RESEARCH ARTICLE | JUNE 01 2008
Activity of Adenosine Receptors Type 1 Is Required for CX CL1-Mediated
Neuroprotection and Neuromodulation in Hippocampal Neurons
3
1
J Immunol (2008) 180 (11): 7590–7596.
https://doi.org/10.4049/jimmunol.180.11.7590
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Clotilde Lauro; ... et. al
The Journal of Immunology
Activity of Adenosine Receptors Type 1 Is Required for
CX3CL1-Mediated Neuroprotection and Neuromodulation
in Hippocampal Neurons1
Clotilde Lauro,* Silvia Di Angelantonio,* Raffaela Cipriani,* Fabrizia Sobrero,*
Letizia Antonilli,* Valentina Brusadin,† Davide Ragozzino,*‡ and Cristina Limatola2*‡
T
he chemokine fractalkine (CX3CL1) and its specific receptor, CX3CR1, are constitutively expressed throughout the
CNS, (1) where in addition to modulating neurotransmission
(2), they play the pathophysiological role of regulating microglial neurotoxicity (3, 4), neuropathic nociception (5), NK cell recruitment (6),
and neuron survival (7, 8). CX3CR1 is expressed by microglia under
physiological conditions (1), and its expression can be induced or
up-regulated by cytokines both on astrocytes and microglia (9, 10). In
contrast, CX3CR1 expression on neurons is controversial, pointing to
microglial cells as privileged target implicated in CX3CL1-induced
activities in the CNS (3, 7, 8, 11, 12). Direct effects of CX3CL1 on
microglia include migration (1, 9, 13, 14), activation (9), proliferation
(15), inhibition of Fas-ligand-induced cell death (16), and inhibition
of cytokine release (3, 17). In recent years, it has become clear that
microglia plays a double role in its contribution to neuronal damage,
which occurs in neurodegenerative diseases and ischemic injury; it
can either drive neurotoxicity or act favoring proregenerative and neuroprotective strategies (18, 19). Different observations described a
*Istituto Pasteur-Fondazione Cenci Bolognetti & Dipartimento di Fisiologia Umana e
Farmacologia, Centro di Eccellenza BEMM, Università Sapienza, Roma, †Universitá
di Bari, and ‡Neuromed Istituto Ricovero & Cura a Carattere Scientifico via Atinese
18, Venafro, Italy
Received for publication November 15, 2007. Accepted for publication March 26, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from Ministry of Health (to Dr. Fabrizio Eusebi,
Rome University, Sapienza) and by the Ministry of University and Research (Programmi di ricercadi Rilevante Interesse Nazionale to C.L.).
2
Address correspondence and reprint requests Dr. Cristina Limatola, Dipartimento di
Fisiologia Umana e Farmacologia, Piazzale A. Moro 5, I00185 Roma. E-mail address:
cristina.limatola@uniroma1.it
www.jimmunol.org
protective role of both exogenous and endogenous microglia toward
ischemic injury (20, 21). Although microglia-induced neurotoxicity
has been, at least in part, explained by the production of cytokines and
reactive oxygen species that follows microglia overactivation (19), the
mechanisms underlying the neuroprotective activity of microglia
likely involve the production of a number of growth factors that possess neurotrophic activity (22); furthermore, the impairment of microglia-neuron communication through the pair CX3CL1/CX3CR1
increases the severity of neurodegeneration, which occurs in different
diseases (4).
