Adenosine A1 receptors and microglial cells mediate CX3CL1-induced
protection of hippocampal neurons against Glu-induced death.
Running title: CX3CL1 is not neuroprotective on A1R-/- hippocampal neurons.
Clotilde Lauro, PhD1,2, Raffaela Cipriani, PhD student1,2, Myriam Catalano, PhD1,2, Flavia
Trettel, PhD1,2, Giuseppina Chece1,2, Valentina Brusadin, PhD2, Letizia Antonilli, PhD2, Nico
van Rooijen, PhD3, Fabrizio Eusebi, PhD1,2,5†, Bertil B. Fredholm, PhD4, Cristina Limatola,
1Istituto Pasteur, Fondazione Cenci Bolognetti & Centro di Eccellenza BEMM, 2Dipartimento di
Fisiologia e Farmacologia Università Sapienza, Piazzale Aldo Moro, 5 00185 Rome, Italy;
3Department of Molecular Cell Biology, Free University Medical Center, Amsterdam, The
Netherlands; 4Karolinska Institutet, Nanna Svartz väg 2, Stockholm S-171 77 Sweden; 5IRCSS
NeuroMed, Via Atinese, Pozzilli Italy
† deceased 26th October 2009
CORRESPONDING AUTHOR: CRISTINA.LIMATOLA@UNIROMA1.IT
Dipartimento di Fisiologia e Farmacologia, Università di Roma Sapienza, Piazzale Aldo Moro, 5
00185 Roma, Italia; phone: +39 06 49690243; fax: +39 06 49910851.
Fractalkine/CX3CL1 is a neuron-associated chemokine, which modulates microglia-induced
neurotoxicity activating the specific and unique receptor CX3CR1. CX3CL1/CX3CR1 interaction
modulates the release of cytokines from microglia, reducing the level of tumor necrosis factor-α
(TNF-α), interleukin-1β (IL1-β) and nitric oxide and induces the production of neurotrophic
substances, both in vivo and in vitro. We have recently shown that blocking adenosine A1 receptors
(A1R) with the specific antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) abolishes
CX3CL1-mediated rescue of neuronal excitotoxic death and that CX3CL1 induces the release of
adenosine from microglia. In this paper we demonstrate that the presence of extracellular adenosine
is mandatory for the neurotrophic effect of CX3CL1 since reducing adenosine levels in
hippocampal cultures, by adenosine deaminase (ADA) treatment, strongly impairs CX3CL1-
mediated neuroprotection. Furthermore, we confirm the predominant role of microglia in mediating
the neuronal effects of CX3CL1, since the selective depletion of microglia from hippocampal
cultures treated with clodronate-filled liposomes causes the complete loss of effect of CX3CL1. We
also demonstrate that hippocampal neurons obtained from A1R-/- mice are not protected by CX3CL1
whereas A2AR-/- neurons are. The requirement of functional A1R for neuroprotection is not unique
for CX3CL1 since A1R-/- hippocampal neurons are not rescued from Glu-induced cell death by
other neurotrophins like brain-derived neurotrophic factor (BDNF) and erythropoietin (EPO), which
are fully active on wt neurons.
Keywords: fractalkine, excitotoxicity, neuroprotection, A1R, microglia, clodronate
Abbreviations: A1R, adenosine receptor type 1; A2AR, adenosine receptor type 2; A3R,
adenosine receptor type 3; AOPCP, α α-β β-Methyleneadenosine 5’-diphosphate; ADA,
adenosine deaminase; BDNF, brain-derived neurotrophic factor; DPCPX, 1,3-dipropyl-8-
cyclopentylxanthine; MEM, minimal essential medium; DMEM, Dulbecco’s modified Eagle
medium; Glu, glutamate; EPO, erythropoietin; FBS, foetal bovine serum; GPCR, G protein
coupled receptor; HBSS, Hank’s balanced salt solution; MTT, 3-(4,5-Dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide; NBTI, S-(4-Nitrobenzyl)-6-thioinosine.
The chemokine family comprises more than 40 members in 4 different subfamilies, whose
expression in the nervous system has been correlated with different pathological conditions (Tran
and Miller, 2003). CX3CL1, also called fractalkine, is the unique member of the CX3C (or δ)
family, and, together with CXCL16, are the only two transmembrane chemokines described until
now. It is converted to a soluble form upon cleavage from the plasma membrane through the action
of metalloproteinases, like a disintegrin and metalloproteinase domains (ADAM) 10 and ADAM17
on leukocytes (Hundhausen et al., 2003) or cathepsin S in the spinal cord (Clark et al., 2007).
CX3CL1 is constitutively expressed in the nervous system, but levels in the brain can be modulated
under diverse pathological conditions (Pan et al., 1997; Hughes et al., 2002; Kastenbauer et al.,
2003; Sunnemark et al., 2005; Huang et al., 2006). The presence and the stimulation (Zujovic et al.,
2000, 2001; Mizuno et al., 2003; Cardona et al., 2006, Lyons et al., 2009) of the CX3CL1 receptor
CX3CR1 has been correlated with a reduced release of interleukin-1β (IL-1β) and tumor necrosis
factor α (TNFα) from microglial cells and a lower rate of neuronal degeneration in different
experimental models of neuropathologies like experimental autoimmune encephalomyelitis (EAE),
1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine hydrochloride (MPTP) striatal injection,
lipopolysaccharide (LPS) administration and superoxide dismutase (SOD1) mutation (Huang et al.,
2006; Cardona et al., 2006). These data attest to a role of the pair CX3CL1/CX3CR1 in reducing
neuronal degeneration upon several types of brain injury. Exceptions are however reported: in
experimental transient brain ischemia the absence of CX3CL1 or CX3CR1 is correlated with
reduced IL-1β and TNF-α production and a better outcome for neurons (Soriano et al., 2002; Dénes
et al., 2008).
