated at a density of 3 3 106 cells per
35 mm dish coated with 10 lg/ml poly-D-lysine. After treat-
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Pozzoli et al.
Fig. 1. Effects of WIN55,212-2 on CGC cultures vitality. A: Cells
were incubated with medium alone (control), with medium containing 0.1% DMSO (vehicle), or with medium containing graded dosed
of the drug for 36 hr. Phase-contrast micrography shows neurons after 36 hr in medium alone (a), or in medium containing 1 lM
WIN55,212-2 (b), or 10 lM WIN55,212-2 (c). Scale bar ¼ 25 lm.
B: Cells were incubated as in (A). Staining with the fluorescent nuclear dye Hoechst 33258 shows neurons after 36 hr in medium alone
(a), or in medium containing 1 lM WIN55,212-2 (b), or 10 lM
WIN55,212-2 (c) or 0.1% DMSO (d). Arrows indicate typical apoptotic nuclei with fragmented/condensed chromatin. C: Dose range
of the effects of WIN55,212-2 on neuronal vitality. Cells were incubated as in (A). Results are from three independent experiments. **P
< 0.01 vs. control; ***P < 0.001 vs. control. Figure can be viewed
in color online via www.interscience.wiley.com.
ment with 10 lM WIN55,212-2, cells were harvested by centrifugation; pellets were resuspended in 300 ll of PBS and
transferred to sterile tubes. Ten microliters of the 30X FLICA
FAM-YVAD-FMK peptide solution, which specifically binds
to caspase-1, were added and cells were then incubated for
1 hr in a humidified atmosphere of 5% CO2, 95% O2 at 378C
in the dark. Two milliliters of wash buffer were added and
the cells were centrifuged at 400 3 g for 5 min at room temperature. The pellets were then washed twice with wash
buffer, centrifuged, and resuspended in final volume of 400 ll
PBS. One hundred microliters of cell suspension for each
sample were read in duplicate on a 96-well fluorescence plate
reader set at 490 nm excitation and 520 nm emission using a
495 cut-off filter. Results are expressed as % of control.
Statistical Analysis
The data were analyzed by one-way ANOVA, followed
by post-hoc Newman-Keul’s test for multiple comparisons
among group means, using a PrismTM computer program
Journal of Neuroscience Research DOI 10.1002/jnr
Cannabinoid and Neurotoxicity in CGC
1061
(GraphPad, San Diego, CA), and differences were considered
statistically significant if P < 0.05. All results are presented as
the mean 6 SEM of at least three different experiments carried out in duplicate, unless otherwise specified.
Drugs
R(þ) - [2,3-dihydro - 5-methyl-3-(4-morpholinylmethyl)
pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenyl methanone mesylate, (R(þ)-WIN55,212-2 mesylate, WIN55,212-2)
and dimethylsulfoxide (DMSO) were purchased from Sigma
Chemical (St. Louis, MO). The cell-permeable caspase-1 inhibitor
Ac-AAVALLPAVLLALLAPYVAD-CHO was purchased from
Calbiochem (La Jolla, CA). N-piperidino-5-(4-chlorophenyl)-1(2,4-dichlorophenyl)-4-methyl-3-pyrazole carboxamide (SR141716A)
was a gift of Sanofi Recherche (Montpellier, France). When
appropriate, the drugs were dissolved in DMSO. The DMSO
concentration to which cell cultures were exposed never
exceeded 0.1%. All other reagents were of analytical grade.
RESULTS
Effects of WIN55,212-2 on CGCs Vitality
CGCs were allowed to fully develop in standard
25 mM KCl medium for 7 days after plating. The effect
on cell viability was then determined by incubating the
cultures with graded doses of WIN55,212-2 (1–10 lM)
and monitored after 36 hr by means of phase-contrast microscopic analysis and by Hoechst 33258 staining (Fig. 1).