Adenosine is an endogenous compound with different physiological activities: it is produced both inside and outside cells, and is a
metabolic product intermediate of different pathways (23). There are
different sources of extracellular adenosine in the nervous system; it
can derive from the degradation of the released adenine nucleotides
through the activity of extracellular nucleotidases, from equilibrative
transporters, or as result of cell damage (24). Moreover, a recent report described adenosine release from the parallel fibers of cerebellum
with an activity-dependent mechanism (25). In the CNS, adenosine
functions mainly involve modulation of neurotransmission and neuroprotection, and its extracellular concentration rapidly increases upon
brain damage, which follows stroke, ischemia, and epileptic seizures
(26). Adenosine-induced activities are mediated through the activation of four different G-protein coupled receptors (adenosine receptor
1 (AR1),3 AR2A, AR2B, and AR3), which are widely distributed
3
Abbreviations used in this paper: AR, adenosine receptor; Glu, glutamate; DPCPX,
1,3-dipropyl-8-cyclopentylxanthine; CCPA, 2-chloro-N6-cyclopentyladenosine;
AMPA, ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; EGFP, enhanced
green fluorescent protein; NES, normal external solution.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
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The chemokine fractalkine (CX3CL1) is constitutively expressed by central neurons, regulating microglial responses including
chemotaxis, activation, and toxicity. Through the activation of its own specific receptor, CX3CR1, CX3CL1 exerts both neuroprotection against glutamate (Glu) toxicity and neuromodulation of the glutamatergic synaptic transmission in hippocampal
neurons. Using cultured hippocampal neuronal cell preparations, obtained from CX3CR1ⴚ/ⴚ (CX3CR1GFP/GFP) mice, we report
that these same effects are mimicked by exposing neurons to a medium conditioned with CX3CL1-treated mouse microglial cell line BV2
(BV2-st medium). Furthermore, CX3CL1-induced neuroprotection from Glu toxicity is mediated through the adenosine receptor 1
(AR1), being blocked by neuronal cell preparations treatment with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a specific inhibitor of
AR1, and mimicked by both adenosine and the specific AR1 agonist 2-chloro-N6-cyclopentyladenosine. Similarly, experiments from
whole-cell patch-clamped hippocampal neurons in culture, obtained from CX3CR1ⴙ/ⴙ mice, show that CX3CL1-induced depression of
␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- (AMPA-) type Glu receptor-mediated current (AMPA-current), is associated
with AR1 activity being blocked by DPCPX and mimicked by adenosine. Furthermore, BV2-st medium induced a similar AMPAcurrent depression in CX3CR1GFP/GFP hippocampal neurons and this depression was again blocked by DPCPX. We also report that
CX3CL1 induced a significant release of adenosine from microglial BV2 cells, as measured by HPLC analysis. We demonstrate that (i)
CX3CL1, along with AR1, are critical players for counteracting Glu-mediated neurotoxicity in the brain and (ii) AR1 mediates neuromodulatory action of CX3CL1 on hippocampal neurons. The Journal of Immunology, 2008, 180: 7590 –7596.
The Journal of Immunology
Materials and Methods
Animals
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). Homozygous CX3CR1GFP/GFP knock-in mice (The Jackson Laboratory) were
obtained from Charles River.
Microglia culture and conditioned media preparation
BV2 cells were routinely maintained in culture in DMEM containing 10%
FBS. Cells were used up to passage 20. CX3CR1 expression was verified
by RT-PCR (data not shown). For medium conditioning, cells were plated
at 2 ⫻ 105 in a 24-multiwell plate at day 0, shifted to vehicle- or 100 nM
CX3CL1-containing 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, and HEPES
5 mM (pH 7.2)) for 30 min at day 1 and then to Neurobasal B27 medium
for different times, from 1 to 18 h. Media were collected, centrifuged at
3.000 ⫻ g for 5 min, and added to the medium of cultured hippocampal
neurons in a 1:1 ratio.
Primary hippocampal neuronal cultures
Hippocampal neuronal cultures were prepared from 1- or 2-day-old (P1P2) Wistar rats, and from C57BL/6 or CX3CR1GFP/GFP mice. In brief, after
careful dissection from diencephalic structures, the meninges were removed and the hippocampi were chopped and digested in 1.25 mg/ml
papain for 20 min at 37°C. Cells were mechanically dissociated and plated
at a density of 2.5 ⫻ 105 in poly-L-lysine coated plastic 24-well dishes in
serum-free Neurobasal medium, supplemented with B27 and 100 g/ml
gentamicin. Successively, cells were kept at 37°C in 5% CO2 for 11 days
with a twice a week medium replacement (1:1 ratio). The percentage of
neuronal cells obtained with this method is around 60%, as determined
with -tubulin III staining.