The neurotrophic activity of exogenously administered CX3CL1 has been related to the
simultaneous production of protective factors from microglial cells and in particular with the
activation of adenosine receptors (ARs, Lauro et al., 2008). Adenosine is a cellular metabolite
whose intracellular and extracellular levels can be rapidly modulated by variation of cellular
metabolic state (see Fredholm 2007). Under physiological conditions, in the brain, ATP can be
released by neurons and glial cells (Pascual et al., 2005; Burnstock et al., 2007). The released ATP
is rapidly degraded to ADP, AMP and adenosine by the sequential activity of extracellular
nucleotidase (Zimmermann et al., 1996). Pathological stimuli, which led to the imbalance of
membrane potential, like energy failure due to a reduced tissue perfusion, prolonged activation of
glutamate (Glu) receptors (Manzoni et al., 1994), transient oxygen and glucose deprivation (Lloyd
et al., 1993), or prolonged electric activity like that observed during seizures (Cunha et al., 1996),
have all been associated with an increased release of adenosine in the extracellular space, in some
cases due to altered activity of adenosine kinase (Boison 2006).
Adenosine is a pleiotropic agent which, in the nervous system, exerts a wide range of effects (see
Fredholm et al., 2005): it has a general inhibitory pre-synaptic activity on glutamatergic
transmission (Dolphin and Archer, 1983), modulates the response to noxious stimuli (de Mendonça
et al., 2000), regulates pain sensation (Sawynok and Liu, 2003), and has been implicated in pre-
conditioning (reviewed in Fredholm, 2007). Adenosine effects are mediated through four G protein
coupled receptors (GPCRs). It has long been recognized that adenosine is a modulator and that
therefore signaling via its receptors occurs together with signaling via other GPCRs like
metabotropic Glu receptors (mGluRs), dopamine, purine and cannabinoid receptors, (Agnati et al.,
2003), as well as tyrosine kinase receptors like the fibroblast growth factor receptors (FGFRs,
Flajolet et al., 2008). For example, ARs enhance the effect of other substances, like FGF (Flajolet et
al., 2008) ATP (Gerwins et al., 1992; Färber et al., 2008), BDNF (Diógenes et al., 2007; Tebano et
al., 2008) and GDNF (Gomes et al., 2009).
In the present study we have further examined the role of adenosine in mediating the neurotrophic
activity of CX3CL1. We have explored the role of microglial cells, the release of adenosine, and the
receptors responsible for the effect.
Materials and methods
Transwell inserts were from BD Labware (Franklin Lakes, NJ); recombinant rat CX3CL1 and
recombinant human BDNF were from Calbiochem/Merck (Nottingham, UK); adenosine deaminase
(ADA) from calf intestinal mucosa, recombinant human EPO, S-(4-Nitrobenzyl)-6-thioinosine
(NBTI), α−β-Methyleneadenosine 5’-diphosphate sodium salt (AOPCP) and poly-L-lysine were
from Sigma-Aldrich (Milan, Italy); all culture media were from Invitrogen Life Technologies (San
Giuliano Milanese, Italy); Cl2MDP (or clodronate) was a gift of Roche Diagnostics GmbH
Animals and cell lines
Procedures using laboratory animals were in accordance with the international guidelines on the
ethical use of animals from the European Communities Council Directive of 24 November 1986
(86/609/EEC). CX3CR1GFP/GFP mice (Jung et al., 2000) were obtained from Jackson Laboratory;
A1R-/- (Johansson et al., 2001) and A2AR-/- (Chen et al. 1999), were backcrossed at least 10 times on
a C57BL/6 background; CX3CL1-/- (Cook et al., 2001) were kindly provided by Dr. Richard M.
Ransohoff (Cleveland Clinic, OH).
Hippocampal neuronal cultures
Primary hippocampal neuronal cultures were prepared from 0-2 day-old (p0–p2) C57BL/6 (wt),
A1R-/-, A2AR-/-, CX3CL1-/- and CX3CR1GFP/GFP mice. Briefly, after careful dissection from
diencephalic structures, the meninges were removed and hippocampal tissues were chopped and
digested for 15 min at 37°C in 0.025% trypsin and Hank’s balanced salt solution (HBSS). Cells
were washed twice with HBSS to remove the excess of trypsin, mechanically dissociated in
minimal essential medium (MEM) with Earl’s Salts and GLUTAMAX supplemented with 10%
dialyzed and heat inactivated foetal bovine serum (FBS), 100 μg/ml gentamycin and 25mM KCl
Cells were plated at a density of 2.5 x 105 in the same medium on poly-L-lysine (100 µg/ml) coated
plastic 24-well dishes. After 1-2 h the medium was replaced with serum-free Neurobasal/B27
medium. Cells were kept at 37°C in 5% CO2 for 11 days with twice a week medium replacement
(1:1 ratio). At this time point we have 2.1 x 105 ± 0.05 x 105 alive cells (which corresponds to about
85 % of initially plated cells); no significant differences were obtained in the number of alive cells
in hippocampal preparations obtained from the brains of wt and genetically modified mice after 11
days in culture. With this method we obtained: 60-70% neurons, 30-35% astrocytes, 4-8%
microglia, as determined with β-tubulin III, GFAP and IBA-I staining. For details see
Supplementary Methods. Cells were used for experiments after 11 days.
