RESEARCH ARTICLE

P2X4 Receptors Control the Fate andSurvival of Activated Microglia

Nuria V�azquez-Villoldo,1 Mar�ıa Domercq,1 Abraham Mart�ın,2 Jordi Llop,3

Vanessa G�omez-Vallejo,3 and Carlos Matute 1

Microglia, the resident immune cells of the central nervous system, responds to brain disarrangements by becoming activatedto contend with brain damage. Here we show that the expression of P2X4 receptors is upregulated in inflammatory foci andin activated microglia in the spinal cord of rats with experimental autoimmune encephalomyelitis (EAE) as well as in the opticnerve of multiple sclerosis patients. To study the role of P2X4 receptors in microgliosis, we activated microglia with LPS invitro and in vivo. We observed that P2X4 receptor activity in vitro was increased in LPS-activated microglia as assessed bypatch-clamp recordings. In addition, P2X4 receptor blockade significantly reduced microglial membrane ruffling, TNFa secre-tion and morphological changes, as well as LPS-induced microglial cell death. Accordingly, neuroinflammation provoked byLPS injection in vivo induced a rapid microglial loss in the spinal cord that was totally prevented or potentiated by P2X4receptor blockade or facilitation, respectively. Within the brain, microglia in the hippocampal dentate gyrus showed particularvulnerability to LPS-induced neuroinflammation. Thus, microglia processes in this region retracted as early as 2 h after injec-tion of LPS and died around 24 h later, two features which were prevented by blocking P2X4 receptors. Together, these datasuggest that P2X4 receptors contribute to controlling the fate of activated microglia and its survival.

GLIA 2014;62:171–184Key words: microglia, P2X4 purinergic receptor, EAE and multiple sclerosis, cell death

Introduction

Microglial cells are the main effectors of the innate

immune response after CNS injury (Kreutzberg, 1996).

The precursors of adult microglia are yolk sac progenitors,

which enter the embryo around E8 and colonize the CNS at

E10 (Ginhoux et al., 2010). Microglia can be found postna-

tally in all regions of the CNS, in the parenchyma and in

perivascular spaces in a nonoverlapping territorial fashion,

and comprise a large proportion of the total cellular makeup

of the CNS, estimated to be as high as 12% of all cells. Per-

haps owing to this origin, microglia share many commonal-

ties with resident tissue macrophages in peripheral organ

systems (Ransohoff and Perry, 2009). Like their peripherals

macrophage counterparts, microglia displays a remarkable

range in both morphology and activity, depending in part on

the state of the surrounding tissue (Ransohoff and Perry,

2009). In their resting state, microglia continuously monitor

the tissue for injury or pathological changes by extending and

retracting highly motile processes on a time scale of minutes

(Davalos et al., 2005; Nimmerjahn et al., 2005; reviewed in

Kettenmann et al., 2013). Exogenous factors (infectious

agents) as well as endogenous factors or neurotransmitters

released by damaged cells or tissues induce progressive

changes in microglia including altered expression of cell sur-

face markers and inflammation related genes, process retrac-

tion and acquisition of an ameboid morphology, migration of

cell bodies, proliferation and increased phagocytic rate (Ket-

tenmann et al., 2011).

The rapidly diffusible neurostransmitter ATP is one of

the main regulators of microglial functions. Low ATP concen-

trations almost exclusively activate chemotaxis, through

P2Y12 receptor activation, in order to recruit cells at the site

View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22596

Published online November 19, 2013 in Wiley Online Library (wileyonlinelibrary.com). Received May 23, 2013, Accepted for publication Oct 16, 2013.

Address correspondence to Carlos Matute, Departamento de Neurociencias, Universidad del Pa�ıs Vasco, E-48940 Leioa, Spain. E-mail: carlos.matute@ehu.es or

Maria Domercq, Departamento de Neurociencias, Universidad del Pa�ıs Vasco, E-48940 Leioa, Spain. E-mail: maria.domercq@ehu.es

From the 1Departamento de Neurociencias, Universidad del Pa�ıs Vasco-UPV/EHU, Barrio Sarriena s/n, 48940, Leioa, Spain, Achucarro Basque Center for Neuro-

science-UPV/EHU, 48170, Zamudio, Spain, Instituto de Salud Carlos III, Centro de Investigaci�on Biom�edica en Red de Enfermedades Neurodegenerativas

(CIBERNED), 48940, Leioa, Spain; 2Molecular Imaging Unit, CIC biomaGUNE, Paseo Miramon 182, 20009, San Sebasti�an, Spain; 3Radiochemistry Department,

Molecular Imaging Unit, CIC biomaGUNE, Paseo Miramon 182, 20009, San Sebasti�an, Spain.

Additional Supporting Information may be found in the online version of this article.

VC 2013 Wiley Periodicals, Inc. 171

of injury or inflammation. When the ATP concentration

increases, additional effector functions, such as phagocytosis

and cytokine secretion, are also triggered. The switch on

microglial ATP responses are sometimes linked to an acute

remodeling of purinoceptor expression, i.e., upregulation of

the Gs-coupled A2A receptor and P2X4 receptor (Beggs et al.,

2012; Orr et al., 2009) and downregulation of P2Y12 (Hay-

nes et al., 2006).

While still a lot has to be learned about the events

affecting microglia throughout the activation process, even

less is known about the period after. Acute activation of

microglia after neural injury rapidly leads to reactive micro-

gliosis, a cardinal feature of expansion of microglia in the

affected CNS region (Wirenfeldt et al., 2005). The state of

reactive microgliosis resolves days to weeks later, according to

an inherently tightly regulated schedule, which has been sug-

gested to involve microglial apoptosis (Shin et al., 2004; Soria

et al., 2011). Like in the immune system, microglial cell

death secondary to its activation (called AICD, activation-

induced cell death) may be a mechanism to resolve brain

inflammation. However, little is known about the underlying

mechanisms responsible for cell death of activated microglia.

Moreover, in addition to apoptosis, other forms of cell death

such as autophagy (Arroyo et al., 2013), pyroptosis, a pro-

grammed cell death triggered by caspase-1 activation by the

inflammasome (Miao et al., 2011), and necroptosis (Fricker

et al., 2013) could contributed to microglia AICD. In this

study, we first showed that P2X4 receptors are upregulated in

multiple sclerosis (MS) samples and in one of its animal

models, encephalitis autoimmune experimental (EAE). To

gain information on the role of P2X4 signaling, we used the

most classical model of neuroinflammation, LPS treatment or

injection. We report here that a microglia can die rapidly

(<24 h) both in vitro and in vivo by a mechanism dependent

on P2X4 activation and independent of signaling by the

inflammasome.