Glutamate- (Glu-) induced excitotoxic experiments
The 11-day-old hippocampal cultures were washed and stimulated in
Locke’s buffer with Glu (100 M) alone or together with CX3CL1 (100
nM) for 30 min. Following stimulation, cells were washed and re-incubated
in Neurobasal medium supplemented with B27 containing 100 g/ml
gentamicin for an additional 18 h. In some experiments, cells were both
pretreated (for 15 min) and re-incubated with different agonists and antagonists of ARs. Neuronal cell preparation was then treated with detergentcontaining buffer (0.5% ethylhexadecyldimethylammonium bromide,
0.28% acetic acid, 0.5% Triton X-100, 3 mM NaCl, and 2 mM MgCl2, in PBS
(pH 7.4) diluted 1/10) and counted in a hemacytometer for viability (8) or was
analyzed with the In Situ Cell Death Detection kit, fluorescein (Roche), to
detect apoptotic cells by fluorescence microscopy (Axioscope 2 Zeiss); the
apoptotic index was calculated as the percentage of these cells relative to total
number of cells (stained with 4⬘,6-diamidino-2-phenylindole).
Statistics
Data were reported as the means ⫾ SEM. Differences among means were
analyzed by one way or two way ANOVA.
Patch-clamp recordings
In brief, hippocampal cultured neurons, bathed in standard external medium, containing (normal external solution (NES): 140 mM NaCl, 2.5 mM
KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES-NaOH, and 10 mM
glucose (pH 7.3), were visualized at ⫻640 with Nomarski optics with an
upright microscope (Zeiss Axioscope). Patch-clamp whole cell recordings
were performed at room temperature (23–25°C) using borosilicate glass electrodes (3–5 M⍀) filled with (in mM): 140 mM Cs-methane sulfonate, 2 mM
MgCl2, 10 mM HEPES, 2 mM MgATP, and 0.5 mM EGTA ((pH 7.3) with
CsOH). Tetrodotoxin (0.2 M), unless otherwise indicated, was routinely
added to the bath solution. Neurons were clamped at ⫺70 mV. The stability of
the patch was checked by repetitively monitoring the input and series resistance during the experiment, and recordings were discarded when any of these
parameters changed by ⬎10%. Membrane currents, recorded with a patchclamp amplifier (Axopatch 200A; Axon Instruments), were acquired with
Clampex 10 software at 2 kHz (Axon Instruments).
Drugs and application procedures
Neuronal cell preparations were continuously superfused using a gravitydriven perfusion system consisting of independent tubes for standard and
agonist-containing solutions, positioned 50 –100 m from the recording
pipette and connected to a fast exchanger system (RSC-100; Bio-Logic).
To activate postsynaptic GluR, the ionotropic GluR agonist ␣-amino-3hydroxy-5-methylisoxazole-4-propionate (AMPA, 100 M; Tocris) was
delivered together with cyclothiazide (50 M; Tocris). CX3CL1 (chemokine domain, human; PeproTech), tetrodotoxin (Ascent Scientific,), and
1,3-dipropyl-8-cyclopentylxanthine (DPCPX; stock solution 5 mM in
DMSO) were similarly applied to neurons by gravity-driven perfusion. For
experiments with microglia conditioned medium, BV2 cells, plated at 2 ⫻
106 in a 100-mm plate, were shifted to NES or 5 nM CX3CL1-containing
NES for 30 min. Media were collected, centrifuged at 3.000 ⫻ g for 5 min,
and continuously superfused to the recorded cell using the gravity-driven
perfusion system. Adenosine, the adenosine agonists, and antagonists
2-chloro-N6-cyclopentyladenosine (CCPA), DPCPX, and triazoloquinazoline (CGS15943), were also purchased from Tocris. All other reagents
were of analytical grade and purchased from Sigma-Aldrich.
HPLC analysis
The 11-day-old rat hippocampal cultures were treated in Locke’s buffer for
30 min with CX3CL1, Glu, or CX3CL1/Glu, while murine BV2 cells were
treated for 30 min with CX3CL1. After this time, cells were washed, reincubated in their original conditioned medium, and, after additional 7.5 h,
the media were collected, added with ice-cold acetonitrile, centrifuged for
5 min at 1,440 ⫻ g, and the resulting supernatants were analyzed by HPLC.