Cortical mixed glia cultures were obtained from p0-p1 mice. Cerebral cortex were chopped and
digested in 20 U/ml papain for 40 min at 37°C. Cells (5.0 x105 cells/cm2) were plated on dishes
coated with poly-L-lysine (100 µg/ml) in DMEM supplemented with 10% FBS, 100 U/ml
penicillin, 0.1 mg/ml streptomycin . After 7-9 days cells were shaken for 2 h at 37°C to detach and
collect microglial cells. These procedures gave almost pure (no more than 2% contamination)
microglial cell populations, as verified by staining with GFAP and Iba I Abs.
To induce excitotoxicity, 11 day-old hippocampal cultures were washed and stimulated in modified
Locke’s buffer (CaCl2 2.3 mM, glucose 5.6 mM, glycine 10 mM, NaCl 154 mM, KCl 5.6 mM,
NaHCO3 3.6 mM, Hepes 5 mM pH 7.2) with 100 μm Glu alone or together with 100 nM CX3CL1
or vehicle, for 30 min. Following stimulation, cells were washed in Locke’s buffer and re-incubated
in the conditioned Neurobasal/B27 medium for additional 18 h. When BDNF (100 nM) and EPO
(40 U/ml, 10 nM) were used, hippocampal neurons were pre-treated for 7 h with drugs or vehicle;
medium was removed and cells were treated as described above with Glu (in the presence or in the
absence of BDNF and EPO), washed and further incubated with the original medium containing
BDNF or EPO for additional 18 h, till the end of the experiment. For experiments with conditioned
medium, microglial cells (obtained from wt mice) were treated with CX3CL1 for 30 min, washed
and re-incubated in growth medium. Eight h after CX3CL1 treatment, media were collected and
used to stimulate neuronal cultures (obtained from CX3CR1GFP/GFP mice) treated with Glu to induce
excitotoxicity. Conditioned media from glia cultures were always diluted 1:1 with the original
medium of neuronal cultures. For experiments with ADA, hippocampal cells were pre-incubated for
1 h with 1 U/ml ADA, treated with Glu or Glu/CX3CL1 in the presence of ADA, washed and
reincubated in the original conditioned medium for 18 h. For experiments with conditioned media,
medium obtained from CX3CL1-stimulated microglia was treated with or without ADA (1 U/ml)
for 1 h before administration to neuronal cells treated with Glu. In these protocols, ADA was
present till the end of the experiments. To evaluate neuron viability, cells were then treated with
detergent-containing buffer (0.05% ethyl hexadecyl dimethylammonium bromide, 0.028% acetic
acid, 0.05% Triton X-100, 0.3mM NaCl, 0.2 mM MgCl2, in PBS pH 7.4) and counted in a
hemacytometer as already described (Lauro et al., 2008). Alternatively, cell viability was analyzed
by the MTT assay: in detail, 5 mg/ml MTT was added 1:10 to the cell medium and incubated for 2
h; the medium was aspired, cells were treated with DMSO and incubated at 37°C for 10 min.
Samples were then analyzed with a microplate reader at 490 nm and 630 nm to subtract
background. In all excitotoxicity experiments, results are expressed as % of cell survival, taking as
100% untreated cells in control conditions. Exactly the same procedures (plated cell number,
volumes of reagents) were applied to experiments with cells obtained from mice of different
genotypes for comparison of cell viability also under basal conditions.
Transwell migration assays
Chemotaxis assay was performed on microglia obtained from mice cortex. Cells were re-suspended
in serum-free medium and plated on poly-L-lysine-treated 12-mm transwells (8-μm pore size
polycarbonate; 5 x 105 cells/well). The lower chambers contained CX3CL1 100 nM, prepared in the
same medium. The chambers were incubated for 2 h at 37°C in a moist 5% CO2 atmosphere. After
incubation, cells were treated with 10% trichloroacetic acid on ice for 10 min and the non migrating
cells, adhering to the upper face of the filters, were scraped off, while cells on the lower side were
stained with a solution containing 50% isopropanol, 1% formic acid, and 0.5% (w/v) brilliant blue
R250 and dried on a glass slide. The number of migrating cells was counted in 20 fields with a 63X
Depletion of microglia with clodronate liposomes
Mixed hippocampal cultures obtained from CX3CR1GFP/GFP mice were treated for different times
with liposomes encapsulating clodronate (Cl2MBP) or as control, with empty liposomes. Clodronate
liposomes as well as control liposomes without clodronate were prepared according to the standard
method (van Rooijen et al. 1994). The resulting standard suspension of clodronate liposomes is
containing 1.2 mg of Clodronate per 1 ml of the suspension. This liposome suspension was diluted
1:10 in the growth medium. At different time points, from 5 h to 72 h, cell cultures were analyzed
with a fluorescence microscope to recognize and count the number of EGFP-labeled microglial
cells. Cultures were stained with Hoechst to visualize total cell nuclei.
