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Molecular Signalling Mediating the Protective Effect of A
1
Adenosine and mGlu3 Metabotropic Glutamate Receptor
Activation against Apoptosis by Oxygen/Glucose Deprivation in
Cultured Astrocytes
Renata Ciccarelli, Iolanda D’Alimonte, Patrizia Ballerini, Mariagrazia D’Auro, Eleonora Nargi,
Silvana Buccella, Patrizia Di Iorio, Valeria Bruno, Ferdinando Nicoletti, and Francesco Caciagli
Department. of Biomedical Sciences, “G. D’Annunzio” University, Chieti, Italy (R.C., I.D.A., P.B., M.D.A., E.N., S.B., P.D.I.,
F.C.); and Department of Human Physiology and Pharmacology, University of Rome “La Sapienza,” Rome, Italy, and
Department of Neuropharmacology, I.N.M. Neuromed, Pozzilli, Italy (V.B., F.N.)
Received October 11, 2006; accepted February 9, 2007
ABSTRACT
Astrocyte death may occur in neurodegenerative disorders and
complicates the outcome of brain ischemia, a condition as-
sociated with high extracellular levels of adenosine and glu-
tamate. We show that pharmacological activation of A
1
adenosine and mGlu3 metabotropic glutamate receptors
with N
6
-chlorocyclopentyladenosine (CCPA) and (⫺)2-oxa-
4-aminocyclo-[3.1.0]hexane-4,6-dicarboxylic acid (LY379268),
respectively, protects cultured astrocytes against apoptosis
induced by a 3-h exposure to oxygen/glucose deprivation
(OGD). Protection by CCPA and LY379268 was less than ad-
ditive and was abrogated by receptor blockade with selective
competitive antagonists or pertussis toxin. Both in control as-
trocytes and in astrocytes exposed to OGD, CCPA and
LY379268 induced a rapid activation of the phosphatidylinosi-
tol-3-kinase (PI3K) and extracellular signal-regulated kinases 1
and 2 (ERK1/2)/mitogen-activated protein kinase (MAPK) path-
ways, which are known to support cell survival. In cultures ex-
posed to OGD, CCPA and LY379268 reduced the activation of
c-Jun N-terminal kinase and p38/MAPK, reduced the levels of the
proapoptotic protein Bad, increased the levels of the antiapoptotic
protein Bcl-X
L
, and were highly protective against apoptotic
death, as shown by nuclear 4⬘-6-diamidino-2-phenylindole stain-
ing and measurements of caspase-3 activity. All of these effects
were attenuated by treatment with 1,4-diamino-2,3-dicyano-1,4-
bis(methylthio)butadiene (U0126) and 2-(4-morpholinyl)-8-phenyl-
1(4H)-benzopyran-4-one hydrochloride (LY294002), which inhibit
the MAPK and the PI3K pathways, respectively. These data sug-
gest that pharmacological activation of A
1
and mGlu3 receptors
protects astrocytes against hypoxic/ischemic damage by stimu-
lating the PI3K and ERK1/2 MAPK pathways.
Astrocytes, the most abundant glial cell types in the brain,
provide metabolic and trophic support to neurons by several
mechanisms that include the clearance of ions and environ-
mental toxins, the supply of energy substrates, and the pro-
duction of trophic factors, and modulate synaptic activity
(Volterra and Meldolesi, 2005). Impairments in these func-
tions critically affect neuronal survival.
Recent studies have shown that ischemic and inflamma-
tory insults induce astrocyte apoptotic death, and this con-
tributes to the pathophysiology of short- and long-term neu-
rodegenerative disorders (Takuma et al., 2004). Apoptotic
astrocytes are found in Alzheimer’s disease (Kobayashi et al.,
This research was supported by funds (to R.C. and F.C.) from the Centre of
Excellence on Aging of the University of Chieti and the Italian Ministry of
Education, University and Research.
R.C. and I.D.A. contributed equally to this article.
Article, publication date, and citation information can be found at
http://molpharm.aspetjournals.org.
doi:10.1124/mol.106.031617.
ABBREVIATIONS: CCPA, N
6
-chlorocyclopentyladenosine; ASK1, apoptosis-signal-regulating kinase 1; DAPI, 4⬘-6-diamidino-2-phenylindole;
DMEM, Dulbecco’s modified Eagle’s medium; DPCPX, 1,3-dipropyl-8-cyclopentyl xanthine; ERK1/2, extracellular signal-regulated kinases 1 and
2; HRP, horseradish peroxidase; JNK, c-Jun N-terminal kinase; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride;
LY341495, (2S,1⬘S,2⬘S)-2(9-xanthylmethyl)-2-(2⬘-carboxycyclo-propyl)glycine; LY379268, (⫺)2-oxa-4-aminocyclo-[3.1.0] hexane-4,6-dicarboxylic
acid; MAPK, mitogen-activated protein kinase; mGluR, metabotropic glutamate receptor; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-
phenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PBS, phosphate-buffered saline; PI3K, phosphatidylinositol-3 kinase; OGD, oxygen glucose depriva-
tion; PTX, pertussis toxin; PKB, protein kinase B; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene; ANOVA, analysis of
variance; RFLU, relative fluorescence units; MEK, mitogen-activated protein kinase kinase; solvent A, KH
2
PO
4
and tetrabutylammonium phos-
phate; solvent B, methanol plus KH
2
PO
4
and tetrabutylammonium phosphate.
0026-895X/07/7105-1369–1380$20.00
M
OLECULAR PHARMACOLOGY Vol. 71, No. 5
Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics 31617/3200018
Mol Pharmacol 71:1369–1380, 2007 Printed in U.S.A.