For HPLC analysis of medium obtained from cocultures of BV2 and hippocampal neuronal preparations, 2 ⫻ 105 BV2 cells were plated on 11day-old hippocampal cultures and, after 8 h, the media were collected and
processed as above. Cells in the dish were washed twice with PBS, scraped,
lysed in NaOH 0.1 M, and analyzed for protein content with a BCA assay
(Pierce). Chromatographic analyses were conducted using a Merck Hitachi
HPLC system equipped with programmable autosampler (model L-7250),
pump (model L-7100), diode array detector (model L-7455), and fluorescence detector (model L-7480). 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 m,
250 ⫻ 4 mm) with precolumn Reprosil-Pur C18-AQ 5 m, 5 ⫻ 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 l. 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.
Dose-response relations
Drug concentration/cell survival relations were obtained by pretreating hippocampal neuronal cell preparations with the antagonists, for ⬃15 min, and
inducing excitotoxicity as already described in the presence of the antagonist. Cells were then left in the presence of the antagonist until analyzed
for survival. The IC50 of the AR antagonist DPCPX was estimated by fitting
the data to the Hill equation, using least-square routines: I/Imax ⫽ IC50nH/
([Antagonist]nH⫹IC50nH), where nH is the Hill coefficient, I is the inhibition of
CX3CL1-induced survival to excitotoxic insult at increasing doses of the agonist, and Imax is the maximum inhibition of CX3CL1-induced survival to
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throughout the brain and the spinal cord (26). Activation of AR1 has
been often correlated with neuroprotection in cell culture and brain
slice experiments (27), but also using knock-out animals for AR1 (28).
In contrast, AR1⫺/⫺ mice show no differences in the brain damage
observed after ischemia in comparison with wild-type animals, probably due to compensatory mechanisms (29).
Using primary hippocampal neuronal cultures from either wildtype or CX3CR1GFP/GFP mice, which express the gene reporter
enhanced green fluorescent protein (EGFP) in place of CX3CR1,
and the mouse microglial cell line BV2, we addressed the experiments to see whether the neuroprotective and neuromodulatory role of
CX3CL1 could be mediated through a cross-talk between glia and
neurons. We demonstrate that CX3CL1 exerts its neurotrophic and
neuromodulatory actions with mechanisms that involve AR1 activity,
and that it is able to induce adenosine release from microglial cells.
Our report provides new findings, involving two very well known
pairs like adenosine/AR1 and CX3CL1/CX3CR1, on the cross-talk
between glia and neurons.
7591
7592
CX3CL1 ACTIVITIES ON HIPPOCAMPAL NEURONS MEDIATED BY AR1
excitotoxic insult. In experiments with adenosine or CCPA, these drugs were
used as described for CX3CL1.
Results
CX3CL1-stimulated microglial cells release neurotrophic factors
We have previously demonstrated (8), and here confirmed, that
CX3CL1 protects hippocampal neurons against Glu-induced excitotoxicity. Since the hippocampal neuronal cultures we used in the
current experiments were composed by a mixed cellular population containing, in addition to neurons, also astroglial and microglial cells (see below), we wanted to analyze the role of microglial
cells in CX3CL1-mediated neuroprotective effect. For this reason,
we decided to use hippocampal neuronal cultures obtained from
CX3CR1GFP/GFP mice, which completely lack the CX3CL1 receptor, CX3CR1 (11). A preliminary analysis of hippocampal neuronal cultures obtained from CX3CR1GFP/GFP mice revealed that (at
11 days in cultures) neither neurons nor astrocytes, evidenced by
MAP2 or GFAP staining, were labeled by EGFP, whereas microglial cells (stained by CD11b) were EGFP positive (data not
shown). Similar indications of a lack of EGFP expression in neurons and astrocytes from these mice were obtained when
CX3CR1GFP/GFP hippocampal slices (obtained from 1- to 2-mo-old
mice) were analyzed using the patch clamp technique; all the
EGFP-expressing cells had the membrane current properties typical (30) of microglial cells (n ⫽ 30, data not shown). First, we
confirmed that CX3CL1 protects mouse CX3CR1⫹/⫹ neurons (8),
whereas it fails to protect CX3CR1⫺/⫺ (CX3CR1GFP/GFP) neurons
exposed to excitotoxic Glu (Fig. 1A). Second, we exposed
CX3CR1GFP/GFP hippocampal neuronal cell preparations to media conditioned with microglial BV2 cells treated either with
vehicle (not stimulated, BV2-ns) or with CX3CL1 (stimulated,
BV2-st), demonstrating that only BV2-st medium-protected
CX3CR1GFP/GFP neurons against Glu-mediated excitotoxicity
(Fig. 1B), likely through microglia released factor(s). The activity of BV2-st medium was time-dependent, starting to significantly protect neurons at ⬃8 h (medium collected 8 h after
CX3CL1 stimulation) and lasting up to 18 h (Fig. 1C). Following this experimental protocol, a complete reversal of events
leading to Glu-induced cell death was obtained, indicating that
at least part of the neuroprotective activity of CX3CL1 accounted for an indirect microglial contribution.
