Ca2+/calmodulin-dependent kinase II signalling cascade mediates P2X7 receptor-dependent inhibition...

Ca2+/calmodulin-dependent kinase II signalling cascademediates P2X7 receptor-dependent inhibition ofneuritogenesis in neuroblastoma cellsRosa Gomez-Villafuertes1, Ana del Puerto2,3,4, Miguel Dıaz-Hernandez1,4, Diego Bustillo5,Juan I. Dıaz-Hernandez1, Paula G. Huerta1, Antonio R. Artalejo5, Juan J. Garrido2,3,4

and Ma Teresa Miras-Portugal1

1 Departamento de Bioquımica y Biologıa Molecular, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain

2 Centro de Biologıa Molecular ‘Severo Ochoa’, CSIC-UAM, Madrid, Spain

3 Departamento de Neurobiologıa Celular Molecular y del Desarrollo, Instituto Cajal, CSIC, Madrid, Spain

4 Centro de Investigacion Biomedica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain

5 Departamento de Toxicologıa y Farmacologıa, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain

Keywords

brilliant blue G; Ca2+ ⁄ calmodulin-dependent

kinase II; neuritogenesis; neuroblastoma;

P2X7 receptor

Correspondence

M. T. Miras-Portugal, Departamento de

Bioquımica y Biologıa Molecular IV, Facultad

de Veterinaria, Universidad Complutense de

Madrid, Av. Puerta de Hierro s ⁄ n, 28040

Madrid, Spain

Fax: +34 91 3943909

Tel: +34 91 3943894

E-mail: [email protected]

(Received 13 May 2009, revised 25 June

2009, accepted 22 July 2009)

doi:10.1111/j.1742-4658.2009.07228.x

ATP, via purinergic P2X receptors, acts as a neurotransmitter and modula-

tor in both the central and peripheral nervous systems, and is also involved

in many biological processes, including cell proliferation, differentiation

and apoptosis. Previously, we have reported that P2X7 receptor inhibition

promotes axonal growth and branching in cultured hippocampal neurons.

In this article, we demonstrate that the P2X7 receptor negatively regulates

neurite formation in mouse Neuro-2a neuroblastoma cells through a

Ca2+ ⁄ calmodulin-dependent kinase II-related mechanism. Using both

molecular and immunocytochemical techniques, we characterized the pres-

ence of endogenous P2X1, P2X3, P2X4 and P2X7 subunits in these cells.

Of these, the P2X7 receptor was the only functional receptor, as its activa-

tion induced intracellular calcium increments similar to those observed in

primary neuronal cultures, exhibiting pharmacological properties character-

istic of homomeric P2X7 receptors. Patch-clamp experiments were also

conducted to fully demonstrate that ionotropic P2X7 receptors mediate

nonselective cation currents in this cell line. Pharmacological inhibition of

the P2X7 receptor and its knockdown by small hairpin RNA interference

resulted in increased neuritogenesis in cells cultured in low serum-contain-

ing medium, whereas P2X7 overexpression significantly reduced the forma-

tion of neurites. Interestingly, P2X7 receptor inhibition also modified the

phosphorylation state of focal adhesion kinase, Akt and glycogen synthase

kinase 3, protein kinases that participate in the Ca2+ ⁄ calmodulin-depen-

dent kinase II signalling cascade and that have been related to neuronal

differentiation and axonal growth. Taken together, our results provide the

first mechanistic insight into P2X7 receptor-triggered signalling pathways

that regulate neurite formation in neuroblastoma cells.

Abbreviations

[Ca2+]i, intracellular free calcium concentration; BBG, brilliant blue G; BzATP, 2¢,3¢-O-(4-benzoyl)-benzoyl ATP; CaMKII, Ca2+ ⁄ calmodulin-

dependent kinase II; CNS, central nervous system; DiBucAMP, dibutyryl-cAMP; FAK, focal adhesion kinase; Fura-2 AM, fura-2

acetoxymethyl ester; GFP, green fluorescent protein; GSK3, glycogen synthase kinase 3; N2a, Neuro-2a; PFA, paraformaldehyde, PI3K,

phosphatidylinositol 3-kinase; shRNA, small hairpin RNA; a,b-meATP, a,b-methylene-ATP.

FEBS Journal 276 (2009) 5307–5325 ª 2009 The Authors Journal compilation ª 2009 FEBS 5307

Introduction

Purinergic signal transduction mechanisms constitute a

complex intra- and intercellular signalling network that

plays an important regulatory role in both the central

(CNS) and peripheral nervous systems. Nucleotides

exert their extracellular effects by acting on specific P2

receptors, which are subdivided into ionotropic P2X

and metabotropic P2Y subtypes [1]. To date, seven

mammalian P2X receptor subtypes (P2X1–7) and eight

mammalian P2Y receptor subtypes (P2Y1, 2, 4, 6, 11–

14) have been cloned and functionally characterized

[2–5].

P2X receptors are nonselective cation channels

formed by the association of three P2X subunits [6].