1369
at ASPET Journals on November 6, 2015molpharm.aspetjournals.orgDownloaded from
2002), ischemic demyelinating lesions in vascular dementia
(Tomimoto et al., 1997), and in the gray matter of frontotem-
poral dementia (Martin et al., 2000). Moreover, astrocyte
damage precedes neuronal death in the spinal cord of mice
carrying mutations of type-1 superoxide dismutase that are
typically found in amyotrophic lateral sclerosis (Bruijn et al.,
1997). Hence, drugs with potential for use in the treatment of
neurodegenerative disorders may target membrane receptors
that support glial cell survival. We have focused on some
adenosine and glutamate receptors that are known to be
expressed by astrocytes and regulate several functions of
glial cells, including the production of trophic factors (Bruno
et al., 1998; Ciccarelli et al., 1999). Activation of A
1
adenosine
receptors produces neuroprotective effects (Ribeiro et al.,
2002) and protects human vascular endothelial cells against
apoptosis induced by low concentrations of ethanol (Liu et al.,
2002); however, whether these receptors regulate processes
of death/survival in glial cells survival is unknown. In con-
trast, deleterious effects are induced by the activation of
other adenosine receptor subtypes, and induction of astrocyte
apoptosis by adenosine is mediated by A
3
receptors (Appel et
al., 2001; Di Iorio et al., 2002).
Glutamate activates both ionotropic (
␣
-amino-3-hydroxy-5-
methyl-4-isoxazolepropionic acid, N-methyl-
D-aspartate, and
kainate) and metabotropic (mGlu1 to mGlu8) receptors. As-
trocytes mainly express
␣
-amino-3-hydroxy-5-methyl-4-isox-
azolepropionic acid/kainate, mGlu3, and mGlu5 receptors,
although other subtypes are occasionally found. Activation of
glial mGlu3 receptors protects neighbor neurons in cultured
cortical cells through a paracrine mechanism mediated by
the production of transforming growth-factor

(Bruno et al.,
1998), and the presence of astrocytes is required for the
neuroprotective effects of mGlu2/3 receptor agonists
(D’Onofrio et al., 2001). The role of mGlu3 receptors in the
regulation of glial cell survival is also unknown. It is note-
worthy that A
1
and mGlu2/3 receptors are both linked to a G
i
protein and functionally interact in modulating glutamate
release from nerve endings (Di Iorio et al., 1996).
The study of these two receptors is now facilitated by the
availability of potent and selective ligands. A
1
receptors are
activated by N
6
-chlorocyclopentyl-ADO (CCPA) and antago
-
nized by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (Jacob-
son and Gao, 2006), whereas mGlu3 receptors are activated
by LY379268 and antagonized by LY341495 (Schoepp et al.,
1999). Using these drugs, we now report that activation of
both receptors protects cultured astrocytes against apoptotic
death and that this effect is mediated by the activation of the
phosphatidylinositol-3 kinase (PI3K) and extracellular sig-
nal-regulated kinase (ERK) 1/2/mitogen-activated protein ki-
nase (MAPK) pathways.
Materials and Methods
Materials. Poly(D-lysine), L-leucine methyl ester, and pertussis
toxin (PTX) were supplied by Sigma (Sigma-Aldrich, Milan, Italy),
whereas LY294002, LY341495, and DPCPX were from Tocris (Bris-
tol, UK). U0126 was purchased from Calbiochem (San Diego, CA),
and CCPA was from Research Biochemical Incorporated (Sigma-
Aldrich). LY379268 was kindly provided by Eli Lilly (Indianapolis,
IN). Disposable materials for tissue cultures were supplied from
Nalge Nunc (Mascia Brunelli, Milan, Italy). Culture medium, anti-
biotics, and serum were from Invitrogen (Milan, Italy). All other
chemicals were of analytical grade or were the best commercially
available.
Cell Culture and Treatments. Primary cultures of rat astro-
cytes were prepared from neonatal rats 2 to 4 days after birth as
described previously (Di Iorio et al., 2004). Cerebral cortices were
collected in growth medium [high-glucose Dulbecco modified Eagle’s
medium (DMEM) supplemented with 10% fetal calf serum and 1%
penicillin/streptomycin (10,000 U/ml penicillin G sodium and 10,000
g/ml streptomycin sulfate in 0.85% saline)]. Then the tissue was
washed in phosphate-buffered saline (PBS), cut in small fragments,
and digested with 0.025% trypsin/0.04% EDTA solution in PBS for
20 min at 37°C. The cells were then dissociated in 0.01% DNase
solution in growth medium for 10 min at 37°C and centrifuged at
1000 rpm for 10 min. The pellet was resuspended in growth medium
containing 5 mM
L-leucine methyl ester to constrain microglia con-
tamination. Cells, seeded on poly(
D-lysine)-coated T75 flasks, were
grown in this medium for the first 24 h and were then maintained in
an identical medium without leucine methyl ester. The medium was
replaced every 3 to 4 days. At the 7th and then at the 13th day in
vitro, cells were shaken for3hat80rpmonaplate shaker to
minimize attachment and hence microglial contamination of the
cultures. Astrocytes were detached from the culture flasks by treat-
ment (5–10 min at 37°C) with 0.025% trypsin/0.04% EDTA. Astro-
cytes were replated onto poly(
D-lysine)-coated 100-mm dishes at a
concentration of 2 ⫻ 10
6
cells/dish for Western blot analyses or they
were plated onto poly(
D-lysine)-coated round-glass coverslips (Ø ⫽ 13
mm) at a concentration of 3 ⫻ 10
4
cells/coverslip and 96-multiwell
plate at a concentration of 1 ⫻ 10
4
cells/well for the apoptosis/
viability experiments. In experiments in which enzyme inhibitors or
receptor antagonists were tested, astrocytes were pretreated with
various agents for 30 min before the addition of CCPA or LY379268,
except for the ADP-ribosylating factor of the inhibitory guanosine
nucleotide binding protein (G
i
), PTX, which was added overnight (16
h). These treatments included the following: selective inhibitors of
PI3K, LY294002, and MAPK kinase U0126; and the selective antag-
onists of the A
1
adenosine receptor, DPCPX, and of the mGlu2/3
receptor, LY341495.