In phase-contrast observation (Fig. 1A), control neurons
seemed healthy, with round cell bodies and a well developed network of neurites throughout the experiments.
After a 36 hr treatment with 1–10 lM WIN55,212-2, the
cell bodies of several neurons were shrunken, with condensed nuclei, and had lost their phase-bright appearance.
Neurites were fragmented and their density had decreased.
A number of cells were starting to detach from the substrate.
WIN55,212-2 induced a concentration-dependent
decrease in cell viability (27.1% of apoptotic cells after
1 lM, and 58.5% of apoptotic cells after 10 lM, (P <
0.01 and P < 0.001 vs. control respectively), as quantified
by microscopic analysis with the fluorescent nuclear dye
Hoechst 33258 (Fig. 1B,C). Degenerated cells exhibited
the typical features of apoptosis, (Bursch et al., 1992),
including chromatin condensation and fragmentation. By
contrast, control cultures showed no change in the number of viable neurons over 36 hr treatment, with <15%
apoptotic neurons. The latter figure accounts for normally
occurring apoptosis in mature CGCs. The effect of 0.1%
DMSO (vehicle) on CGCs was also tested. As shown in
Figure 1B, DMSO did not modify cell survival.
Effects of WIN55,212-2 on bcl-xL mRNA Levels
To investigate the putative molecular mechanism
underlying the pro-apoptotic activity of WIN55,212-2,
we assessed the gene expression of a number of pro- and
anti-apoptotic factors in CGCs exposed to plain medium
or medium containing 10 lM of the drug. Specific bands
Journal of Neuroscience Research DOI 10.1002/jnr
Fig. 2. Time course of the effects of WIN55,212-2 on bcl-xL
mRNA levels in CGC cultures. Cells were incubated with medium
alone (control) or with medium containing WIN55,212-2 (10 lM)
for the indicated times. Results are from three independent experiments. **P < 0.01 vs. control. A representative experiment is shown
in the insert.
for FAS, bcl-xL, caspase-1, caspase-2, caspase-3, and bax
were detected in controls and treated cells. We found that
WIN55,212-2 decreased the accumulation of mRNA of
the anti-apoptotic gene bcl-xL after 24 hr of treatment (to
45.6% of control, P < 0.01, Fig. 2). The other genes
detected showed changes lower than 5%, that were considered not significant.
Effects of CB1 Receptor Antagonist on CGCs
Vitality and bcl-xL mRNA Levels
To determine whether the WIN55,212-2-induced
neurotoxicity was mediated by the activation of CB1
receptors, CGCs were incubated with 10 lM WIN55,
212-2 for 36 hr in the presence of a specific CB1 receptor
antagonist, SR141716A, used at the concentration of
1 lM. As shown in Figure 3, the latter antagonized the
effects of WIN55,212-2 on CGCs vitality. In addition,
CB1 receptor antagonist was also able to completely abolish WIN55,212-2-induced decrease in bcl-xL mRNA
accumulation after 24 hr (Fig. 4).
Effects of Caspase-1 Inhibitor on WIN55,212-2Induced Decrease in CGCs Vitality
The activation of caspase pathways is a critical event
in apoptosis; therefore it was of interest to determine
whether caspase activation occur in CGCs after WIN55,
212-2 exposure. When CGCs were incubated for 36 hr
with 10 lM WIN55,212-2 in the presence of a specific
caspase-1 inhibitor (ICE, 50 nM), the latter was able to
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Pozzoli et al.
Fig. 3. Effects of CB1 receptor antagonist on WIN55,212-2-induced
decrease in vitality of CGC cultures. Cells were incubated with
medium alone (control) or with medium containing 10 lM
WIN55,212-2 and 1 lM SR141716A for 36 hr. Results are from
three independent experiments. ***P < 0.001 vs. control.