Eleven-day old rat hippocampal cultures were pre-treated in Locke’s buffer for 10 min with the
transporter inhibitor NBTI or with the ectonucleotidase inhibitor AOPCP, and then stimulated 30
min with CX3CL1 100 nM or vehicle, while primary microglial cell cultures were only treated in
Locke’s buffer for 30 min with CX3CL1 or vehicle. After this time, cells were washed and
reincubated in their original conditioned medium, in the presence or in the absence of the inhibitors
and, after additional 6 h (or 7.5 h for microglia), the media were collected, added with ice-cold
acetonitrile, centrifuged for 5 min at 1.440 x g and the resulting supernatants were analyzed by
HPLC. Cells remaining in the dish were analyzed for protein content with a BCA assay.
Chromatographic analyses were conducted using a Merck Hitachi HPLC system equipped with
programmable autosampler (model L-7250), pump (model L-7100), and diode array detector (model
L-7455). Data were stored and processed using appropriate software (D-7000 HPLC System
Manager Ver. 3.1; Hitachi). Separation was achieved by using a column Reprosil-Pur C18-AQ (5
µm, 250x4 mm) with precolumn Reprosil-Pur C18-AQ 5 µm, 5 x 4 mm (Dr. Maisch, Ammerbruch,
Germany). Elution was performed isocratically with a mobile phase consisting of 10 mM potassium
phosphate (pH 6) and acetonitrile (90:10). The pump flow rate was set at 1.0 ml/min, and the
injection volume was 40 µl. Adenosine was monitored by UV diode array detection at 260 nm, and
was identified on the basis of its retention time (3.90 min) and spectral data relative to reference
standards. All separations were conducted at room temperature. The limit of detection and
quantification for adenosine were found to be 18.7 nM and 187 nM, respectively.
Statistical data analysis
For all the experiments shown in the manuscript, significance was evaluated with t-test analysis and
differences between groups of data were considered highly significant with P≤0.01 (**) and
significant with P≤0.05 (*).
Microglia depletion with clodronate liposomes impairs the neuroprotective activity of CX3CL1.
To study the neuroprotective role of CX3CL1, we used an injury model involving glutamate (Glu)-
induced excitotoxicity in hippocampal cultures obtained from p0-p2 mice. Upon treatment with Glu
(100 μM, 30 min), we consistently obtained about 40-50 % of cell death in comparison with
untreated control cultures. This corresponded to about 70 % of total neuronal loss upon Glu
treatment, as assessed by immunofluorescence analysis with β-tubulin III staining (data not shown).
We confirm, in this manuscript, that CX3CL1 protects hippocampal neurons from Glu-induced
excitotoxicity (Fig. 1A) similarly to what already shown in neuronal preparations which contain
different ratios of neurons:astrocytes:microglia (Limatola et al., 2005; Lauro et al., 2008). Given
that CX3CR1 are predominantly expressed in microglial cells, it is likely that microglial cells
mediate the neurotrophic effect of CX3CL1. To substantiate this, hippocampal neuronal cultures
were treated with clodronate liposomes to specifically kill microglia (van Rooijen et al., 1996;
Marín -Teva et al., 2004). We first performed a kinetic analysis on hippocampal cultures obtained
from CX3CR1GFP/GFP mice, where microglial cells are labeled by EGFP (Jung et al., 2000). Data
reported in Fig. 1B indicate that the number of EGFP-labeled microglial cells selectively decreases
with time in hippocampal cultures treated with clodronate-filled liposomes, while it is not affected
in cultures treated with empty liposomes. At the same time points chosen for microglia cell
viability, liposome-treated hippocampal cultures were analyzed for CX3CL1 responsiveness in
terms of protection from Glu-induced toxicity. To this end, cells were treated with empty or
clodronate-filled liposomes, washed, stimulated with Glu and analyzed for viability 18 h later.
Results in Fig. 1C demonstrate that, after 72 h of treatment with clodronate liposomes and
microglial cells in culture have almost completely disappeared (see Fig. 1B), CX3CL1 is not able to
reduce Glu-induced toxicity. Unexpectedly, the same effect is observed as early as 5 h of clodronate
liposome treatment (Fig 1C), when most of microglial cells are still present, suggesting that
microglia are strongly affected well before they are eliminated and in such a way that response to
CX3CL1 is strongly impaired at this time point. Note that, in basal conditions (C), clodronate-filled
liposomes did not significantly modify total cell survival in culture, suggesting that neither neurons
nor astrocytes, which together account for more than 95% of total cell population (see methods), are
significantly affected by these treatments. Neuronal cell treatment with empty liposomes did not
affect CX3CL1-induced neuroprotection (Fig. S1).