Oxygen Glucose Deprivation Protocol. Apoptosis was induced
in cultured astrocytes by exposing cells to a combined deprivation of
oxygen and glucose (OGD). Twenty-four hours after replating, cells
were serum-starved for a further 24 h. Then a glucose-free bicarbon-
ate-buffered DMEM (Sigma-Aldrich) was added to the cultures after
a gentle cell washing with the same buffer. This medium was bub-
bled previously with 95% N
2
/5% CO
2
at 3 l/min for 5 min and
prewarmed at 37°C. Hypoxia was induced by placing cells in a
humidified, sealed chamber (Billups-Rothenberg, Del Mar, CA) at
37°C, which was flushed with 95% N
2
/5% CO
2
for 5 min. In this
condition, all but 0.3% oxygen tension could be removed, as indicated
byapO
2
meter (oxygen analyzer OM-11; Beckman Coulter, Milan,
Italy). At the end of the OGD period (3 h), cultures were returned to
standard condition for the indicated periods. In each experiment,
cultures exposed to OGD were always compared with normoxic con-
trols, supplied with DMEM containing glucose, and maintained in
standard incubation condition.
Evaluation of Apoptosis. DNA fragmentation was evaluated
histochemically by 4⬘-6-diamidino-2-phenylindole (DAPI) staining
(Roche Molecular Biochemicals, Mannheim, Germany) and fluores-
cence microscopy. Cells were seeded onto poly(
D-lysine)-coated glass
coverslips, and part of them was exposed to OGD after 24-h serum
starvation in the presence or absence of CCPA or LY379268, for 3 h
in a glucose-free DMEM without fetal calf serum. Cells were then
maintained in normal DMEM without serum, like control cells (not
subjected to OGD), and then astrocytes were fixed with 3.7% para-
formaldehyde in PBS for 25 min at room temperature and then
incubated with 70% ethanol for 15 min at room temperature. DAPI
Antifade ES solution (0.125
g/ml) was added for 5 min at room
temperature to fixed astrocytes. Observations were carried out using
a fluorescence microscope (Leica DMRXA2) (excitation, 358 nm;
1370 Ciccarelli et al.
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emission, 461 nm). The number of apoptotic (i.e., showing frag-
mented nuclei with condensed chromatin) and viable astrocytes was
counted in five fixed fields/coverslip of up to five separate cultures.
The percentage of apoptotic cells was calculated as follows: percent-
age of apoptotic cells ⫽ (total number of cells with apoptotic nuclei/
total number of counted cells) ⫻ 100.
MTS Assay. The number of viable cells was determined using the
CellTiter 96 AQ
ueous
One Solution Cell Proliferation Assay (Promega
Corporation, Madison, WI) according to the manufacturer’s instruc-
tions. In brief, cell cultures (1 ⫻ 10
4
cells/well) were added with 20
l
of CellTiter 96 AQ
ueous
One Solution Reagent containing a tetrazo
-
lium compound, 3-(4,5–dimethylthiazol-2-yl)-5-(3-carboxymethoxy-
phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), and an electron-
coupling reagent (phenazine ethosulfate). The plate was incubated at
37°C for2hinahumidified atmosphere. The reduction of MTS in the
presence of cellular dehydrogenases yielded formazan crystals at the
bottom of the plate. The absorbance was measured at 490 nm using
a microtiter plate reader (Spectracount; PerkinElmer Life and Ana-
lytical Sciences, Boston, MA).
Western Blot Analysis. Western blot analysis was used to detect
phosphorylated extracellular signal-regulated kinases 1 and 2
(ERK1/2), p38 MAPK, c-Jun N-terminal kinase (JNK), Akt/protein
kinase B (PKB), apoptosis signal-regulating kinase 1 (ASK1), and
Bad protein as well as procaspase 3, Bad, and Bcl-X
L
protein content.
Cultured astrocytes were serum-deprived for 24 h before pharmaco-
logical treatments (as reported in the figures). At the end of drug
incubation, astrocytes were washed twice with ice-cold PBS and then
harvested at 4°C in a lysis buffer (25 mM Tris buffer, pH 7.4,
containing 150 mM NaCl, 100
M sodium orthovanadate, 1.5 mM
MgCl
2
, 1.0 mM EDTA, 1.0 mM EGTA, 1% Nonidet P-40, 10% glyc
-
erol, 1 mM phenylmethylsulfonyl fluoride, 5
g/ml leupeptin, and 5
g/ml aprotinin). Cells were disrupted by sonication and then were
centrifuged at 14,000 rpm for 5 min at 4°C. Aliquots (20
l) were
removed from the supernatants for the determination of protein
concentration by the Bio-Rad method. Samples were diluted in SDS-
bromphenol blue buffer and boiled for 5 min. Cell lysates were
separated on 12% SDS-polyacrylamide gels and electrophoretically
transferred to a polyvinylidene fluoride membrane (Bio-Rad Labora-
tories, Milan, Italy).