Fig. 5. Effects of caspase-1 inhibitor (ICE) on WIN55,212-2induced decrease in CGC cultures vitality. Cells were incubated with
medium alone (control) or with medium containing 10 lM
WIN55,212-2 and 50 nM ICE for 36 hr. Results are from three independent experiments. *P < 0.05 vs. control; ***P < 0.001 vs.
control; ##P < 0.01 vs. WIN þ ICE.
Fig. 6. Time course of the effects of WIN55,212-2 on caspase-1 activity in CGC cultures. Cells were incubated with medium alone
(control) or with medium containing WIN55,212-2 (10 lM) for the
indicated times. Results are from three independent experiments.
*P < 0.05 vs. control.
pase-1 activity after 24 hr of exposure (þ30.1% with
respect to control, P < 0.05) (Fig. 6).
Fig. 4. Effects of CB1 receptor antagonist on WIN55,212-2-induced
decrease in bcl-xL mRNA levels in CGC cultures. Cells were incubated with medium alone (control) or with medium containing 10
lM WIN55,212-2 and 1 lM SR141716A for 24 hr. Results are
from three independent experiments. **P < 0.01 vs. control. A representative experiment is shown in the insert.
reduce significantly the WIN55,212-2-induced decrease
in CGCs vitality (Fig. 5).
Effects of WIN55,212-2 on Caspase-1 Activity
CGCs were exposed to 10 lM WIN55,212-2 in 8and 24-hr experiments. The cannabinoid increased cas-
DISCUSSION
Experimental data on the effects of cannabinoids on
neuronal survival are still highly controversial. Several studies have shown that D9-THC can induce neurotoxic effects
in cultured cell systems, including hippocampal neurons
(Chan et al., 1998), cortical neurons (Campbell, 2001), and
glioma cells (Sánchez et al., 1998). In addition to its antiproliferative effects in neuronal cultures, D9-THC has also
been shown to inhibit neuronal cell growth in vivo (GalveRoperh et al., 2000). Furthermore, AEA induces apoptosis
in PC-12 cells (Sarker et al., 2003) and lymphocytes
(Schwarz et al., 1994). D9-THC-induced apoptosis in hipJournal of Neuroscience Research DOI 10.1002/jnr
Cannabinoid and Neurotoxicity in CGC
pocampal (Chan et al., 1998) and cortical (Downer et al.,
2001, 2003) neuron cultures is CB1 receptor-dependent.
Proposed mechanisms of cannabinoid-induced neurotoxicity include the generation of reactive oxygen species (Chan
et al., 1998); activation of the caspase-3 cell-death pathway
(Campbell, 2001; Downer et al., 2001); sphingomyelin hydrolysis (Sánchez et al., 1998); sustained ceramide accumulation (Galve-Roperh et al., 2000; Rueda et al., 2000); activation of the JNK cascade (Downer et al., 2003; Sarker
et al., 2003); inhibition of protein kinase A and the K-ras
oncogene product p21ras; and activation of the p42/p44
ERK pathway (for a review, see Maccarrone and FinazziAgrò, 2003). Activation of the vanilloid receptor (VR1) by
AEA, which binds to an intracellular site of the receptor,
can provoke a series of pro-apoptotic events that include
the release of cytochrome c and the activation of caspase-3
and caspase-9 (Maccarrone et al., 2000).
Other studies suggest, however, that cannabinoids
may have neuroprotective properties (Guzmán et al.,
2002; Mechoulam et al., 2002). Acute in vivo administration of WIN55,212-2 seems to protect against global and
focal ischemic injury in both the hippocampus and cortex
(Nagayama et al., 1999); in an in vivo model of excitotoxicity induced by ouabain, the application of D9-THC (van
der Stelt et al., 2001a) or AEA (van der Stelt et al., 2001b)
reduced infarct volumes via a CB1-dependent mechanism.
Some investigators have even postulated that the endogenous cannabinoid system plays a role in physiologic neuroprotection (Guzmán et al., 2001; Marsicano et al.,
2003).