Materials and Methods

Human Tissue SamplesPostmortem optic nerve samples from 13 long-standing MS patients

and 12 control subjects (who died from non-neurological diseases)

were obtained at autopsy under the management of the Netherlands

Brain Bank. All patients and controls had previously given written

approval for the use of their tissue, according to the guidelines of

the Netherlands Brain Bank. Clinical characteristics for control and

patient groups have been previously described (see Vallejo-

Illarramendi et al., 2006; Pampliega et al., 2011). We used clinical

data together with macroscopic tissue analysis to classify MS samples

as normal appearing (NAON) or damaged optic nerves (DON),

when showing macroscopic plaques, atrophy and/or optic neuritis.

For comparisons, MS samples were matched with control samples

for age, sex, and postmortem delay.

Induction of Acute EAE and NeuroinflammationIn vivo experiments in rats were performed in accordance with the

approved protocols and guidelines of the University of the Basque

Country Ethics Review Committee for animal experiments. Animals

were handled in accordance with the European Communities Coun-

cil Directive and all possible efforts were made to minimize animal

suffering and the number of animals used. Young adult male Lewis

rats, (200–220 g weight) were each injected subcutaneously in both

hind feet with inoculum containing 100 lg of guinea pig myelin

basic protein (Sigma) diluted in water, emulsified in equal volumes

of Freund’s incomplete adjuvant (Sigma), supplemented with 500 lg

of heat-inactivated M. tuberculosis H37Ra (DIFCO Laboratories).

The neurological deficits started 10 days postimmunization (dpi),

peaked after 14 days and remitted by 20 dpi. Neurological impair-

ment was monitored and scored daily for clinical signs of EAE

according to the following scale: 0, normal; 1, flaccid tail; 2, tail

paralysis; 3, loss of muscle tone in hind limbs; 4, hind limbs hemi-

paralysis; 5, complete hind limbs paralysis; 6, moderate paraparesis;

7, tetraparalysis; 8, death. The tissues and samples examined in this

study were obtained at 14 days after immunization when rats devel-

oped peak EAE (score 3–6).

Lipopolysaccharide (LPS) or phosphate-buffered saline (PBS)

was injected intraperitoneally (ip; 3 mg kg21) and stereotaxically in

the lateral ventricule (icv; 1 mg kg21, 10 lL). Sprague–Dawley rats

(200 g weight) were anesthetized with a mixture of ketamine (80 mg

kg21) and xylazine (10 mg kg21), both ip. The stereotaxic injection

(21 mm from Bregma; 2 mm lateral; 4 mm deep) was performed

at a rate of 1 lL min21, with the use of a microinjection autopump

and a 25-lL Hamilton microsyringe. TNP-ATP (10 mg kg21) was

administered intraperitoneally immediately and at 12 h after LPS

injection.

RNA Isolation and Quantitative Real Time PCRTotal RNA was extracted from rat and human samples using TRIzol

reagent kit (Invitrogen) and purified using the RNeasy MniKit (Qia-

gen). No DNase I treatment was performed in RNA obtained from

rat and human samples. The integrity of RNA was analyzed by ethi-

dium bromide agarose gel or using a Bioanalyzer (Agilent). For

cDNA synthesis, 2 lg of total RNA was used using random hexamer

primers and Super Script II reverse transcriptase (Invitrogen). Mock

reverse transcription (without enzyme) for each sample served as neg-

ative controls.

The relative expression level of genes was measured according

to the manufacturer’s protocol with quantitative real time PCR as

previously described (Vallejo-Illarramendi et al., 2006). Briefly,

qPCR reactions were carried out with 20–80 ng of reverse tran-

scribed RNA and 50–300 nM of forward and reverse primers (Gen-

otek, Sabadell, Spain) diluted in SYBRGreen PCR universal master

mix reagent (Applied Biosystems) in an ABI PRISM 7000 Sequence

Detection System instrument (Applied Biosystems). The expression

level was normalized with a normalization factor obtained using the

Genorm software (Vandesompele et al., 2002) based on the expres-

sion of three to six housekeeping genes (see Supp. Info. Table 1).

Primers were designed in exon–exon junction regions to avoid

gDNA amplification with the PrimerExpress software (Applied

172 Volume 62, No. 2

Biosystems; see Supp. Info. Table 1). We verified that generated fluo-

rescence was not overestimated by contamination resulting from

residual genomic DNA amplification (using RT negative controls)

and from primer dimer formation or external DNA contamination

(no template controls). qPCR products were also subjected to a dis-

sociation protocol to ensure that a single amplicon of the expected

melting temperature was indeed obtained.

Western BlotTotal protein was extracted from microglia cell cultures by scraping

the cells in SDS/sample buffer. Samples (10 lg of protein per lane)

of microglia cell cultures and rat spinal cord were boiled for 5 min,

loaded and size separated in 12.5% SDS/PAGE polyacrylamide gels.

After electroblotting on nitrocellulose membranes, proteins were

visualized using primary antibodies to P2X4 (1 lg mL21; Alomone

Labs, Jerusalem, Israel) and b-actin (1:1,000; Sigma, Madrid, Spain),

followed by secondary peroxidase-coupled goat anti-rabbit antibodies

(1:5,000; Sigma). After washing, the blots were developed using an

enhanced chemiluminescence detection kit according to the manu-

facturer’s instructions (Super Signal ULTRA, Pierce). Images were

acquired with a ChemiDoc MP system (BioRad) and quantified

using Scion Image Software.

ImmunochemistryMicroglial expression of purinergic receptors was examined by

immunofluorescence applied to cultured microglia, BV-2 cells and

tissue from rat spinal cord and cerebrum as well as human spinal

cord. Cells were fixed in 4% p-formaldehyde in PBS and processed

for immunocytochemistry as previously described (Domercq et al.,

1999). For tissue, adult Sprague–Dawley and Lewis rats were deeply

anesthetized with chloral hydrate (500 mg kg21, i.p) and transcar-

dially perfused with 0.1 M sodium phosphate buffer, pH 7.4, fol-

lowed by 4% p-formaldehyde in the same buffer. Human spinal

cord samples from MS and control subjects were obtained at autopsy

from the Netherlands Brain Bank. Characteristics of spinal cord sam-

ples are described previously (Pampliega et al., 2011). Frozen tissue

was kept at 280�C until use.