Membranes were incubated overnight at 4°C with specific primary
antibodies [polyclonal rabbit phospho-ERK1/2, phospho-Akt, phos-
pho-p38, phospho-JNK, phospho-ASK1 (Ser83), and phospho-Bad
(Ser112 or Ser136)] from Cell Signaling Technology (Danvers, MA)
diluted 1:1000, or with polyclonal rabbit anticaspase-3, anti-Bad, or
anti-Bcl-X
L
(final dilution 1:200; Santa Cruz Biotechnologies, Santa
Cruz, CA). Membranes were then exposed to a secondary antibody
for1hatroom temperature [donkey anti-rabbit horseradish perox-
idase (HRP)-conjugated; GE Healthcare, Milan, Italy]. To confirm
that equal amounts of protein were loaded in each lane, the mem-
branes were incubated in stripping buffer (62.5 mM Tris-HCl, pH
6.7, 2% SDS, and 100 mM

-mercaptoethanol) at 50°C for 30 min to
remove the primary/secondary antibody complex. The blots were
then reprobed with nonphosphorylated form of the antibodies men-
tioned above (dilution, 1:1000; Cell Signaling) or with goat polyclonal
anti-

actin (dilution 1:100; Santa Cruz Biotechnologies) (incubation,
1 h at room temperature). Membranes were then exposed to a
secondary antibody for1hatroom temperature (donkey anti-
rabbit HRP-conjugated from GE Healthcare, or donkey anti-goat
HRP-conjugated from Santa Cruz Biotechnologies, respectively, both
diluted 1:2500), according to the manufacturer’s instructions. Immu-
nocomplexes were visualized using the enhancing chemilumines-
cence detection system (GE Healthcare). Densitometric analysis was
performed for the quantification of the immunoblots using the
Molecular Analyst System (Bio-Rad Laboratories) program.
Assay of Caspase-3 Activity. The activity of caspase-3 was
determined using the Apo-ONE Homogenous Caspase 3/7 Assay
according to manufacturer’s instruction (Promega). Cell cultures
(1 ⫻ 10
4
cell/well) were added with Homogeneous Caspase Reagent
containing the fluorescent caspase substrate rhodamine 110, bis(N-
CBZ-
L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide and were
incubated for 4 h. After shaking at 300 rpm for 30 s, the plate was
incubated at room temperature for 30 min to 18 h. The fluorescence
at different time points was measured at an excitation wavelength of
485 ⫾ 20 nm and an emission wavelength of 530 ⫾ 25 nm using a
Fig. 1. Evaluation of OGD-induced
apoptosis in rat cultured astrocytes.
Astrocytes were cultured in serum-
free medium for 24 h and then ex-
posed to OGD for 3 h. Apoptosis was
assessed by DAPI nuclear staining
(A), the MTS assay (B), and caspase-3
activity (C). Values are means ⫾
S.E.M. of three independent experi-
ments performed in duplicate. ⴱⴱ, p ⬍
0.01 versus controls (unpaired Stu-
dent’s t test). D, immunoblot analysis
of procaspase 3 in control astrocytes
and in astrocytes exposed to OGD. At
the end of OGD, cells were main-
tained in DMEM without serum for
further 9 h. Densitometric values are
means ⫾ S.E.M. from three indepen-
dent experiments. ⴱⴱⴱ, p ⬍ 0.001 ver-
sus controls (unpaired Student’s t
test).
A
1
and mGlu3 Receptor Antiapoptotic Effect in Astrocytes 1371
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microtiter plate reader (Fluorocount; PerkinElmer Life and Analyt-
ical Sciences). The values were expressed as the relative fluorescence
units (RFLU) measured at each time point.
Measurements of Extracellular Adenosine and Glutamate.
To evaluate the endogenous adenosine and glutamate released from
astrocytes, culture medium was replaced with Krebs’ solution con-
taining 118.5 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl
2
, 1.2 mM MgSO
4
,
1.2 mM KH
2
PO
4
, 10 mM glucose, and 25 mM NaHCO
3
equilibrated
with 95% air/5% CO
2
at 37°C (pH adjusted at 7.3–7.4). The cultures
were maintained in Krebs’ solution for 60 min to evaluate the purine
and glutamate release in a steady-state condition, and then this
medium was renewed for a further 60 min. Then, some cultures were
gently washed with glucose-free bicarbonate-buffered Krebs, placed
in the apparatus described above, and incubated for 1 h under
hypoxic-hypoglycemic conditions. The extracellular adenosine and
glutamate levels in medium samples from cells under basal condi-
tions and 1 h after OGD exposure were measured by high-perfor-
mance liquid chromatography as described previously (Di Iorio et al.,
1996). Adenine purine separation was carried out with a reverse-
phase analytical column (LiChrospher 100 RP-18 5
m; Merck,
Darmstadt, Germany). Elution was performed by applying a linear
gradient from 100% solvent A (60 mM KH
2
PO
4
and 5 mM tetrabu
-
tylammonium phosphate, pH 6.0) to 100% solvent B (30% methanol
plus 70% solvent A) for 15 min at a flow rate of 1.5 ml/min. Adenosine
Fig. 2. Molecular pathways activated
by OGD in cultured astrocytes. Cul-
tures were grown in serum-free me-
dium for 24 h and then exposed to
OGD for different times. Western blot
analyses of phosphorylated (p-)
ERK1/2 (A), p-Akt (B), p-ASK1-ser83
(B), p-JNK (C), p-p38 (D), p-Bad-
ser112 and -ser136 (F), total Bad, and
Bcl-X
L
(E) are shown. Densitometric
values are means ⫾ S.E.M. from three
independent experiments. ⴱ, p ⬍ 0.05;
ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001 (one-way
ANOVA plus Dunnett’s test) versus
control astrocytes.
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was revealed by absorbance at 254 nm. To evaluate extracellular
glutamate levels, samples were subjected to precolumn derivatiza-
tion with o-phthaldialdehyde/2-mercaptoethanol reagent. Glutamate
was separated on a Waters Pico/TAG column with a linear gradient
from 100% 100 mM potassium acetate, pH 7.1/methanol (80:20)
(solvent A) to 100% methanol/100 mM potassium acetate, pH 7.1
(80:20) (solvent B) and detected fluorometrically.
Statistical Analysis. All data are presented as the means ⫾
S.E.M. for a series of n experiments. Statistical analyses were per-
formed by Prism software version 3 (GraphPad Software Inc., San
Diego, CA), using unpaired unpaired Student’s t test or one-way
analysis of variance (ANOVA) followed by Dunnett’s post hoc com-
parison test. Group differences with P ⬍ 0.05 were considered sta-
tistically significant. Dose- or time-response curves were calculated
by using nonlinear regression (Prism software).