Hippocampal neurons from CX3CL1-/- mice are not more vulnerable to Glu injury in comparison
with wt neurons. To investigate the role of endogenous CX3CL1 as neuroprotective agent upon
Glu-induced excitotoxicity, hippocampal cultures were obtained from CX3CL1-/- mice, treated with
different Glu concentrations (from 1μM to 1 mM) and analyzed for cell viability. No significant
differences in neuron death were observed between wt and CX3CL1-/- mice at all tested Glu
concentrations (Fig. S2). This suggests that endogenous levels of CX3CL1, neither before nor after
Glu treatment (Chapman et al., 2000; Erichsen et al., 2003; Limatola et al., 2005), are sufficient to
protect neurons by excitotoxicity under our in vitro conditions. To analyze if the effect of the
administration of the soluble form of CX3CL1 could be different in wt vs CX3CL1-/- mice,
evidencing a possible cooperative role of the endogenous CX3CL1, excitotoxicity experiments were
performed as shown in Fig. 2. Data obtained indicate that in the absence of endogenous (membrane
bound and shed forms) CX3CL1, exogenous administration of soluble CX3CL1 is still able to
reduce Glu-induced cell death (CX3CL1-/- mice: Glu 48.6% ± 3.5% vs Glu/CX3CL1 73.5 % ± 3.0%
p≤0.001) albeit at lower levels. No difference in cell viability were observed, in the absence of Glu,
between wt and CX3CL1-/- cultures (data shown in the legend to Fig. 2).
Role and origin of extracellular adenosine. We have previously shown that CX3CL1 induces the
release of adenosine from the murine microglial cell line BV2 and from mixed hippocampal
cultures (Lauro et al., 2008). However, since immortalized cell lines may differ from primary cells,
we wanted to investigate: i) if primary microglia also release adenosine upon CX3CL1 treatment
and ii) if the reduction of extracellular adenosine levels, by treating cultured cells with adenosine
deaminase (ADA, the enzyme which degrades adenosine to inosine), is sufficient to prevent
CX3CL1 neuroprotection against Glu-induced excitotoxicity. Figure S3 shows that CX3CL1
treatment of primary cultures of murine microglia induces adenosine release, as previously shown
with BV2 cells (Lauro et al., 2008). Results in Fig. 3A demonstrate that ADA treatment (1 U/ml, 1
h, 37°C) of Glu-treated hippocampal cultures completely abolished CX3CL1-mediated
neuroprotection. Interestingly, ADA treatment per se already results in some cell toxicity (19.5 % ±
0.9 % reduction of cell viability), suggesting that basal adenosine levels contribute to keep cells
We next used the medium conditioned by CX3CL1-stimulated (st) primary wt microglia (at the
same time point shown in Fig. S3), to reduce Glu-induced cell death of CX3CR1GFP/GFP neurons
(confirming previous data with the microglia cell line BV2, Lauro et al., 2008); in the absence of
CX3CL1 (not-stimulated cells, ns), this medium is not able to prevent Glu-induced cell death (Fig.
3B). When st medium was pre-treated with ADA (1 U/ml, 1 h, 37°C) and then given to
CX3CR1GFP/GFP hippocampal neurons, the neuroprotective properties were completely lost (Fig.
3B). Extracellular adenosine, which accumulates upon CX3CL1 stimulation of hippocampal
cultures (Lauro et al., 2008 and Fig. 4), likely derived from released ATP since, in the presence of
the specific ectonucleotidase inhibitor, AOPCP (1 μM, 10 min pre-incubation), the level of
extracellular adenosine was not increased by CX3CL1 treatment (Fig. 4). When higher levels of
AOPCP were used (5μM), the same results were obtained upon CX3CL1 treatment (data not
shown). However, in those conditions, the basal extracellular levels of adenosine were reduced at
the limit of method detection. In contrast, the presence of the equilibrative transporter inhibitor
NBTI did not significantly alter extracellular adenosine accumulation upon CX3CL1 treatment (Fig.
Hippocampal neurons obtained from A1R-/- mice are not protected by CX3CL1 against Glu
excitotoxicity. We recently showed that the protective effect of CX3CL1 against Glu-induced
hippocampal neuron injury could be eliminated by the A1R antagonist DPCPX (Lauro et al., 2008).
Although selective, this antagonist is not absolutely selective and, to prove the involvement of A1R
in the neurotrophic activity of CX3CL1, hippocampal cultures obtained from A1R-/- mice were
treated with Glu (100 μM, 30 min) to induce excitotoxicity in the presence or in the absence of
CX3CL1. We demonstrated that, in contrast with data obtained in wt mice, A1R-/- cultures were not
protected from Glu-induced cell death by CX3CL1 treatment (100 nM, Fig. 5) thus providing
further evidence that A1Rs are required for the neuroprotective effect of the chemokine (Lauro et
al., 2008). To exclude that the lack of neuroprotective effects of CX3CL1 on A1R-/- neurons was
due to an impairment of CX3CR1 functional properties on these specific genetically modified mice,
experiments were addressed to investigate CX3CL1-induced chemotaxis on microglial cells
obtained from A1R-/- mice. Data reported in Fig. S4 demonstrate that A1R-/- microglia responds to
CX3CL1 similarly to wt microglia in terms of transwell migration. Similarly, A1R-/- and wt cultured
hippocampal neurons responded to CX3CL1 treatment with comparable levels of ERK
phosphorylation (data not shown). These data indicate that there is no gross functional impairment
of CX3CR1 pathway when A1R are absent. Considering the physical and functional interaction
described for A1R/A2AR pair (Ciruela et al., 2008), we wanted to analyze the possible involvement
of A2AR in the neurotrophic effect of CX3CL1 using A2AR-/- mice. Data, reported in Fig. 5,
demonstrate the selective involvement of A1R, with no participation of A2AR in CX3CL1-mediated
neuroprotection from Glu-excitotoxicity. Furthermore, the number of cells that survived in the
controls (C) was not significantly different between wt, A1R-/- and A2AR-/- mice (respectively 41 ±
4.3; 41 ± 2.9; 42 ± 1.1 cells per microscopic field (10 x)).