Primary antibodies were as follows to: P2X1, P2X2, P2X4,

and P2X7 (1–2 lg mL21; Alomone Labs, Jerusalem, Israel), P2X3

(1 lg mL21; Chemicon, Temecula, CA), CD11b (OX42, 1:50,

Serotec), Iba-1 (1:1,000; Wako) and CD68 (1:50; DakoCytomation)

For secondary antibodies, we used Alexa 488-conjugated goat anti-

bodies to rat IgG (Molecular Probes), and Alexa546-conjugated goat

antibody to mouse IgG (Molecular Probes). Controls were carried

out by omitting the primary antibody. Images were acquired using a

laser scanning confocal Olympus Fluoview FW500 microscopy or a

bright field Axiovision microscopy (Zeiss). Images were acquired

with the same settings for all samples within one experimental

group. P2X4 immunoreactivity per cell was calculated with the

ImageJ software (NIH, http://rsb.info.nih.gov/ij/) (n 5 40–50 cells

from at least three different experiments or samples). Morphology

analysis of microglia was performed with ImageJ software as

described before (Fontainhas et al., 2011). Microglial cells were out-

lined with the CD11b immunostaining as the defining parameter

for the region of interest. Dendritic tree area as a morphological

parameter was determined by circumscribing the area outlined by

the ends of dendritic processes using a smooth polygon tool in NIH

ImageJ. The 3D reconstructions were performed with ImageSurfer

(http://www.imagesurfer.org). CD11b and Iba11 cells were counted

blindly in longitudinal sections of spinal cord using a 403 objective

in an Axiovision microscopy (Zeiss). At least 40 different fields from

three slices per animal were counted and each treatment was per-

formed in duplicate in three different experiments.

Microglia Cell Cultures and In Vitro AssaysMicroglial cultures derived from cortical tissue from postnatal P0

rats. Briefly, cortices were dissociated and plated with serum-

supplemented Iscove’s modified Dulbecco’s medium (Gibco). After 2

weeks, confluent monolayer of cultured astrocytes was depleted from

microglia by mechanical shaking, according to standard protocols.

Free-floating microglia were collected from shaken astrocyte flasks

and purified by plating on noncoated plastic Petri dishes (Sterilin).

After 24 h, nonadhered cells (progenitors cells) were eliminated and

microglial cells were re-plated on PDL-coated coverslips or wells.

The purity of cultured microglia was higher than 99% pure under

these conditions. The immortalised murine microglial cell line BV-2

was also used as an alternative model to primary microglia. BV-2

cells were grown in serum-supplemented Dulbecco’s modified Eagle

medium (Gibco). Cells were treated with lipopolysaccharide (LPS,

E. coli O11:B4) for 24 h unless otherwise indicated.

Membrane ruffling was determined on the basis of phalloidin

immunostaining. Briefly, after LPS treatment, cells were fixed in 4%

PFA in PBS for 10 min, permeabilized for 20 min with PBS con-

taining 0.1% Triton X-100 and stained for 1 h with 2 U mL21

Texas-Red-conjugated phallodin (Invitrogen) diluted in PBS contain-

ing 1% bovine serum albumin. Phalloidin staining was observed

under the fluorescence microscopy (Axioplan, Zeiss) and blind-eye

quantified in at least 15 different fields from n 5 4 experiments per-

formed in triplicate. Cell viability was determined by measuring

LDH release to the extracellular medium after 24-h treatment with

LPS in the presence or absence of P2XR receptor antagonists. Cell-

free supernatants from microglia cultures were collected after incuba-

tion with LPS and release of rat TNFa and mature IL-1b was moni-

tored using highly sensitive ELISA kits (Invitrogen) according to the

manufacturer instructions.

Patch-Clamp ElectrophysiologyStandard whole-cell recordings of microglia (Vhold 5 270 mV)

were performed at room temperature on an inverted microscope

(CK-40, Olympus) using the EPC-7 patch-clamp amplifier (HEKA,

Lambrecht, Germany). Recordings were low-pass filtered at 2 kHz,

digitized at 5 kHz and stored as data files on a computer using the

pClamp 8.2 program (Axon Instruments, CA) for later analysis. The

extracellular bath solution contained (in mM): NaCl 140, KCl 5.4,

CaCl2 2, MgCl2 1, Hepes 10, pH 7.3. Divalent cation-free extracel-

lular solutions were obtained by omitting Ca21 and Mg21. Patch

clamp pipettes (3–5 MX) were filled with a solution containing (in

mM): CsCl 140, CaCl2 2, MgCl2 2, Hepes 10, EGTA 11, Na-ATP

2, pH 7.3. Local microperfusion of agonist was performed with a

V�azquez-Villoldo et al.: P2X4 Receptor Activation Kills Microglia

February 2014 173

multibarreled apparatus connected to an electronically driven rota-

tory motor (RSC-100, Biologic, France).

Radiochemistry[11C]CH4 was directly generated in an IBA Cyclone 18/9 cyclotron

and transferred to a TRACERlab FXC Pro synthesis module (GE

Healthcare) where [11C]CH3I was generated. [11C]PK11195 was

synthesized with the captive solvent method (Wilson et al., 2000)

using DMSO as the solvent and KOH as the base (reaction time 5

5 min). Average decay corrected radiochemical yield and specific

activity were 40% and 125 GBq lmol21, respectively. Radiochemi-

cal purity was higher than 99%.

Positron Emission Tomography Scans, DataAcquisition, and Image AnalysisPositron emission tomography was performed in rats treated with

LPS (n 5 5) in the absence or in the presence of TNP-ATP (n 5

5) and vehicle (control, n 5 5) rats at 6 h after administration to

assess the peripheral benzodiazepine receptor/translocator protein (18

kDa) TSPO (PBR/TSPO) binding. Animals were placed into a rat

holder compatible with PET acquisition systems and PET images

acquired on a General Electric eXplore Vista CT camera (GE

Healthcare) during 63 min after intravenous injection of 20 MBq of

[11C]PK11195. The attenuation correction factors were calculated

from the CT acquisition. Dynamic acquisitions were reconstructed

(decay and CT-based attenuation corrected) with filtered back pro-

jection (FBP) using a Ramp filter with a cutoff frequency of 1 Hz.

PET images were analyzed using PMOD image analysis soft-

ware (PMOD Technologies, Z€urich, Switzerland). To verify the ana-

tomical location of the signal, PET images were coregistered to the

anatomical data of the CT of the same animal for the analysis of the

cervical spinal cord and the MRI rat brain template for the analysis

of the brain and cerebellum. Likewise, three type of VOIs were

established as follows: (i) A first set of volumes of interest (VOIs)

was defined in the inner part of the spine using the CT scan of the

same rat defined to study the [11C]PK11195 binding in the cervical

cord. A VOI was manually defined in the inner part of the spine

using the CT scan of the same rat. (ii) A second set of VOIs was

automatically generated in both (ii) the brain and (iii) the cerebel-

lum, by using the regions proposed by the PMOD rat brain tem-

plate. For quantification of both VOIs, summed uptake during the

last 15 minutes of acquisition was expressed as percent of injected

dose per cc of tissue (ID/cc).