Results
Apoptosis Induced by OGD in Cultured Astrocytes
Cultured astrocytes that had been maintained in serum-free
medium for 24 h were exposed to OGD for 3 h. This treatment
caused apoptotic cell death, which was detectable after 3 to 6 h
by combining microscopic analysis after DAPI nuclear staining
(Fig. 1A), the MTS assay (Fig. 1B), and measurements of
caspase 3 activity (Fig. 1C) and procaspase 3 levels by immu-
noblotting (Fig. 1D). The extent of apoptotic death reached a
plateau between 12 and 24 h after OGD exposure, when ⬎50%
of cell nuclei bore chromatin condensation or pyknosis, MTS
activity decreased by ⬎50%, caspase 3 activity increased by
approximately 3-fold, and procaspase 3 levels decreased by
⬎80% (Fig. 1). OGD induced a sustained activation of the JNK
and p38/MAPK pathways, which peaked between 30 min and
2 h and could still be detected after 3 h (Fig. 2, A and B). In
contrast, OGD had no effect on the ERK1/2 MAPK pathway and
caused only a small and delayed stimulation of the PI3K path-
way, as assessed by immunoblot analysis of phosphorylated-
ERK1/2 and phosphorylated-Akt, respectively (Fig. 2, C and D).
Stimulation of PI3K was associated to a transient increase in
the levels of phosphorylated-(Ser83)/inactivated ASK1, which is
a MAP kinase kinase kinase upstream to JNK and p38 MAPK
in the death cascade (Sumbayev and Yasinska, 2005) and is
under the inhibitory control of the PI3K pathway (Kim et al.,
2001a). Finally, OGD altered the balance between pro- and
antiapoptotic members of the Bcl-2 family, causing a remark-
able increase in the Bcl-2-associated death protein, BAD, with-
out affecting the levels of the antiapoptotic factor Bcl-X
L
(Fig.
2F).
Extracellular adenosine levels were 23 ⫾ 6 nM under basal
conditions and 62 ⫾ 5 nM after 1-h exposure to OGD. Glu-
tamate levels were 0.35 ⫾ 0.07
M under basal conditions
and 1.5 ⫾ 0.22
M after 1-h exposure to OGD (means ⫾
S.E.M., n ⫽ 4).
Pharmacological Activation of A
1
or mGlu3 Receptors
Protects Astrocytes against Apoptosis by
Oxygen/Glucose Deprivation
The A
1
adenosine receptor agonist CCPA (2.5–75 nM)
and/or the mGlu2/3 receptor agonist LY379268 (0.25–7.5
M) was added to the cultures 1 h before OGD exposure and
was maintained in the medium throughout OGD exposure
(i.e., for the following 3 h). Both drugs reduced the percentage
of apoptotic cells in a concentration-dependent manner (Fig.
3A), with maximal antiapoptotic effect at 30 nM CCPA and 5
M LY379268. The calculated EC
50
values were 15.4 ⫾ 2.3
nM for CCPA and 2.23 ⫾ 0.7
M for LY379268. Similar
results were obtained using the MTS assay (data not shown).
Maximal concentrations of CCPA or LY379268 reduced the
increase in caspase 3 activity by approximately 50%, as as-
Fig. 3. Protective effect of CCPA and LY379268 against astrocyte apo-
ptosis induced by OGD. In A, different concentrations of CCPA (2.5–75
nM) or LY379268 (0.25–7.5
M) were applied to the cultures 1 h before
OGD exposure. In B and C, cells were pretreated with DPCPX or
LY341495 30 min before the addition of CCPA or LY379268, respectively.
Pretreatment with PTX was carried out for 16 h. In D, concentrations of
CCPA or LY379268 corresponding to their IC
25
and IC
50
values were
applied alone or in combination 1 h before OGD. All drugs were main-
tained during the3hofOGDexposure. Apoptosis was assessed 24 h after
the beginning of the experiment by DAPI staining. Results are expressed
as the percentage of apoptotic cells and are the means ⫾ S.E.M. of eight
values from four independent experiments. ⴱⴱⴱ, p ⬍ 0.001 (one-way
ANOVA plus Dunnett’s test) versus control astrocytes.
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1
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sessed 12 h after OGD exposure (caspase 3 activity expressed
as RFLU ⫻ 10
3
⫽ 70 ⫾ 8.1 in untreated cultures, 35 ⫾ 4.7 in
cultures treated with 30 nM CCPA, and 33 ⫾ 5.1 in cultures
treated with 5
M LY379268, respectively). The antiapo-
ptotic effect of CCPA was largely attenuated by the A
1
recep
-
tor antagonist DPCPX (100 nM) and by a pretreatment with
Fig. 4. Pharmacological activation of A
1
or mGlu3 receptors stimulates the PI3K
pathway and induces ASK1 phosphory-
lation in cultured astrocytes. Astrocytes
were starved for 24 h and then exposed
to increasing concentrations of CCPA or
LY379268 for 30 min (A) or to 30 nM
CCPA or 5
M LY379268 for different
times (B). In C, astrocytes were treated
with either the PI3K inhibitor
LY294002 or the receptor antagonists
DPCPX or LY341495 30 min before the
addition of CCPA or LY379268, whereas
pretreatment with PTX was carried out
for 16 h. Levels of phosphorylated Akt/
PKB (pAkt) and ASK1 at serine 83
(pASK1-Ser83) were determined by
Western blot analysis (60
g of proteins
was loaded per lane). Densitometric val-
ues are means ⫾ S.E.M., from three in-
dependent experiments. ⴱ, p ⬍ 0.05; ⴱⴱ,
p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001 (one-way
ANOVA plus Dunnett’s test) versus con-
trol astrocytes.