Glu-injured hippocampal A1R-/- cultures are not rescued by other neurotrophins. To investigate if
A1R presence was a specific requirement for the neurotrophic activity of CX3CL1 or if it was
shared by other neurotrophins, hippocampal A1R-/- cultures were treated with Glu to induce
excitotoxicity in the presence of BDNF (100 nM) or EPO (40 U/ml). Data shown in Fig. 6 indicate
that, under these conditions, both these substances protect wt neurons but fail to preserve A1R-/-
neurons, thus suggesting that the presence of functional A1R on neuronal cells is permissive for the
activity of different neurotrophic factors. These results further underline that the elimination of the
neuroprotective effect of the chemokine cannot be simply explained by a specific loss of ARs and
indicate that the mediation of neuroprotective effect by adenosine acting at A1Rs is quite a general
In this paper we demonstrate that microglial cells are required for the neuroprotective activity of
CX3CL1, that they release adenosine which activates A1R and that A1R presence is necessary for
the neurotrophic activity of CX3CL1 on Glu-injured neurons. We also describe for the first time
that the expression of A1R is necessary for the neurotrophic activity against excitotoxicity of other
neurotrophins, like BDNF and EPO. These findings will be discussed in turn.
The role of adenosine in neuroprotection is very well established: experimental evidence indicates
that activation of A1R or inhibition of A2AR improves neuronal recovery upon brain injury (Cunha,
2005), while the role played by A3R and A2BR in neuroprotection is less clear-cut (Michel et al.,
1999; Fedorova et al., 2003; Chen et al., 2006; Pugliese et al., 2007). In the present experimental
conditions we did not observe any clear effect of eliminating A2A receptors. It has been reported
that, in the brain, the level of adenosine and A1R strongly increases upon trauma like brain ischemia
(Pearson et al., 2006), where adenosine is mainly released by astrocytes (Martín et al., 2007) and
participates in the protective effect of ischemic preconditioning (Heurteaux et al., 1995) or upon
repetitive seizures, where the rapid modulation of adenosine kinase has been reported (reviewed by
Boison, 2006). Even the basal level of extracellular adenosine, and the corresponding tonic
activation of ARs, can be responsible of the modulatory activity on synaptic transmission (see
Fredholm et al., 2005). Furthermore, AR activation is necessary, as co-receptor requirement, either
to permit or to enhance neuronal and glial response to purines (ATP, Gerwins and Fredholm, 1992;
Färber et al., 2008), neuropeptides (VIP, Cunha-Reis et al., 2007; CGRP, Sebastião et al., 2000;
GDNF, Gomes et al., 2009), cytokines (IL6, Biber et al., 2008), growth and trophic factors (FGF,
Flajolet et al., 2008; BDNF, Diógenes et al., 2004) and chemokines (CX3CL1, Lauro et al., 2008).
Data reported in this paper demonstrate that adenosine, in addition to its well known direct
neuroprotective effect on neurons (see above) and indirect protective effects via CCL2, IL-6 and S-
100b release by astrocytes (Schwaninger et al., 1997; Ciccarelli et al., 1999; Wittendorp et al.,
2004), appears to enable the neurotrophic activity of different neurotrophins to occur, thereby
extending the repertoire of actions for adenosine in brain homeostatic control.
We previously demonstrated that A1R are probably involved in the neurotrophic activity of
CX3CL1 as it was blocked by a relatively selective antagonist (Lauro et al. 2008). We now strongly
support this conclusion in experiments where the protective effect is absent in A1R-/- mice. It might
be argued that this is due to some functional impairment of CX3CR1 activation in A1R-/- mice.
However, another effect of CX3CL1, namely direct induced by receptor stimulation, like microglia
migration, is similarly activated by CX3CL1 both in wt and A1R-/- mice. Furthermore, the
neuroprotection induced by other agents was also reduced. Together these observations make it very
unlikely that the reason why mice that lack A1Rs are not protected by CX3CL1 is that they are
unable to respond to the chemokine.
One might argue that the present data are compatible with A1Rs modulating the neuroprotective
effect of endogenous CX3CL1. However, we report that lack of endogenous CX3CL1 (in CX3CL1-
/- mice) does not change hippocampal neuron response to Glu but reduces the protective effects
induced by exogenous CX3CL1, suggesting a protective effect of the endogenous protein. We also
show that this is not due to some major adaptive response to the targeted deletion of CX3CL1.