Statistical AnalysisData are presented as mean 6 SEM. Comparisons between two

groups were analyzed using Student’s two tailed t test. Comparisons

among multiple groups were analyzed by one-way ANOVA followed

by Bonferroni post hoc test.

Results

P2X4 is Overexpressed in EAE Rats and in MSSamplesWe previously demonstrated that purinergic signaling is

altered in chronic EAE, an animal model of MS, and that it

is involved in oligodendroglial cell death and axonal damage

(Matute et al., 2007). We have analyzed here the expression

of purinergic ionotropic receptors in the spinal cord of rats

after induction of acute EAE, which reproduces the neuroin-

flammatory component of the disease. At 14 days postimmu-

nization, when animals showed maximal motor deficits, we

detected a high increase in the expression of P2X4 mRNA in

spinal cord samples from rats with EAE versus control ani-

mals (Fig. 1A). Accordingly, P2X4 protein levels were signifi-

cantly increased in EAE rats (Fig. 1B). We also analyzed the

expression of P2X4 in the spinal cord by double immunoflu-

orescence using antibodies to OX-42, a marker of microglia

lineage. P2X4 was expressed at low levels in OX421 micro-

glial cells from control rats (Fig. 1C) but the expression was

increased after EAE induction in parenchymal and perivascu-

lar microglia (Fig. 1C, P2X4 grey level units in control and

EAE rats were 32.8 6 3.2 and 74.8 6 3.74, respectively, n

5 6 per group, P < 0.01).

We next studied the expression of P2X4 in post-

mortem optic nerve samples of MS patients. This cohort of

samples has been previously characterized and showed astro-

glial reactivity, inflammation and oligodendroglial cell death

(Vallejo-Illarramendi et al., 2006). We observed that the levels

of P2X4 mRNA were significantly higher in MS than in con-

trol optic nerves (1.34 average fold increase; Fig. 1D). Sur-

prisingly, we found that the increased expression in P2X4 was

higher in the normal appearing optic nerves (NAON)

(�1.56-fold; Fig. 1D) than in the damaged optic nerves

(DON) characterized by the presence of macroscopic plaques,

atrophy and/or optic neuritis (Vallejo-Illarramendi et al.,

2006; Fig. 1D). Double immunofluorescence analysis revealed

a higher expression of P2X4 in CD681 microglia/macro-

phages in the spinal cord of MS patients versus controls (Fig.

1E, P2X4 gray level units in control and MS samples were

30.2 6 1.9 and 60.1 6 3.6, respectively, n 5 4, P < 0.01).

These results are consistent with the increase of P2X4 expres-

sion in EAE animals, and suggest that altered P2X4 expres-

sion may have a pathophysiological role in MS.

P2X4 Expression and Function is Increased inActivated MicrogliaWe next investigated P2X receptor expression using in vitroas well as in vivo systemic administration of bacterial lipo-

polysaccharide, an endotoxin that signals through Toll-like

receptor 4 (Lehnardt et al., 2003), as a model of neuroinflam-

mation. In BV-2 cells, all receptors analyzed (P2X1, 2, 3, 4,

and 7) were expressed in control conditions (data not shown).

After LPS treatment (1 lg mL21, 24 h) we found an increase

in the expression of P2X4 receptors (Fig. 2A). Similar

changes after LPS treatment were observed in primary cul-

tures of microglial cells, as revealed by immunocytochemistry

174 Volume 62, No. 2

(data not shown) and Western blot (Fig. 2B), in accordance

with previous reports (Raouf et al., 2007). In control spinal

cord, microglial cells express low levels of P2X3 and P2X4

receptors and high levels of P2X7 receptors (Fig. 2C). The

other P2X receptors analyzed, P2X1 and P2X2, were located

exclusively in axons and oligodendrocytes, respectively (Fig.

2C). We did not detect any change in P2X3 and P2X7

expression after injection of LPS (3 mg kg21, i.p.; 24 h). In

contrast, there was a robust increase in P2X4 in LPS-

activated microglia (Fig. 2D; P2X4 gray level units in control

and in LPS-treated animals were 68.3 6 3.6 and 126.3 6

5.5, respectively, P < 0.001).

We next used electrophysiological recordings to analyze

P2X4 function in control and LPS-stimulated microglial cells.

ATP at low concentrations (30–100 lM), to avoid activation

of low–affinity P2X7 receptor, induced an inward current in

almost all microglial cells (n 5 18; Fig. 3A). ATP-induced

currents were greatly reduced by TNP-ATP (10 lM; n 5 12;

Fig. 3B), an antagonist of P2X1-4 receptors, by paroxetine

(10 lM, n 5 11; Fig. 3B), an antidepressant that specifically

inhibits P2X4 receptor function (Nagata et al., 2009) and by

the selective P2X4 receptor antagonist 5-BDBD (25 lM; n5 10; *P < 0.05; Fig. 3C; Bal�azs et al., 2013). Moreover,

ivermectin (IVM, 2 lM), an allosteric modulator and endo-

cytosis inhibitor of P2X4 receptor (Khakh et al., 1999;

Toulme et al., 2006), potentiated ATP (30 lM)-induced

inward currents in microglia (n 5 12; *P < 0.05; Fig. 3C).

Treatment with LPS (1 lg mL21, 24 h; n 5 18; Fig. 3A)

FIGURE 1: Increased expression of P2X4 receptors in microglia of EAE rats and of MS patients. (A–B) P2X4 mRNA (A) and protein (B) isincreased in the spinal cord of EAE rats at the peak of neurological disability (14 days after immunization). n 5 6–7 per group; *P <0.05, **P < 0.01. (C) Double immunofluorescence of P2X4 (green) and CD11b (red) in the spinal cord of EAE rats reveal an increasedexpression of P2X4 in perivascular (arrowheads) as well as parenchymal (arrows) microglial cells. Scale bar 5 25 lm (top) and 10 lm(middle and bottom). (D) P2X4 mRNA expression is increased in human optic nerves from MS patients as compared with matched con-trols. Normal appearing optic nerves (NAON), lacking macroscopic plaques, atrophy and/or optic neuritis have a significantly higherincrease in P2X4 mRNA. Controls, n 5 13; Total MS, n 5 13; MS with normal appearing optic nerves (NAON), n 5 6; MS with damagedoptic nerves (DON), n 5 7. *P < 0.05. (E) Representative images showing an increase in the level of P2X4 receptors in CD681 microglia/monocytic cells from human MS spinal cord. Scale bar 5 10 lm.