1374 Ciccarelli et al.
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PTX (200 ng/ml). The antiapoptotic effect of LY379268 was
instead sensitive to the preferential mGlu2/3 receptor antag-
onist LY341495 (1
M) and to PTX (Fig. 3, B and C). We also
combined CCPA and LY379268 at concentrations close to the
IC
25
or the IC
50
values in cultures exposed to OGD. Protec
-
tion was lower than that expected if the antiapoptotic effects
of the two drugs were additive (Fig. 3D). This suggests that
the activation of A
1
and mGlu2/3 receptors converges into a
common, G
i
-mediated intracellular pathway, which is ulti
-
mately responsible for the antiapoptotic effect in cultured
astrocytes challenged with OGD.
Signaling Pathways and Downstream Effector Molecules
Mediating the Antiapoptotic Effect of A
1
and mGlu3
Receptors in Cultured Astrocytes
Effect of A
1
and mGlu3 Receptor Activation in Con
-
trol Cultures. In control cultures, addition of CCPA (5–60
nM) induced a concentration-dependent activation of the
PI3K pathway, which showed a rapid and long-lasting kinet-
ics. The increase in Akt phosphorylation was detectable at 5
min and reached a long plateau between 30 min and 4 h (Fig.
4, A and B). Stimulation was abrogated by DPCPX, PTX, and
the PI3K inhibitor LY294002 (Fig. 4C). Stimulation of the
PI3K pathway was associated with a parallel increase in
ASK1 phosphorylation, which also peaked after 30 min (Fig.
4D). CCPA also stimulated ERK1/2 phosphorylation with a
slightly different kinetics. Stimulation peaked after 5 min,
remained stable up to 4 h (Fig. 5, A and B), and was sensitive
to DPCPX, PTX, and the MEK inhibitor U0126 (Fig. 5C). It is
interesting that stimulation of the ERK1/2 pathway by CCPA
was also reduced by LY294002 (Fig. 5C), suggesting that the
PI3K pathway precedes the MAPK pathway in the cascade of
reactions triggered by A
1
receptors in astrocytes. Similar
results were obtained with the mGlu2/3 receptor agonist
Fig. 5. Activation of A
1
or mGlu3 re
-
ceptors stimulates the ERK1/2 MAPK
pathway in cultured astrocytes. As-
trocytes were starved for 24 h and
then exposed to increasing concentra-
tions of CCPA or LY379268 for 30 min
(A) or to 30 nM CCPA or 5
M
LY379268 for different times (B). In
C, astrocytes were treated with either
the MEK inhibitor U0126 or the re-
ceptor antagonists DPCPX or
LY341495 30 min before the addition
of CCPA or LY379268, whereas pre-
treatment with PTX was carried out
for 16 h. Levels of phosphorylated
ERK1/2 (pERK1/2) and total ERK1/2
were determined by Western blot
analysis (10
g of proteins was loaded
per lane). Densitometric values are
means ⫾ S.E.M. from three indepen-
dent experiments. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍
0.01; ⴱⴱⴱ, p ⬍ 0.001 (one-way ANOVA
plus Dunnett’s test) versus control as-
trocytes.
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1
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LY379268, which, however, stimulated both pathways with a
different kinetics compared with CCPA. Phosphorylation of
Akt/PI3K and ERK1/2 MAPK peaked after 60 min of expo-
sure to LY379268, whereas phosphorylation of ASK1 in-
creased linearly from 30 min to 3 h. The action of LY379268
was sensitive to the mGlu2/3 receptor antagonist LY341495
(Figs. 4 and 5). Neither CCPA nor LY379268 had any effect
on the JNK and the p38 MAPK pathways in control cultures
(data not shown). However, both drugs increased the levels of
the phosphorylated/inactivated forms of the proapoptotic pro-
tein Bad (Fig. 6, A and B) at serine residues 136 and 112,
which are phosphorylated by PI3K and ERK1/2, respectively
(Datta et al., 1997; Scheid et al., 1999). As expected, phos-
phorylation at serine 136 was prevented by the PI3K inhib-
itor LY294002, whereas phosphorylation at serine 122 was
prevented by the MEK inhibitor U0126 (Fig. 6, A and B).
CCPA and LY379268 also increased the levels of the anti-
apoptotic protein Bcl-X
L
, as assessed by Western blotting.
This effect was abolished by LY294002 and attenuated by
U0126 (Fig. 6C).
Effect of A
1
or mGlu3 Receptor Activation in Cul
-
tures Exposed to OGD. In cultures challenged with OGD,
the two receptor agonists CCPA and LY379268 retained the
ability to stimulate the MAPK and PI3K pathways and at-
tenuated the pathological activation of the JNK and p38
MAPK pathways (Fig. 7, A–D). The two drugs could still
activate Bad phosphorylation (Fig. 8, A and B), thus reducing
the increase in Bad levels caused by OGD (Fig. 8C). CCPA
and LY379268 could still enhance Bcl-X
L
levels in cultures
exposed to OGD (Fig. 8D). Both effects were largely attenu-
ated by the PI3K inhibitor LY294002 and the MEK inhibitor
U0126 (Fig. 8, C and D). Finally, U0126 and LY294002
Fig. 6. Pharmacological activation of A
1
or
mGlu3 receptors promotes Bad phosphorylation
and increases the cytosolic content of the anti-
apoptotic protein Bcl-X
L
. Astrocytes were
starved for 24 h and then treated with 30 nM
CCPA or 5
M LY379268 for 1 (A and B) or 4 h
(C). At the end of the treatment, cells were
switched into fresh serum-free medium for addi-
tional 2 h. In some experiments, astrocytes were
treated with either LY294002 or U0126 30 min
before the addition of CCPA or LY379268. Levels
of phosphorylated Bad and levels of Bcl-X
L
were
examined by Western blotting (50
g of proteins
was loaded per lane). Values are means ⫾ S.E.M.
from three independent experiments. ⴱⴱ, p ⬍
0.01; ⴱⴱⴱ, p ⬍ 0.001(one-way ANOVA plus Dun-
nett’s test) versus control astrocytes.