The specific AR1 antagonist DPCPX abolishes CX3CL1-induced
neuroprotective activity
Trying to identify the soluble factor(s) released from microglial
cells upon CX3CL1 stimulation, we looked at adenosine as a possible candidate, because its neurotrophic activities are very well
known (26). We report that the AR1 antagonist, DPCPX (100 nM)
and the non-specific AR antagonist triazoloquinazoline
(CGS15943, 100 nM), efficiently blocked neuroprotection against
Glu-mediated excitotoxicity induced either by BV2-st medium in
CX3CR1GFP/GFP neuronal cell preparations (Fig. 2A) or by
CX3CL1 in CX3CR1⫹/⫹ neuronal cell preparations (Fig. 2, B and
C, respectively, mouse and rat). Comparable results were obtained
with a TUNEL assay to monitor cell death on rat hippocampal
neurons (Fig. 2D). The dose-dependency of the inhibitory effect of
DPCPX in rat hippocampal neurons disclosed an IC50 value of
31.3 ⫾ 2.5 nM (Fig. 3, n ⫽ 4), which is far below the IC50 reported
for receptor subtypes other than AR1 (31). Furthermore, the specific antagonists for AR2A (SCH58261, 5 nM), AR2B (MRS1706,
20 nM), and AR3 (MRS3777, 50 nM) were ineffective on
CX3CL1-mediated neuroprotection at the indicated concentrations
(Table I). Considered together, these findings show an involvement of AR1 on the CX3CL1-induced neuroprotective activity.
CX3CL1 induces adenosine release from both hippocampal
cultures and microglial cell line BV2
To analyze whether adenosine was produced upon CX3CL1 stimulation, HPLC analysis was performed on the media conditioned with rat
hippocampal cultures treated as for the excitotoxic assay. Fig. 4A
shows that, after 8 h, CX3CL1 doubled adenosine accumulation in the
extracellular medium (44.3 ⫾ 3.3 vs 23.6 ⫾ 2.8 nmoles adenosine/mg
protein in unstimulated cells, p ⬍ 0.01, n ⫽ 8; adenosine concentration achieved in the culture medium upon CX3CL1 treatment was
6.9 ⫾ 2.4 M), and that significant increases occurred with Glu (to
35.5 ⫾ 2.9 nmoles adenosine/mg protein, p ⬍ 0.05, n ⫽ 8) and with
Glu/CX3CL1 (to 45.8 ⫾ 6.3 nmoles adenosine/mg protein, p ⬍ 0.01,
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FIGURE 1. CX3CL1-induced neurotrophic effect is mimicked by BV2-st medium. A, Excitotoxic cell death inhibited by CX3CL1 in CX3CR1⫹/⫹, not
in CX3CR1GFP/GFP mice. Hippocampal neurons cultured for 11 days and then stimulated with Glu (100 M, 30 min) in the presence or in the absence of
CX3CL1 (100 nM). After 18 h, cells were analyzed for death as described in Materials and Methods. B, Glu-induced excitotoxicity inhibited by BV2-st
medium, not by BV2-ns. Eighteen hours after incubation with or without CX3CL1, media were collected from BV2 cells and added in a 1:1 ratio to
hippocampal cultures obtained from CX3CR1GFP/GFP mice, previously treated with Glu (see Materials and Methods). After 18 h, cell death was analyzed.