V�azquez-Villoldo et al.: P2X4 Receptor Activation Kills Microglia

February 2014 175

induced a significant increased in P2X4-mediated inward cur-

rents. In addition, currents in activated microglia cells were

similarly blocked by paroxetine and TNP-ATP (Fig. 3B).

Therefore, the pharmacological profile of ATP responses in

microglia under the conditions examined indicates that they

are mainly mediated by P2X4 receptors, and that microglial

activation leads to enhanced P2X4 receptor function.

Role of P2X4 in Microglial ActivationTo investigate the role of P2X4 receptors in microglial activa-

tion, we stimulated microglial cells with LPS in the presence

or absence of TNP-ATP. LPS treatment induced membrane

ruffling, increased in TNFa release, and morphological

changes associated with microglial activation (Fig. 4A–C). All

these LPS-induced effects were significantly inhibited by the

P2X4 receptor antagonist TNP-ATP (Fig. 4A–C). In contrast,

TNP-ATP treatment did not change the phagocytic ability of

activated microglia which was reduced by the wide spectrum

P2X antagonist PPADS (data not shown), suggesting that

other P2X receptors are responsible for regulating this func-

tion. All this data suggest that ATP release in response to

LPS treatment contributes to the phenotypic responses of

activated microglia through P2X4 receptor activation.

Activated microglia eventually undergo apoptosis by a

process known as activated-induced cell death (Mayo et al.,

2008; Takeuchi et al., 2006; Yun et al., 2011). Because of

that, we next analyzed microglia cell death after activation

with LPS. Using BV-2 cells, we observed that addition of

LPS (24 h) kill BV-2 cells in dose-dependent manner

(Fig. 4D). Cell death was blocked by TNP-ATP (10 lM),

but not in the presence of PPADS (100 lM), antagonist of

P2X1, 2, 3, 5, and 7, or BBG (50 nM), at a concentration

that preferentially blocked P2X7 receptors (Anderson and

Nerdergaard, 2006) (Fig. 4D). This data suggested that LPS-

activated microglia release ATP that induces cell death pre-

dominantly by acting at P2X4 receptor, but not at the cytoly-

tic P2X7 receptor. Accordingly addition of ATP was toxic to

BV-2 cells, an effect inhibited in the presence of TNP-ATP

(10 lM; Fig. 4E). Cell death was similarly induced in pri-

mary microglial cells after 24 h exposure to LPS and signifi-

cantly reduced in the presence of TNP-ATP (Fig. 4F) and

the P2X4 specific antagonist 5-BDBD (25 lM; Fig. 4H).

Previous reports have shown that P2X4 receptors in

neurons activate the inflammasome by promoting caspase-1

cleavage as well as the processing and maturation of IL-1b(de Ribero Vaccari et al., 2012). Caspase-1 activation by the

inflammasome leads to a form of programmed cell death

called pyroptosis, which is present in macrophages and den-

dritic cells (Miao et al., 2011). Therefore, we next analyzed

whether microglia cell death was caused by inflammasome

activation. Microglial activation with LPS showed an increase

in IL-1b production. However, IL-1b production was not

FIGURE 2: Increased expression of P2X4 receptors in microglia activated with LPS. (A) P2X4 receptors in control and LPS-treated (1 lgmL21, 24 h) BV-2 cells. Original pixel intensities from 0 to 255 are represented as a gradient (right). Scale bar 5 50 lm. (B) Western blotanalysis shows an increase in P2X4 expression in microglial cells after LPS stimulation (1 lg mL21, 24 h). n 5 3, *P < 0.05.(C,D) Immuno-staining of P2X receptors in spinal cord sections from control rats. P2X3 (C), P2X4 (D) and P2X7 (C) (green) receptors are located in thesurface of CD11b1 (red)-microglial cells, whereas no expression of P2X1 and P2X2 receptors is detected in microglia. Note the presenceof P2X4 receptors also in spinal cord motor neurons (arrowheads in D). Representative images in D of vehicle (PBS)- and LPS-injectedrats show an increased expression of P2X4 receptors (green) in microglia (red, CD11b) 24 h after LPS injection. Scale bar 5 10 lm.Insets, higher magnification of microglia showing P2X4 receptor as viewed by original pixel intensity shown in A.

176 Volume 62, No. 2

altered in the presence of TNP-ATP (Fig. 4G), excluding any

role of P2X4 receptors in inflammasome signaling. In turn,

LPS (100 ng mL21)-induced microglial cell death was

blocked by inhibiting caspase-1 with YVAD, a protective

effect that added to that of TNP-ATP, suggesting that down-

stream signaling by P2X4 did not converge with caspase-1

activation pathways (Fig. 4H). Together, these data indicates

that P2X4 receptor-mediated toxicity in microglia does not

activate the inflammosome.

P2X4 Receptors Contribute to Microglial Cell DeathIn VivoWe next analyzed the behavior of microglia in vivo 24 h after

injecting LPS (3 mg kg21, i.p.) in the presence or in the

absence of TNP-ATP (10 mg kg21, i.p.). Spinal cord sections

were stained with antibodies to CD11b (OX42), a marker of

microglia and infiltrating monocytes. CD11b1 microglia in

LPS-treated rats showed thicker and shorter processes, a hall-

mark of activated microglia and of CNS inflammation, a fact

which was more evident in grey matter microglia (Fig. 5A,B).

In addition, LPS induced a significant reduction in CD11b1

cells both in white and grey matter (Fig. 5A). Administration

of TNP-ATP completely blocked the retraction of microglial

processes and cell death (Fig. 5A,B). To further assess micro-

glial cell loss, we imaged microglia in vivo by PET using

[11C]PK11195, a radioligand that binds to peripheral benzo-

diazepine receptor PBR/TSPO, a mitochondrial transmem-

brane protein that is mainly expressed in the cerebral

monocytic lineage (microglia and monocyte). We compared

[11C]PK11195 PET signal in control and LPS-injected rats.

LPS induced a reduction in [11C]PK11195 specific binding

in spinal cord and cerebellum whereas no significant change

was detected in total brain (Fig. 5C,D). Moreover, TNP-ATP

completely prevented LPS-induced reduction in

[11C]PK11195 PET signal in the spinal cord (P < 0.05) and

cerebellum (P 5 0.06) (Fig. 5C,D).