1376 Ciccarelli et al.
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prevented the antiapoptotic effect of CCPA and LY379268 in
cultures exposed to OGD, as assessed by DAPI staining and
caspase 3 activity (Fig. 9, A and B).
Discussion
Apoptosis is a phenotype of death common to virtually all
cell types during development, senescence, and a variety of
pathological conditions. In the central nervous system, most
studies have focused on mechanisms regulating apoptotic
death in neurons as potential targets for protective agents in
neurodegenerative disorders. We switched the attention to
astrocytes because astrocyte death also occurs in neurode-
generative disorders and may have a profound impact on
synaptic transmission and neuronal viability (Takuma et al.,
2004). We challenged cultured astrocytes by OGD in an at-
tempt to mimic cell damage occurring under hypoxic/isch-
emic conditions (Giffard and Swanson, 2005). A 3-h exposure
to OGD induced approximately 50% of cell death under our
conditions. This extent of death allowed a reliable assess-
ment of the underlying mechanisms and, at the same time,
was considered optimal for the identification of protective
strategies, which could have been obscured by a more severe
insult.
OGD induced a number of processes that are causally
related to apoptotic death, such as the activation of the p38
MAPK and JNK pathways and an increase in the levels of
Bad (Chen et al., 2005; Sumbayev and Yasinska, 2005). The
increase in Bad levels was not apparently related to a reduc-
tion of protein phosphorylation. We rather observed a tran-
sient increase in Bad phosphorylation in cultures exposed to
OGD, which may represent a compensatory mechanism
aimed at avoiding excessive increases in Bad levels.
Our major finding was that pharmacological activation of
A
1
or group II mGlu receptors was highly protective against
astrocyte death. Both types of receptors have an established
protective function against neuronal damage in a variety of
in vitro and in vivo models of neurodegenerative disorders
(Bruno et al., 2001; Ribeiro et al., 2002; Chong et al., 2003).
At least a component of neuroprotection is mediated by A
1
and mGlu3 receptors present in astrocytes, which control the
production of neurotrophic factors such as transforming
growth factor-

and nerve growth factor (Bruno et al., 1998;
Ciccarelli et al., 1999; D’Onofrio et al., 2001). Thus, pharma-
cological activation of A
1
and mGlu3 receptors may provide a
dual strategy of protection limiting the death of neurons and
astrocytes at the same time.
CCPA and LY379268 have nanomolar affinity for A
1
and
mGlu3 receptors (Schoepp et al., 1999; Jacobson and Gao,
2006), respectively, but their intrinsic efficacy is not greater
than that of adenosine and glutamate. This suggests that A
1
and mGlu3 receptors present in astrocytes were not satu-
rated by the relatively high amounts of extracellular adeno-
sine and glutamate found during OGD. Whether glial recep-
tors are also responsive to pharmacological agonists in vivo is
Fig. 7. Pharmacological activation of A
1
or mGlu3 receptors stimulates the PI3K
and ERK1/2 MAPK pathways and re-
duces the stimulation of the p38MAPK
and the JNK pathways in cultured astro-
cytes exposed to OGD. Astrocytes were
starved for 24 h and then pretreated
with 30 nM CCPA or 5
M LY379268 1 h
before exposure to OGD in the absence or
presence of LY294002 (30
M) or U0126
(10
M) (both added 30 min before recep-
tor agonists). Levels of p-ERK1/2 (A), p-
Akt (B), p-JNK (C), and p-p38 (D) were
evaluated by Western blot analysis (20 or
50
g of proteins was loaded per lane in
A or in B–D, respectively). Values are
means ⫾ S.E.M. from three independent
experiments. ⴱⴱⴱ, p ⬍ 0.01 versus basal
values (unpaired Student’s t test); E, p ⬍
0.05; EE, p ⬍ 0.01; EEE, p ⬍ 0.001 (one-
way ANOVA plus Dunnett’s test) versus
OGD (control) values.
A
1
and mGlu3 Receptor Antiapoptotic Effect in Astrocytes 1377
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unknown, and one should take into account that neurons
release high amounts of purines and glutamate under hy-
poxic/ischemic conditions (Franceschini et al., 2006; Franke
et al., 2006). However, it is noteworthy that at least glial
mGlu3 receptors are not present on the surface of astrocytes
facing the synaptic cleft (Shigemoto et al., 1999) and might
not be accessible to synaptic glutamate.
A combined treatment with CCPA and LY379268 at the
respective EC
25
or EC
50
values was protective to a lower
extent than that predicted if the two drugs were additive.
Colocalization and interactions between A
1
receptors and
mGlu or other neurotransmitter receptors have been de-
scribed previously (Ciruela et al., 2001; Torvinen et al.,
2002). Because group II mGlu receptor agonists to do not
influence [
3
H]CCPA binding to A
1
receptors on astrocyte
membranes (R. Ciccarelli, F. Nicoletti, and F. Caciagli, un-
published observations), it is possible that group-II mGlu and
A
1
receptors converge in activating mechanisms that are
relevant to cytoprotection. They might recruit the same pool
of G
i
proteins or converge in the activation of the MAPK or
the PI3K pathways (see below). Thus, the combination be-
tween A
1
and mGlu2/3 receptor agonists may not be partic
-
ularly helpful in in vivo models.