C, Time dependence of BV2-st medium-induced neurotrophic action on Glu-treated CX3CR1GFP/GFP neurons. Experiments performed as in B, but media
from BV2 cells taken at the indicated time points. Throughout all the experiments, results represent mean ⫾ SEM of four independent duplicate experiments, and are expressed as percentage of hippocampal cell survival in untreated cultures, taken as 100%. In this and in the following figures: ⴱ, p ⬍ 0.05;
ⴱⴱ, p ⬍ 0.01.
The Journal of Immunology
7593
n ⫽ 8). To determine whether adenosine was produced by the microglial cells that, in the neuronal cell culture from CX3CR1GFP/GFP
mice, were present at the percentage of 7.8 ⫾ 1.3% of total cell population, HPLC analysis was performed on the media obtained from
BV2-ns and BV2-st cells. Results shown in Fig. 4B indicate that, after
8 h, CX3CL1 significantly ( p ⬍ 0.001, n ⫽ 5) enhanced adenosine
accumulation to 28.5 ⫾ 3.5 vs 6.7 ⫾ 1.3 nmoles adenosine/mg protein in control, BV2-ns cells (adenosine concentration achieved in the
culture medium upon CX3CL1 treatment was 7.5 ⫾ 2.0 M).
To analyze a potential role of neuronal expressed endogenous
CX3CL1 for its ability to induce adenosine release upon CX3CR1
engagement, the medium conditioned by a mixed microglialneuronal enriched cell population was analyzed by HPLC for
adenosine content. In these cocultures, extracellular adenosine,
after 8 h, was 9.5 ⫾ 3.1 nmoles adenosine/mg protein (four
independent quadruplicate experiments). This value was about
three times less than the algebraic sum of extracellular adenosine measured for sibling separate cultures (30.1 ⫾ 2.7 nmoles
adenosine/mg protein), indicating that the direct cell-to-cell (and
likely neuronal CX3CL1-microglial CX3CR1) contact did not mimic
the effect of exogenous CX3CL1 on adenosine release. Nevertheless,
we cannot exclude an effect of endogenous CX3CL1 on extracellular
adenosine accumulation since the coculture likely activates additional
interactions which may have opposite effect.
Furthermore, we demonstrated that both adenosine and the specific
AR1 agonist CCPA significantly protected neurons from Glu-mediated excitotoxicity, their effect being maximal at 1 nM (adenosine,
105 ⫾ 0.6%; adenosine/Glu, 101 ⫾ 8.1%, p ⬍ 0.005; CCPA, 93.8 ⫾
0.3%; and CCPA/Glu, 93.3 ⫾ 0.3% of control, p ⬍ 0.005, n ⫽ 4. The
data reported represent cell survival as % of control).
DPCPX inhibits CX3CL1- and BV2-st medium-mediated
AMPA-current depression
Since we have found that the neuroprotective action of CX3CL1 on
hippocampal neurons is dependent on AR1 activation, we were
interested to investigate whether AR1 could be also involved in the
previously described CX3CL1-induced neuromodulation (2, 8).
We report that the CX3CL1-mediated inhibition of the amplitude
of AMPA-currents in cultured hippocampal neurons was abolished
by DPCPX treatment. As illustrated in Fig. 5, A and B, AR1 activity was required for CX3CL1-induced depression of AMPATable I. The neurotrophic activity of CX3CL1 is not influenced by A2A-,
A2B-, and A3-specific AR antagonistsa
FIGURE 3. Dose-response relationship of DPCPX inhibitory effect on
CX3CL1-mediated cell survival. Rat hippocampal neurons were cultured
and treated for excitotoxicity as in Fig. 1. Before Glu-treatment, cells were
preincubated with increasing doses of DPCPX, as indicated; the drug was
present during all the experiment. After 18 h, cells were analyzed for death
and the results are expressed as % of maximal inhibitory effect obtained at
100 nM DPCPX. Data were fitted to a Hill plot (superimposed curve)
showing a half maximal dose of 31.3 ⫾ 2.5 nM (n ⫽ 4).