To exclude any role of peripheral immune system, we

evaluated microglial cell number in the spinal cord at 2, 6,

and 24 h after intracerebroventricular (icv) LPS injection.

Cells were identified using two markers of microglia, CD11b

FIGURE 3: P2X4 receptor-mediated responses increase in LPS-activated microglia. (A) ATP (100 lM, 1 mM) evokes inward, nondesensi-tizing currents in microglia, which are potentiated in the presence of LPS (1 lg mL21, 24 h). (B) ATP (100 lM)-induced currents areblocked by TNP-ATP (10 lM) and paroxetine (10 lM), two antagonists of P2X4 receptors, in control and LPS-activated microglia. (C)Ivermectin (IVM, 3 lM), an allosteric modulator of microglia, potentiates ATP (30 lM)-induced inward currents whereas 5-BDBD (25 lM)another antagonist of P2X4 receptors, blocks ATP-inward currents.

V�azquez-Villoldo et al.: P2X4 Receptor Activation Kills Microglia

February 2014 177

(OX42) and Iba-1 (ionized calcium-binding adapter molecule

1), which is not expressed in infiltrating monocytes (Ajami

et al., 2011). A reduction of microglial cells (CD11b1,

Iba11) was similarly detected in spinal cord (white and gray

matter) as early as 6 h after icv injection of LPS, an effect

that was increased at 24 h, and P2X4 receptor blockage com-

pletely prevented microglial cell loss (Fig. 6A). Microglial loss

at 24 h was further increased by potentiating P2X4 receptor

function with ivermectin (IVM, 1 mg kg21, i.p.; Fig. 6B)

and inhibited by the P2X4 receptor antagonist 5-BDBD (0.7

mg kg21; i.c.v.; Fig. 6B), thus confirming the role of P2X4

receptors in microglia cell death in vivo.

Surprisingly, when we analyzed the cerebrum after icv

LPS administration, we observed clear region heterogeneity

FIGURE 4: P2X4 receptors regulate LPS-induced microglial activation and cell death. (A–C) P2X4 receptor blockade with TNP-ATP (10lM) prevents LPS-induced microglia ruffling (A, 24 h), attenuates secretion of inflammatory mediators such as TNFa (B, 24 h) and themorphological changes accompanying microglial activation (C; analyzed at 4 h). *P < 0.05 vs. LPS-treated cultures from at least three dif-ferent experiments performed in triplicate. Scale bar 5 20 lm. (D–F) LPS (24 h) induces dose-dependent cell death of BV-2 cells (D) andmicroglia (F). BV-2 cell death is inhibited by TNP-ATP (10 lM), but not by PPADS, antagonist of P2X1, 2, 3, 5, and 7, or BBG (50 nM), aselective blocker of P2X7 at this concentration (D). ATP (1 mM, 24 h) induces BV-2 cell death that is significantly inhibited in the pres-ence of TNP-ATP (E). Microglia cell death is significantly reduced in the presence of TNP-ATP and YVAD, an inhibitor of caspase-1 (F).(G) Mature IL-1b secretion, a measure of inflammasome activation, induced by LPS (24 h) is not modulated by P2X4 blockade. (H) Simul-taneous blocking of P2X4 receptors and caspase-1 with YVAD (50 lM) further reduces LPS-induced microglia cell death currents, indicat-ing that both signaling pathways in microglia are independent. Cell death is also reduced by the P2X4 antagonist 5-BDBD (25 lM).Curves and histograms in D–H represents mean 6 SEM from at least three different experiments performed in triplicate. *P < 0.05.[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

178 Volume 62, No. 2

on the microglial response with the hippocampal dentate

gyrus being most sensitive. Thus, at 2 h after LPS injection,

microglia showed retracted processes in this area, a hallmark of

activated microglia, as quantified by measuring microglia area

and process length (Fig. 7A,B). At 24 h, not at 6 h, microglia

in dentate gyrus appeared dystrophic and showed fragmented

processes, a phenomenon called cytorrhexis and which associ-

ated to aging in human brains (Streit et al., 2004) and to neu-

rodegenerative diseases (Fendrick et al., 2007; Streit et al.,

2009; Xue et al., 2011) (Fig. 8A). Double immunostaining

with Hoechst showed nuclei loss, piknosis and DNA fragmen-

tation in microglia (Fig. 8B), further confirming the degenera-

tion of microglia. In contrast to dentate gyrus, microglia in

CA3 at 24 h showed ameboid and phagocytic morphology

(Fig. 8C). In other regions like cortex, no clear signs of activa-

tion were detected at the earlier stages examined and rare

degenerating microglial cells were observed close to the cortical

surface. The different response of microglia in dentate gyrus

could reflect differences in the vulnerability of cells in this area

and/or differences in LPS exposure. However, we also observed

microglia cell death in the dentate gyrus 24 h after i.p. LPS

injection (Fig. 8D), suggesting that microglia in this region

FIGURE 5: P2X4 receptor blockade prevents microglial loss in the spinal cord after i.p. injection of LPS. (A) Top, Photographs of spi-nal cord sections of vehicle (saline)-, LPS-, and LPS 1 TNP-ATP-injected rats stained for CD11b. Scale bar 5 50 lm. Bottom, Histo-gram illustrates that rats receiving LPS show a significant decrease in the number of CD11b1 cells compared with vehicle-treatedcontrols, and that TNP-ATP administration (i.p.) reverts this effect. Similar results were obtained in three independent experimentsperformed in duplicate. **, ##P < 0.01 vs. control (*) or vs. LPS-treated rats (#). (B) Higher magnification images showing representa-tive microglia morphology in the different experimental conditions. Note the change in cell shape after LPS injection, which isreverted by TNP-ATP treatment. Scale bar 5 20 lm. (C,D) Representative images of [11C]PK11195 PET signal in the spinal cord (C)and cerebrum (D) of vehicle (PBS)-, LPS-, and LPS 1 TNP-ATP-injected rats. PET images were coregistered with an MRI (T2W) rat tem-plate to localize anatomically the PET signal. The percentage of injected dose per cubic centimeter (%ID/cc; mean 6 s.d.) was quanti-fied in ROI areas defined in the cervical spinal cord, in the cerebellum and in total brain. [11C]PK11195 PET signal decreasessignificantly in the spinal cord (C) and cerebellum (D) of LPS-injected rats versus control rats. Administration of TNP-ATP significantlyblocks the LPS-induced decrease in [11C]PK11195 PET signal. n 5 5 per group. *, #P < 0.05 and **, ##P < 0.01 vs. control (*) or vs.LPS-treated rats (#). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