CB1 receptors seem to be the main mediators of cannabinoid neurotoxicity, whereas their neuroprotective
effects are mediated by CB1 receptors along with other
molecular targets, which are characterized by differential
responses to the various cannabinoids. Cannabinoid-induced neuroprotection has been attributed to the inhibition of excitatory amino-acid release (Shen et al., 1996),
to the blockade of voltage-dependent Ca2þ channel activity, to noncompetitive blocking of NMDA receptors
(Eshhar et al., 1995), to antioxidant activity (Hampson
et al., 1998), to the inhibition of NO release (Gallily et al.,
1997), as well as to inhibition of the production of cytokines and nuclear factor kappa B (Jüttler et al., 2004).
Although many of the studies conducted thus far
seem to support a neuroprotective role for the endocannabinoids (Guzmán, 2003), it is important to recall that several cannabinoids have been shown to exert different types
of effects on neuronal survival. The actual effect of a given
compound may depend on the nature of the toxic insult,
the cell type being studied, and the experimental conditions adopted.
In the present study, we analyzed the effects of the
synthetic cannabinoid, WIN55,212-2, on the survival of
cultured CGCs, in terms of apoptosis itself and the
expression of genes involved in this phenomenon.
WIN55,212-2 acts as a full agonist at CB1 receptors,
which are widely expressed in brain regions involved in
the regulation of movement, such as the basal ganglia
and the cerebellum (Chaperon and Thiebot, 1999). The
Journal of Neuroscience Research DOI 10.1002/jnr
1063
CB1 receptors expressed in the cerebellar cortex are
located predominantly on axon terminals of fibers providing excitatory input to Purkinje cells, i.e., parallel
fibers originating from granule cells and climbing fibers
originating in the inferior olive (Matsuda et al., 1993).
The endocannabinoids released by Purkinje cells cause
retrograde inhibition of presynaptic Ca2þ influx and suppress the release of neurotransmitter at excitatory afferent
synapses, such as those of the cerebellar granule cells
(Kreitzer and Regehr, 2001). The cerebellar granule cell/
Purkinje cell system is thus a useful system to study the
effects of cannabinoids.
Our data demonstrate that WIN55,212-2 induces
apoptosis in cultured CGCs. Fluorescence microscopy of
cells stained with Hoechst 33258 revealed morphologic
changes typical of apoptotic degeneration (i.e., condensation, nuclear fragmentation) after 36 hr of exposure to
WIN55,212-2. Apoptosis was observed in 27.1% of the
cells exposed to WIN55,212-2 at a concentration of 1 lM
and 58.5% of those exposed to 10 lM. Proapoptotic
effects of the synthetic cannabinoid were also reflected in
the diminished expression of the anti-apoptotic protein
bcl-xL. The bcl-2 family includes proteins that inhibit apoptosis (e.g., bcl-2, bcl-xL) and others that are proapoptotic (e.g., bax, bcl-xS, bak). Like bcl-2, bcl-xL prevents
the release of apoptotic factors from mitochondria, e.g.,
cytochrome c (Jordan et al., 2004), whereas bax and bak
enhance mitochondrial permeability and increase the
release of cytochrome c into the cytoplasm. The bcl-x/bax
ratio can thus determine the fate of the cell in terms of its
survival (Roth and Reed, 2002).
The apoptosis observed in cells exposed to 10 lM
WIN55,212-2 for 36 hr was completely abolished by simultaneous exposure to SR141716A (1 lM), a specific
CB1-receptor antagonist. The fact that this concentration
of SR141716A was sufficient to reverse the apoptotic
effect of 10 lM WIN55,212-2 suggests that the apoptosis
is mediated by activation of CB1 receptors. The vanilloid
receptors do not seem to be involved. Functional experiments conducted by other investigators indicate that AEA
may induce apoptosis through VR1-dependent mechanisms whereas its neuroprotective effects might be mediated by the CB1 receptor (Maccarrone et al., 2000). Our
findings, however, are more consistent with the results of
Downer et al. (2001), who showed that D9-THC induces
apoptosis in cultured cortical neurons via a CB1 receptordependent mechanism. More recently, this effect was also
shown to depend on activation of the JNK cascade
(Downer et al., 2003).