In a previous paper we demonstrated that CX3CL1 induces adenosine release from hippocampal
cultures and from a murine microglia cell line (Lauro et al., 2008). Since the mechanisms
underlying adenosine release might vary between different cell types of brain parenchyma, being
mostly due to equilibrative transporters in neurons and to extracellularly released ATP,
subsequently hydrolyzed by ectonucleotidases (Parkinson et al., 2005), in glia cells, we were
interested in defining the potential mechanisms implicated in adenosine release by CX3CL1 in
hippocampal mixed cultures. Our evidence that only the specific inhibitor of ectonucleotidases is
able to strongly reduce CX3CL1-mediated adenosine release, whereas the inhibitor of equilibrative
transport was not, could suggest a predominant involvement of glial cell dependent nucleotide
release in this process. The conclusion that glial cells are particularly important is also corroborated
by our observations that the simultaneous treatment of hippocampal cultures with ADA and
CX3CL1 completely abolished CX3CL1-mediated neurotrophic effect, and that the same result is
obtained when ADA treatment is performed on medium collected from CX3CL1-stimulated
primary microglia, 1 h before administration to Glu-treated hippocampal cultures.
It is proposed that microglia has a prominent role in mediating the neuroprotective effects of
CX3CL1 (Mizuno et al., 2003; Huang et al., 2006; Cardona et al., 2006) and we have recently
demonstrated that CX3CL1-stimulated microglia releases neuroprotective substances which reduce
Glu-induced cell death (Lauro et al., 2008). However CX3CL1 does not protect against all types of
neuronal damage because in transient brain ischemia (Soriano et al., 2002; Denés et al., 2008) and
in a rat model of Parkinson’s disease, intra-striatal CX3CL1 injection induced both microglia-
dependent depletion of dopaminergic cells and motor dysfunction (Shan et al., 2009). In this paper
we demonstrate that the selective ablation of microglia from hippocampal cultures, using
clodronate-encapsulating liposomes, has the effect to fully abolish the neuroprotective activity of
CX3CL1 toward excitotoxic death of hippocampal neurons, confirming that these cells represent
the first target which primes the functional effects of CX3CL1. It is interesting to note how a few
percentage of microglial cells (such as that present in our hippocampal neuronal preparation) can
massively influence neuronal response to CX3CL1. This could explain the reported neuroprotective
effect of CX3CL1 in almost pure neuronal cultures (Limatola et al., 2005).
Thus, we hypothesized that soluble factors released by microglia, like adenosine, could also
activate astrocytes to release neurotrophic substances (Schwaninger et al., 1997; Ciccarelli et al.,
1999; Wittendorp et al., 2004) which contribute to neuroprotection.
In conclusion, these data support the notion that CX3CL1 has neurotrophic activity on hippocampal
and cortical neurons through its activity on microglia, which release soluble factors, among which
adenosine, determining for neuron protection. CX3CL1-mediated neuroprotection is only possible
in the presence of functional A1R, whose activity is also required for the neuroprotective effect of
other neuroactive factors, like BDNF and EPO, thus demonstrating that A1R co-activation is
necessary as permissive signaling which might reinforces or consent to the accomplishment of
survival responses. The relevance of these conclusions remain to be confirmed in more
physiological systems, like neurons obtained from mature brains, or by in vivo studies.
Disclosure/Conflict of Interest
The authors do not have conflicts of interest.
This work was granted by Ministero Università & Ricerca scientifica (PRIN to C.L.), by
Fondazione Cenci Bolognetti (to C.L.) by Ministero Salute (Ricerca finalizzata to C.L. and F.E.)
and by Swedish Science Research Council (to B.B.F.). The authors thank Dr. Knut Biber for
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Titles and legends to figures
Figure 1 Effect of microglia depletion with clodronate liposomes on CX3CL1-mediated
neurotrophic activity. (A) Hippocampal cultures were treated with Glu (100 μM, 30 min) or vehicle
in the presence or in the absence of CX3CL1 (100 nM) and analyzed for viability 18 h later as
described in the text. Results are expressed as % of cell survival in treated (Glu and Glu/ CX3CL1)
vs untreated (C) cells (taken as 100%) and are the mean ± SE of twelve duplicate experiments. (B)
Hippocampal cultures from CX3CR1GFP/GFP mice were treated with empty or clodronate filled
liposomes for the indicated times and analyzed for the presence of EGFP-positive cells under
fluorescence microscopy. Results are expressed as percentage of EGFP-positive cells (microglia) in
the liposome-treated cultures vs control (C) untreated cells, and represent the mean of two duplicate
independent experiments. (C) Alternatively hippocampal cultures, treated with clodronate-filled
liposomes, were analyzed for Glu-induced excitotoxicity in the presence or in the absence of
CX3CL1. Results are expressed as percentage of cell survival, taking 100% as untreated cells at
time 0. Data represent the mean ± SE of four duplicate experiments. For each time point, statistical
significance was analyzed in treated (Glu and Glu/ CX3CL1) vs untreated (C) samples. *, p ≤ 0.05;
**, p ≤ 0.01.
Figure 2 Endogenous levels of CX3CL1 are not sufficient to protect neurons by excitotoxicity.
Eleven day-old hippocampal cultures obtained from wt or CX3CL1 -/- mice were treated with Glu
(100 μM, 30 min) or Glu/CX3CL1 and analyzed for viability 18 h later. Results are expressed as %
of cell survival taking as 100% the control (C), untreated cells, for each mouse strain and are the
mean ± SE of five duplicate experiments. Statistical significance is analyzed between treated and
untreated cells for each mouse strain, unless differently indicated. The number of cells in wt and
CX3CL1-/- mice was not significantly different in untreated samples (41 ± 4.3 and 45 ± 2.4,
respectively, per microscopic field, 10 x). *, p ≤ 0.05; **, p ≤ 0.01.