Searching for intracellular mechanisms that mediate the
antiapoptotic effect of A
1
and mGlu3 receptors in cultured
astrocytes, we focused on the PI3K/Akt and the ERK/MAPK
pathways. Akt/PKB is a multifunctional mediator of PI3K-
dependent signaling that promotes cell survival and exerts in
models of neuronal or astrocyte death (Dudek et al., 1997; Di
Iorio et al., 2004). ERK1/2 activation can also produce neu-
roprotection (Xia et al., 1995). Both intracellular pathways
are activated by A
1
receptor stimulation in non-neuronal
(Germack and Dickenson, 2000) and neuronal cells (Angulo
et al., 2003) and in brain tissue (Gervitz et al., 2002). Acti-
vation of mGlu2/3 receptors can also stimulate Akt/PKB and
ERK1/2 in astrocytes and in brain tissue (D’Onofrio et al.,
2001). The following observations demonstrate that A
1
and
mGlu3 receptors protect cultured astrocytes through the ac-
tivation of PI3K/Akt and ERK/MAPK pathways: 1) CPPA
and LY379268 activated both pathways in a dose- and time-
dependent manner; 2) activation persisted in cultures ex-
posed to OGD; and 3) selective inhibitors of the two pathways
largely reduced the protective activity of A
1
or mGlu3 recep
-
tor agonists in astrocytes challenged with OGD.
Akt or ERK1/2 can phosphorylate several proapoptotic pro-
teins, leading to suppression of death signals. One of these is
ASK1, which triggers an apoptogenic kinase cascade causing
the phosphorylation/activation of JNK and p38 MAPK (Sum-
bayev and Yasinska, 2005). CCPA or LY379268 induced an
early and sustained phosphorylation/inhibition of ASK1 at
serine 83, a site that is phosphorylated by PI3K/Akt (Kim et
al., 2001a). As expected, both drugs partially reduced the
activation of the stress-related JNK and p38 MAPK path-
ways in cultures challenged with OGD. This might be one of
the PI3K/Akt-dependent mechanisms whereby activation of
A
1
and mGlu3 receptors protect astrocytes against apoptosis.
In addition, CCPA and LY379268 changed the balance be-
tween the proapoptotic protein Bad and the antiapoptotic
protein Bcl-X
L
. Bad is the only member of the Bcl-2 family
whose expression is up-regulated significantly during the
Fig. 8. Pharmacological activation of
A
1
or mGlu3 receptors reduces Bad
and increases Bcl-X
L
levels in cultured
astrocytes exposed to OGD. Astrocytes
were starved for 24 h and then treated
with 30 nM CCPA or 5
M LY379268
1 h before OGD exposure. When
present, LY294002 (30
M) or U0126
(10
M) was added 30 min before re-
ceptor agonists. Levels of Bad (A),
Bcl-X
L
(B), and phosphorylated Bad at
serine 112 or 136 (C and D) were eval-
uated by Western blotting. Values are
means ⫾ S.E.M. from three indepen-
dent experiments. ⴱⴱⴱ, p ⬍ 0.01 versus
basal values (unpaired Student’s t
test); E, p ⬍ 0.05; EE, p ⬍ 0.01; EEE,
p ⬍ 0.001 (one-way ANOVA plus Dun-
nett’s test) versus OGD (control) val-
ues.
1378 Ciccarelli et al.
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early stages of an ischemic insult in astrocytes (Chen et al.,
2005). Bad associates with Bcl-X
L
preventing Bcl-X
L
from
exerting antiapoptotic effects in ischemic astrocytes (Chen et
al., 2005). CCPA or LY379268 increased the expression of
Bcl-X
L
and reduced the OGD-induced increase in Bad levels
by phosphorylating Bad at sites targeted by PI3K/Akt and
ERK1/2. Phosphorylated Bad is inactivated and is no longer
able to counteract Bcl-X
L
antiapoptotic activity.
It is interesting that activation of the ERK1/2 MAPK path-
way by CCPA or LY379268 was greater in cultures exposed
to OGD than in control cultures, and this is noteworthy
because the ERK1/2 MAPK is considered one of the major
protective pathways in ischemic astrocytes (Chen et al.,
2005). Why OGD amplifies the activation of the ERK1/2
MAPK pathway mediated by A
1
or mGlu3 receptors is un
-
known. Part of this activation was sensitive to LY294002,
suggesting that activation of PI3K is upstream to ERK1/2
MAPK. Other mechanisms are likely to be involved. Activa-
tion of the PI3K and ERK1/2 pathways may be triggered by
the
␥
subunits of the G
i
protein, as reported for other G
protein-coupled receptors (Marinissen and Gutkind, 2001).
Inhibition of cAMP formation (i.e., the canonical transduc-
tion pathway coupled to A
1
and mGlu3 receptors) may also be
involved because cAMP limits membrane localization of
phosphoinositide-dependent kinase-1 (Kim et al., 2001b),
which is a direct effector kinase of Akt.
In conclusion, our data suggest that activation of A
1
and
mGlu3 receptors produces a strong prosurvival effect in as-
trocytes degenerating in response to “ischemic-like” condi-
tions. At least in culture, these receptors effectively respond
to pharmacological activation despite the large amounts of
adenosine and glutamate released during OGD. A
1
or mGlu3
receptor agonists cater the potential to exert a variety of
beneficial effects during ischemia. These drugs can protect
astrocytes and neurons at the same time and can also stim-
ulate astrocytes to produce neurotrophic/neuroprotective fac-
tors (Bruno et al., 1998; Ciccarelli et al., 1999; D’Onofrio et
al., 2001). LY379268 is a member of a growing list of brain-
permeant and highly potent mGlu2/3 receptor agonists that
are under preclinical development for the treatment of anx-
iety, drugs addiction, and other central nervous system dis-
orders (Schiefer et al., 2004; Kim et al., 2005). These drugs
might be highly effective in limiting astrocyte death in neu-
rodegenerative disorders. A systemic use of A
1
receptor ago
-
nists has long been precluded because of the occurrence of
peripheral side effects. However, innovative delivery systems
are now available that may allow a systemic use of A
1
recep
-
tor agonists in neurodegenerative disorders (Dalpiaz et al.,
2005).
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Address correspondence to: Dr. Renata Ciccarelli, Department of Biomed-
ical Sciences, Section of Pharmacology, University of Chieti, Medical School,
Via dei Vestini 29, pal. B, 66013 Chieti, Italy. E-mail: r.ciccarelli@dsb.unich.it
1380 Ciccarelli et al.
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