SCH58261
(5 nM, A2A)
MRS1706
(20 nM, A2B)
MRS3777
(50 nM, A3)
Glu
59.5 ⫾ 2.5 61.7 ⫾ 7.7
96.1 ⫾ 1.6 100.0 ⫾ 7.0
Glu plus
CX3CL1
65.2 ⫾ 1.8
111.3 ⫾ 7.3
71.6 ⫾ 3.6
92.2 ⫾ 4.5
AR
Antagonists
Nil
a
Results shown as percentage ⫾ SEM of cell survival in comparison with control
cells for each treatment. Variations among different control conditions never exceeded
8%. For details see Materials and Methods and text.
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FIGURE 2. Antagonists of adenosine receptors
block BV2-st medium- or CX3CL1-induced neurotrophic effects on cultured hippocampal neurons. BV2-stand CX3CL1-induced neurotrophic actions were suppressed by either DPCPX (100 nM) or CGS15943
(CGS, 100 nM) cotreatment. Excitotoxity experiments
were performed with same protocol as Fig. 1, A and B,
on neurons obtained, respectively, from CX3CR1GFP/GFP
mice (A), CX3CR1⫹/⫹ mice (B), or rats (C and D). Neurons were preincubated for 15 min with the indicated
drugs that were also present in medium during all the
experiment. In A, B, and C, results reported as in Fig. 1.
In D, results are reported as TUNEL positive cells as
percentage of total cells in a ⫻40 optic field (counting
at least 15 fields for each sample).
7594
CX3CL1 ACTIVITIES ON HIPPOCAMPAL NEURONS MEDIATED BY AR1
FIGURE 4. CX3CL1 induces extracellular accumulation of adenosine. HPLC analysis of adenosine from
growth media of hippocampal cultures (A) and from
BV2-ns (control) or BV2-st (CX3CL1) cells (B). Cells
were stimulated with CX3CL1 (100 nM) for 30 min in
Locke’s buffer, washed and reincubated in conditioned medium (see Materials and Methods). After
7.5 h medium was collected and immediately analyzed for adenosine content. Results are expressed as
nmoles extracellular adenosine accumulated per mg
of cellular proteins and are the mean ⫾ SEM of three
independent quadruplicate experiments.
4; DPCPX), indicating that adenosine itself modulates AMPA-receptors function. Furthermore, using neurons from CX3CR1GFP/GFP
mice, we found that the BV2-ns medium (Fig. 6A) induced a reduction of the AMPA-currents (fall to 85 ⫾ 5%, n ⫽ 8) insensitive
to DPCPX (fall to 83 ⫾ 4%; n ⫽ 8; p ⬎ 0.1 in the presence of
DPCPX). The additional reduction of the AMPA-currents induced
by BV2-st medium (fall to 68 ⫾ 4%; n ⫽ 9 in the absence of
DPCPX; Fig. 6B) compared with BV2-ns medium, was instead
suppressed by DPCPX, the AMPA-currents being recovered to
FIGURE 5. CX3CL1-induced depression of AMPA-currents is suppressed by the adenosine receptor antagonist DPCPX. A, Time course of
the effect of CX3CL1 at indicated dose (horizontal bar) on the amplitude of
AMPA-currents (AMPA 100 M; CTZ 50 M; 250 ms application;
AMPA signal, black arrow) recorded in hippocampal cultured neurons obtained from CX3CR1⫹/⫹ mice in control medium (f; n ⫽ 5), or in the
presence of DPCPX (E; 100 nM; n ⫽ 5), as indicated. Data represent
mean ⫾ SEM of single determinations on separate cells. Superimposed
current traces at right, recorded in the presence of 100 nM DPCPX (top, E)
or in control medium (bottom, f) before and after 10 min of CX3CL1
application. B, Time course of the effect adenosine (at indicated dose) on
AMPA-currents as A (control medium, f; n ⫽ 5; DPCPX 100 nM, E; n ⫽
4). Right, as A before (control) and at maximum adenosine effect (adenosine) (top, E, 100 nM DPCPX; bottom, f, control medium).
FIGURE 6. Depression of AMPA-currents induced by BV2-st medium
is suppressed by the specific AR1 antagonist DPCPX. A, Time course of
the effect of BV2-ns medium (horizontal bar) on the amplitude of AMPAevoked currents (as Fig. 5) in cultured hippocampal neurons from
CX3CR1GFP/GFP mice; control (f; n ⫽ 8), DPCPX (E; 100 nM; n ⫽ 8).