It is important to note that the neuroprotective
effects of cannabinoids are usually more evident in intact
animals than in cultured cells. In the former setting, the
cannabinoid can produce different effects in different cell
types (neurons, glia, vascular endothelial cells). Furthermore, in cell cultures, the cannabinoids can exert opposite
effects on cell survival that depend on the characteristics of
exposure. In general, high cannabinoid concentrations
and long exposure times inhibit cell growth or provoke
cell death. Cultured neuron survival also depends on the
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Pozzoli et al.
specific cell type or its stage of differentiation (Guzmán,
2003). In our study, apoptosis of cultured CGCs was
observed after prolonged exposure to high concentrations
of WIN55,212-2. The lowest concentration of WIN55,
212-2 that induced apoptosis in vitro in our study (1 lM)
is undoubtedly high. Azorlosa et al. (1992) have shown,
however, that marijuana smoking can produce plasma D9THC levels in humans as high as 1 lM, and D9-THC can
be concentrated 15- to 20-fold in some tissues (Johansson
et al., 1989). This means that recreational use of marijuana
can produce levels up to 20 lM in some tissues, and due
to the lipophilia of D9-THC, the central nervous system is
a prime target for high concentrations. For this reason, in
vitro evaluation of the effects of high cannabinoid concentrations is by no means irrelevant.
The terminal steps of programmed cell death are often carried out by the caspases, so often, in fact, that their
activation is considered one of the principal biochemical
characteristics of apoptosis. CB1 receptor-mediated apoptosis in cultured cerebral neurons is reportedly associated
with activation of caspase-3 (Campbell, 2001), which is
also involved in cell death induced by AEA in cultured
PC-12 cells (Sarker et al., 2000). Other authors have
shown that apoptosis induced by D9-THC in macrophages
and lymphocytes is blocked by an inhibitor of caspase-1
(Zhu et al., 1998). In contrast, in a recent knock-out study
conducted in mice, the absence of CB1 receptors was
found to be associated with an increase in caspase-3 activity (Jackson et al., 2005). Our findings indicate that
WIN55,212-2-induced apoptosis in CGCs is at least partially caspase-1-mediated. The apoptosis induced by 10 lM
WIN55,212-2 was attenuated when cells were incubated
in the presence of a cell-permeant caspase-1 inhibitor.
WIN55,212-2 was also able to increase caspase-1 activity
in these cells. We did not observe, however, any increase
in caspase-1 mRNA accumulation. This leads to suggest a
post-transcriptional modulation in enzyme activity which
follows WIN-induced bcl-xL downregulation. Caspase-1
is generally categorized as a cytokine-processing caspase.
In fact, it is responsible for the proteolytic processing that
transforms premature interleukin-1b (IL-1b) to its mature
form. Nonetheless, it has also been shown to induce apoptosis and may function in this way in various developmental stages (Zhang et al., 2003). Our data indicate that
caspase-1 is at least one of the mediators of WIN55,212-2
induced apoptosis in CGCs.
In conclusion, our results suggest that the activation
of CB1 receptors on cerebellar granule cells induces apoptotic cell death, which is associated with downregulation
of the anti-apoptotic protein, bcl-xL, and caspase-1 activation. Endogenous and exogenous cannabinoid ligands
may play general roles in cell survival/death decisions.
Many of their effects seem to depend on the activation of
specific membrane receptors; others seem to be mediated
by interactions with intracellular signalling cascades. Identification of the neuroprotective and neurotoxic properties
of individual ligands could increase our understanding of
how the endogenous cannabinoid system regulates cell
survival and cell death.
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