V�azquez-Villoldo et al.: P2X4 Receptor Activation Kills Microglia

February 2014 179

shows higher vulnerability. As described above for the spinal

cord, TNP-ATP treatment prevented microglial activation and

cell death in the hippocampus of icv LPS-injected rats. These

observations are in accordance with the in vitro data and

FIGURE 6: P2X4 receptors control microglial loss in the spinalcord following intracerebroventricular LPS injection. (A–C) Histo-grams showing the time course of microglial loss in the spinalcord after intracerebroventricular injection of LPS. Spinal cord sec-tions of vehicle (saline)- and LPS-injected rats were stained withIba1 and positive cells counted at 2 h (A), 6 h (B), and 24 h (C)after LPS injection. TNP-ATP, administered (i.p.) immediately afterLPS injection, significantly blocks microglial loss induced by LPS.Similar effects were found both in grey and white matter. (D) Iver-mectin, a positive modulator of P2X4 receptor function, signifi-cantly increases microglial loss induced by LPS whereas the P2X4antagonist 5-BDBD has the opposite effect. Values in A–D repre-sents mean 6 SEM from n 5 4 rats per group. *P < 0.05. *, #P <0.05 and **, ##P < 0.01 vs. control (*) or vs. LPS-treated rats (#).

FIGURE 7: P2X4 receptors determine early microglial activationin hippocampal dentate gryus at 2 h after LPS injection (icv). (A)CD11b immunoreactivity in hippocampus in vehicle (saline)-, LPS(icv)-injected rats in the absence or presence of the P2X4 recep-tor antagonist TNP-ATP. Scale bar 5 200 (left) and 50 (right) lm.(B) Reconstruction of microglia morphology in hippocampal den-tate gyrus in two-dimensional image projections of z-stacks con-focal images. Scale bar 5 20 lm. Two parameters were analyzedusing Image J software (see methods): (1) arborization area (areacircumscribed by the polygonal object defined by connecting theouter points of the ramified arbor), (2) Length of the segmentsidentified in a skeletonization of the arbor. Blockage of P2X4receptors with TNP-ATP abolishes microglial process retractioninduced by LPS. **, ##P < 0.01 vs. control (**) or vs. icv LPS-treated rats (##) (n 5 4 rats per group). [Color figure can beviewed in the online issue, which is available atwileyonlinelibrary.com.]

180 Volume 62, No. 2

suggest that LPS activates microglia which subsequently leads

to cell death in vivo as a result of P2X4 receptor stimulation

by ATP released in the brain.

Discussion

Control of microglial cell activation and the number of acti-

vated microglial cells is crucial for the regulation of the

inflammatory response in the CNS. The data reported here

provide evidence that enhanced P2X4 receptor-mediated

activity in microglial cells is associated with brain neuroin-

flammation and, that these receptors are a key determinant in

controlling the initial stages of microglial activation and later

on the induction of microglial cell death.

Microglial P2X4 receptor upregulation has been

described in acute insults including spinal cord injury (Schwab

et al., 2005), trauma (Zhang et al., 2007), and ischemia (Cava-

liere et al., 2003) as well as in chronic neurodegenerative dis-

eases including amyotrophic lateral sclerosis and its animal

model expressing mutant superoxide dismutase 1 (D’Ambrosi

et al., 2009) (for a recent review, see Kettenmann et al., 2011;

Domercq et al., 2013). In addition, a specific microglia

response phenotype characterized by de novo expression of the

purinergic receptor P2X4, the P2X41 state, is critical for the

pathogenesis of pain hypersensitivity caused by injury to

peripheral nerves (reviewed in Begss et al., 2012).

In the current study, we observed an increase in P2X4

receptor expression in microglia and in peri-vascular cells of

rats with acute EAE which is in accordance with previous

observations (Guo and Schluesener, 2005). In addition, we

also found higher levels of P2X4 receptors in human post-

mortem samples of MS patients. However, in contrast to the

EAE model, the increase in P2X4 receptors in MS is not

restricted to microglia/macrophages since it also occurs in

peri-vascular CD682 cells. Although these cells have been not

characterized, they could correspond to lymphocytes since

they express high levels of P2X4 receptors (Woehrle et al.,

2010). Notably, CD681 microglia/monocytic cells in normal-

appearing tissue in MS samples also display higher P2X4

receptor levels which strongly suggest that elevated P2X4

receptor activity is associated with early neuroinflammatory

FIGURE 8.

FIGURE 8: P2X4 receptors control microglia cell death in hippo-campal dentate gyrus (DG) after 24 h of LPS injection (icv). (A)CD11b immunoreactivity in hippocampus in vehicle (PBS)- andLPS (icv)-injected rats in the absence or presence of P2X recep-tor antagonist TNP-ATP. Left, panoramic view of the DG, andright, higher magnification of the DG medial part. Note that athigher magnification microglia appear dystrophic and frag-mented in LPS injected rats, an effect which is prevented byP2X4 receptor blockade with TNP-ATP. Scale bar 5 200 (left)and 25 (right) lm. (B) The 3D-reconstructions of microglia cells indentate gyrus stained with Iba1 antibodies and Hoechst. LPSinduces microglia cell death in vivo as evidenced by the presenceof pyknotic (arrows) and apoptotic nuclei (arrowhead) or nucleiloss. Scale bar 5 20 lm. (C,D) Differential damage to microgliain CA3 and DG regions. Representative images of microgliastained with Cd11b (C) or with Iba1 (D) in CA3 and DG regionsat 24 h after icv (C) or ip (D) LPS injection. Note that CA3 micro-glia show a more active and phagocytic morphology, comparedwith dystrophic microglia in DG (C). Similar differences wereobserved after ip LPS injection (D). n 5 4 rats per group. Scalebar 5 20 lm. [Color figure can be viewed in the online issue,which is available at wileyonlinelibrary.com.]

V�azquez-Villoldo et al.: P2X4 Receptor Activation Kills Microglia

February 2014 181

events in this disorder. Determination of these early molecular

alterations in normal appearing tissue in MS can be useful to

elucidate the mechanisms of lesion formation and to develop

new treatments.

Purinergic receptors control several microglial functions,

including the motility of their fine processes, migration,

cytokine release, and phagocytosis (Domercq et al., 2013;

Kettenmann et al., 2013). Thus, P2X4 receptors control

microglial chemotaxis in conjunction with P2Y12 receptors

(Ohsawa et al., 2007), and stimulating P2X4 receptors ini-

tiates a core signaling pathway that mediates the synthesis

and release of brain-derived neurotrophic factor (Trang et al.,

2009), which produces a disinhibition of nociceptive trans-

mission (Tsuda et al., 2003; reviewed in Beggs et al., 2012).