Superimposed current traces at right, recorded in the presence of 100 nM
DPCPX (top, E) or in control medium (bottom, f) before and at maximum
BV2-ns medium effect. Note AMPA-current depression, induced by unknown microglia released factors, not influenced by DPCPX. B, Time
course of the effect BV2-st medium on AMPA-currents as (A) (control, f;
n ⫽ 9; DPCPX 100 nM, E; n ⫽ 7). Right, as A before (control) and at
maximum BV2-st medium effect as indicated (top, E, 100 nM DPCPX;
bottom, f, control). Note additional AMPA-current depression induced by
BV2-st medium, abolished by DPCPX.
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currents, a mechanism counteracting Glu-mediated excitoxicity
(8). Specifically, Fig. 5A shows that AR1 antagonist DPCPX (100
nM) blocked the depression of AMPA-currents induced by
CX3CL1 in CX3CR1⫹/⫹ mouse neurons (average depression to
50 ⫾ 5%; n ⫽ 5, control medium; and p ⬍ 0.01; to 101 ⫾ 3%; n ⫽
5, DPCPX). Fig. 5B shows that adenosine (50 M), in the same
neurons, mimicked the CX3CL1-induced AMPA-currents depression that was again sensitive to DPCPX (average depression to
69 ⫾ 8%; n ⫽ 5; p ⬍ 0.05, control medium; and 97 ⫾ 2%,; n ⫽
The Journal of Immunology
values observed with BV2-ns medium (85 ⫾ 4%, n ⫽ 7 in the
presence of DPCPX). These findings point to an indirect effect on
AMPA-currents by CX3CL1 again mediated by AR1 activity.
Discussion
as a negative feedback signal aimed at the reduction of neuronal
loss (46). Our observation that CX3CL1 and Glu induce comparable levels of extracellular adenosine accumulation in hippocampal neuronal cell cultures is suggestive of an additional contribution of CX3CL1 in this mechanism, further amplified by the
previously described Glu-induced CX3CL1 cleavage from neurons
(8, 13).
We and others have previously shown that (i) CX3CL1 modulates glutamatergic AMPA-currents revealed as a reduction of the
amplitude of both synaptic and agonist-evoked currents; (ii)
CX3CL1 action on AMPA-currents is stringently and specifically
related to the activation of CX3CR1 (2, 8, 32). In this study, we
provide evidence that CX3CL1-induced depression of AMPA-currents in hippocampal neurons is suppressed by a specific antagonist of AR1, DPCPX. Furthermore, we report that a similar current
depression is induced on hippocampal neurons treated with a medium conditioned by CX3CL1-stimulated microglia cells and is
again suppressed by DPCPX. We speculate that CX3CL1 acts on
its own receptor CX3CR1, expressed on glial cells, inducing the
release of adenosine that, in turn, interacts with neuronal AR1 influencing the functional properties of AMPA receptors depressing
their channel gating with mechanisms to be ascertained. The role
of adenosine as modulator of synaptic transmission is very well
investigated; its main effect on synaptic transmission is presynaptic
and consists of the inhibition of neurotransmitter release (47), but
postsynaptic membrane hyperpolarization is reported (48). Our
findings provide evidence for another inhibitory effect of adenosine, mediated through AR1, and involving the postsynaptic modulation of AMPA receptor function.
In conclusion, our findings demonstrate that the activity of AR1,
possibly regulated by adenosine released from glial cells, is involved in CX3CL1-induced neuroprotection and modulation of
glutamatergic neurotransmission in hippocampal neurons. The
identification of AR1 as downstream element responsible for beneficial effects of CX3CL1 on neuron survival against Glu neurotoxicity may represent an important challenge for management of
neurological disorders including acute brain ischemia.
Acknowledgments
We thank Giuseppina Chece for skillful technical expertise, Dr. Myriam
Catalano for help with coculture experiments, Drs. Fabrizio Eusebi, Knut
Biber, and Flavia Trettel for helpful discussions, and Dr. Sergio Visentin
(Istituto Superiore Sanità, Roma) for providing BV2 cells.
Disclosures
The authors have no financial conflict of interest.
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13.
CX3CL1 ACTIVITIES ON HIPPOCAMPAL NEURONS MEDIATED BY AR1