Figure 3 ADA treatment abolishes the neuroprotective effect of CX3CL1. (A) Eleven day-old
hippocampal cultures were pre-incubated or not with ADA (1U/ml) for 1 h and then co-stimulated
with Glu or Glu/CX3CL1. Results represent the mean ± SE of five independent duplicate
experiments and are expressed as percentage of cell survival taking as 100% untreated cells in the
absence of ADA. For each experimental condition, statistical significance was analyzed in treated
(Glu and Glu/ CX3CL1) vs untreated (C) samples. *, p ≤ 0.05; **, p ≤ 0.01. (B) Glu-injured
CX3CR1GFP/GFP hippocampal neurons were treated with the medium conditioned by primary
microglia not stimulated (ns) or stimulated with CX3CL1 (st). Alternatively, the conditioned (st)
medium was collected, incubated for 1 h with ADA (1 U/ml; st/ADA) and given to CX3CR1GFP/GFP
hippocampal neurons treated with Glu (100 μM) as described in the experimental section. Cell
survival was analyzed after 18 h. Results represent the mean ± SE of three independent duplicate
experiments, and are expressed as percentage of cell survival taking, as 100%, untreated cells in
control condition. For each experimental condition, statistical significance is analyzed between
treated (Glu) and untreated (C) cells. *, p ≤ 0.05; **, p ≤ 0.01
Figure 4 Adenosine produced by CX3CL1-stimulated hippocampal cultures is reduced by
ectonucleotidases inhibition with AOPCP. Hippocampal neurons were treated with 100 nM
CX3CL1 or vehicle for 30 min, in the presence or in the absence of NBTI (1 μM) and AOPCP (1
μM), washed and re-incubated in growth medium. After 6 h medium was analyzed by HPLC for
adenosine content. Results are expressed as nmoles of adenosine produced for mg of cellular
proteins and significance is analyzed, for each condition, between CX3CL1-treated vs
corresponding control samples. Data are the mean ± SE from 5 independent quadruplicate
Figure 5 Excitotoxic cell death of hippocampal neurons is not inhibited by CX3CL1 in A1R-/-
mice. Hippocampal neurons obtained from wt, A1R-/- or A2AR-/- mice were cultured for 11 days and
then stimulated with Glu (100 μM, 30 min) in the presence or in the absence of CX3CL1 (100 nM).
Cell death was analyzed after 18 h. Results represent the mean ± SE of four independent duplicate
experiments and are expressed as % of cell survival taking as 100% the control (C), untreated cells,
for each mouse strain. Statistical significance is analyzed between treated and untreated cells for
each mouse strain. *, p ≤ 0.05; **, p ≤ 0.01. The number of untreated cells in the control (C) are not
significantly different between wt, A1R-/- and A2AR-/- mice (see data in the text).
Figure 6 The neuroprotective effect of BDNF and EPO is abolished in A1R-/- mice. Hippocampal
neurons obtained from wt or A1R-/- mice were cultured for 11 days and then stimulated with Glu
(100 μM, 30 min) in the presence or in the absence of BDNF (100 nM) or EPO (10 nM). Results
represent the mean ± SE of four independent duplicate experiments and are expressed as in Fig. 5.
Supplementary Figure 1 Neuroprotective effect of CX3CL1 is unaffected by liposome treatment.
CX3CR1GFP/GFP hippocampal cultures, treated with empty liposomes for the indicated times, were
analyzed for Glu-induced excitotoxicity in the presence or in the absence of CX3CL1. Results are
expressed as percentage of cell survival, taking 100% as untreated cells at time 0. Data represent
the mean ± SE of four duplicate experiments. For each time point, statistical significance was
analyzed in treated (Glu and Glu/ CX3CL1) vs untreated (C) samples. *, p ≤ 0.05; **, p ≤ 0.01.
Supplementary Figure 2 Wt and CX3CL1 -/- hippocampal cultures are similarly vulnerable to
Glu-induced injury. Eleven day-old hippocampal cultures obtained from wt or CX3CL1-/- mice
were treated with different Glu concentrations (from 1μM to 1 mM) or vehicle and analyzed for
viability 18 h later. Results are the mean ± SE of four duplicate experiments and are expressed as %
of cell survival taking as 100% the untreated cells in the absence of Glu. Statistical analysis is
performed, for each Glu dose, between wt and CX3CL1-/- cultures.
Supplementay Figure 3 CX3CL1 induces extracellular adenosine accumulation in primary
microglia cultures. Media obtained from microglia treated with vehicle (C) or CX3CL1 (100 nM,
30 min), as described in the Method section, were analyzed by HPLC for adenosine content.
Results are expressed as nmoles of extracellular adenosine accumulated per mg of cellular proteins
and are the mean ± SE of four independent triplicate experiments.
Supplementary Figure 4 Wt and A1R-/- microglia respond similarly to CX3CL1-induced
chemotaxis. Transwell chemotaxis assay toward CX3CL1 (100 nM, 2 h) was performed on purified
microglia obtained from the cerebral cortex of wt and A1R-/- mice. For each mice strain, results are
expressed as percentage of cell migration taking as 100% control (C), untreated cells. Data
represent the mean ± SE of four duplicate experiments. Statistical significance is analyzed for each
mice strain between CX3CL1- and vehicle-treated samples. **, p ≤ 0.01.
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