In turn, our data indicates that P2X4 receptor activity con-

tributes to LPS-induced microglial activation in vitro since

P2X4 antagonists reduce microglia ruffling, TNFa secretion,

morphological changes and cell death. Indeed, the effect of

blocking P2X4 receptors is striking in vivo since it prevents

morphological changes of microglia associated to their activa-

tion and microglia cell death in the spinal cord and hippo-

campus. Differences between microglia in culture and in vivocould be due to differences in P2X4 receptor expression in

microglia from newborn rats versus microglia from adult rats

since purinergic receptors are developmentally regulated

(Xiang and Burnstock, 2005).

Consistent with our findings, microglial cell death sec-

ondary to microglial activation by LPS or tissue injury has

been reported both in vitro and in vivo (Arroyo et al., 2013;

Fricker et al., 2013; Jeong et al., 2010; Ji et al., 2007; Jun

et al., 2011; Min et al., 2012; Shin et al., 2004). However, to

our knowledge the data described in the current study pro-

vides the first evidence that activation of P2X4 receptors, sec-

ondary to ATP released by LPS stimulation, induces

microglia cell death. In contrast to our results, a recent study

in cultured microglia and BV-2 cells demonstrated that pro-

longed activation of P2X4 receptors in microglia by ATP

induces opening of a pore permeable to large molecules that

does not lead to cell death (Bernier et al., 2012). This appa-

rent discrepancy may be due to differences on the age (2- to

4-day old vs. newborn) and species (mice vs. rats) of the ani-

mals used for culturing microglia, and to the time window

examined for monitoring microglia cell death after ATP or

LPS stimulation in vitro (90–180 min vs. 24 h).

We observed that, within the brain, microglia in the

dentate gyrus were particularly vulnerable to LPS stimulation.

Microglia in dentate gyrus are more densely populated, acti-

vated and proliferative because they are located in a neuro-

genic niche and neural progenitor cells have a secretory

protein profile distinct from other brain cells that regulates

microglial activation state (Mosher et al., 2012). Thus, it is

conceivable that this higher level of basal activation renders

microglia more vulnerable to LPS stimulation and the ensu-

ing activation of P2X4 receptors. However, we did not detect

higher P2X4 receptor expression in dentate gyrus microglia

by immunohistochemistry (data not shown) though electro-

physiological recordings will be needed to analyze whether

these cells have enhanced responses to ATP.

ATP homeostasis is compromised in most CNS pathol-

ogies. Immediately after acute CNS injury, damaged cells

release ATP as a “find-me” signal of death to recruit phago-

cytes. The main mechanism of ATP release is the plasma

membrane channel pannexin 1, which opens in most patho-

logical conditions (Domercq et al., 2010; MacVicar and

Thompson, 2010; Pelegrin and Surprenant, 2006; Thompson

et al., 2006; for a review see Domercq et al., 2013). Our

results suggest that microglia releases ATP in response to LPS

stimuli. Indeed, microglia releases ATP after activation with

LPS, leading to an increase in excitatory neurotransmission

(Pascual et al., 2012). However, the expression of pannexins

in microglia and their possible influence on ATP release

under normal conditions and after microglial activation has

not been characterized. Other mechanisms of ATP release in

microglia have been proposed, including ATP release via zinc

uptake by zinc transporters (Higashi et al., 2011) and Ca21-

dependent exocytosis from lysosomes (Dou et al., 2012).

The physiological significance of microglia cell death

remains to be determined. It has been proposed that the

extent and duration of inflammation in the CNS may be

controlled by removal of activated microglia. According to

this hypothesis, microglia proliferates and then undergo apo-

ptosis to achieve tissue homeostasis, as described in chronic

EAE (Ajami et al., 2011). However, microglial cell death

occurs within hours after the insult in the acute neuroinflam-

mation experimental paradigms used in our study (6 and 24

h after ip and icv LPS injection, respectively). Similarly, early

microglial cell death was also observed in the penumbra of

spinal cord lesions where ramified Iba-11 cells (resident

microglia) die earlier (3–6 h) than neurons (12–24 h) (Min

et al., 2012). Moreover, microglia die as early as neurons

within the core region after ATP injection into the substantia

nigra pars compacta, indicating that microglia are as vulnera-

ble to this type of insult as neurons (Jeong et al., 2010). Con-

sequently, early microglia cell death appears to be a direct

consequence of some insults and determines a previously

unnoticed susceptibility of microglia to CNS damage, infec-

tion or altered homeostasis. Early microglia cell death is fol-

lowed by the infiltration and proliferation of circulating

monocytes with stronger phagocytic activity that could con-

tribute to resolve and repair the damage tissue by removing

apoptotic cells and facilitating angiogenesis (Ji et al., 2007;

Jeong et al., 2010).

182 Volume 62, No. 2

Classically, microglia are considered as the immune cells

of the brain and key players in brain injury and disease.

However, in the last decade our view of microglia has dramat-

ically changed after discovering that resting microglia are

extremely dynamic in vivo, perpetually changing their mor-

phology by extending and retracting highly motile processes,

a mechanism that allows microglia to regulate important

functions such as postnatal development, adult neuronal

plasticity, and synaptic excitability (Kettenmann et al., 2013;

Li et al., 2012; Tremblay et al., 2011). Therefore, primary

microglial loss at the initial stages of disease may contribute

to impairing brain function. This may indeed be the case of

microglial apoptosis in perilesional white matter in X-linked

adrenoleukodistrophy (Eichler et al., 2008) as well as of dys-

trophic (fragmented) microglia associated with tau pathology

which precedes the spread of tau pathology and the neuro-

degeneration in Alzheimer’s disease (Streit et al., 2009; Xue

and Streit, 2011). The findings reported here illustrating

that P2X4 receptors mediate early microglial cell death dur-

ing neuroinflammation provide new avenues to control the

fate of activated microglia, and possibly to manipulate

microgliosis towards a beneficial phenotype in CNS injury

and disease.

Acknowledgment

Grant sponsor: CIBERNED; the Spanish Ministry of Educa-

tion and Science; Eranet-Neuron (Nanostroke); the Education

Department and the Industry Department of the Basque

Government.

We would like to thank to M. Gonzalez, A. Leukona and

M. Errasti for technical support in the radiosynthesis, and to

A. Cano and B. Szczupak for technical assistance in the the

PET studies.

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