Each P2X subunit consists of two transmembrane-

spanning segments separated by an N-glycosylated

extracellular loop containing 10 conserved cysteine res-

idues [7,8]. The N- and C-termini are intracellular. The

distribution and relative abundance of P2X receptors

have been studied in different brain areas and cell

types [9]. P2X receptor activation results in Na+ and

Ca2+influx across the cell membrane, which leads to

depolarization of the plasma membrane that can, in

turn, activate voltage-gated channels. Using phar-

macological tools and gene knockout ⁄knockdownapproaches, it has become clear that P2X receptors

are involved in a wide and growing range of physio-

logical processes [10]. In the CNS, ATP directly medi-

ates fast excitatory synaptic transmission, acting via

P2X receptors in mammalian neurons [11–13]. Fur-

thermore, presynaptic P2X receptors can modulate

neurotransmitter release: ATP enhances glutamate

release in the hippocampus [14,15], spinal cord [16],

and midbrain [17]; 4-aminobutyrate release is facili-

tated by P2X activation in cultured spinal cord dorsal

horn neurons [18], midbrain synaptosomes [19] and

lateral hypothalamic neurons [20]; and glycine release

is enhanced by ATP from interneurons in the dor-

sal horn region [21], trigeminal nucleus pars caudalis

neurons [22] and spinal cord substantia gelatinosa

neurons [23].

Interestingly, purinergic signalling is already present

at the early stages of embryogenesis, being involved in

cell proliferation, migration and the differentiation of

a wide variety of structures [24–26]. Moreover, ATP

promotes cell proliferation, acting through P2X3 and

P2X4 receptors in murine embryonic stem cells [27],

and participates in the neuronal maturation of P19

mouse embryonic carcinoma cells via P2X2 and P2X6

receptors, suggesting the presence of purinergic signal-

ling in the initiation and direction of cell differentia-

tion [28]. A trophic effect of purines in regeneration

and neurite outgrowth has also been reported in vari-

ous cell systems, such as PC12 phaeochromocytoma

cells [29,30], retinal ganglion cells [31] and striatal neu-

rons [32]. P2X receptors also participate in the forma-

tion of neuronal networks during hippocampal

development [33,34]. Recently, our group has reported

that the inhibition of P2X7 receptors promotes axonal

growth and branching in cultured hippocampal neu-

rons [35]. Furthermore, ATP, acting via P2X7 recep-

tors, sustains the growth of human neuroblastoma

cells through a substance P-dependent mechanism [36],

whereas a functional decrease in P2X7 receptors seems

to be associated with retinoic acid-induced differentia-

tion of neuroblastoma cells [37]. However, little is

known about the signalling pathways that lead to the

differentiation of neuroblastoma cells following P2X7

receptor inhibition.

In this study, the neurotrophic effect of P2X7 recep-

tors was further examined in mouse Neuro-2a (N2a)

neuroblastoma cells. Pharmacological inhibition and

interference with P2X7 receptor expression were asso-

ciated with neuritogenesis in N2a cells cultured in low

serum-containing medium, whereas P2X7 overexpres-

sion significantly reduced neurite formation. Moreover,

we linked the activation of the P2X7 receptor with the

regulation of Ca2+ ⁄ calmodulin-dependent kinase II

(CaMKII) activity and some of its downstream effec-

tors, including focal adhesion kinase (FAK), phospha-

tidylinositol 3-kinase (PI3K), Akt and glycogen

synthase kinase 3 (GSK3). Together, our results

support a role of P2X7 receptors in the regulation of

neuritogenesis and in the fine control of the balance

between proliferation ⁄differentiation of neuroblastoma

cells.

Results

Molecular characterization of purinergic P2X

receptors in N2a cells

In order to investigate the presence of native puriner-

gic receptors in N2a cells, the expression of mouse

P2X subunits was analysed at both the transcriptional

and protein levels. RT-PCR experiments demonstrated

that most P2X subunits are expressed in N2a cells

(Fig. 1A). To characterize the relative amount of each

P2X transcript, quantitative real-time PCR assays

were performed. Four main P2X mRNAs coding for

P2X1, P2X3, P2X4 and P2X7 subunits were found in

this cell line, with P2X1 and P2X4 transcripts being

the most abundant. The expression of P2X2, P2X5

P2X7 negatively controls neuritogenesis via CaMKII R. Gomez-Villafuertes et al.

5308 FEBS Journal 276 (2009) 5307–5325 ª 2009 The Authors Journal compilation ª 2009 FEBS

and P2X6 was practically undetectable (Fig. 1B). Wes-

tern blot analyses were performed on total protein

extracts obtained from N2a cells using commercial

subunit-specific antibodies. Specific neutralizing pep-

tides were employed to guarantee the specificity of

these bands (data not shown). As expected, the expres-

sion of P2X proteins correlated well with the results

obtained by quantitative PCR (Fig. 1C). Thus, bands

corresponding to monomeric P2X1, P2X3, P2X4,

P2X5 and P2X7 proteins were immunodetected, the

strongest corresponding to P2X3 and P2X7 subunits.

A high level of heterogeneity in the size of P2X su-

bunits has been reported in the literature, probably

caused by differences in glycosylation and ⁄or oligo-

merization. The predicted sizes for unglycosylated

P2X subunits have been reported to range between 43

and 68 kDa, whereas the molecular masses of glycosy-

lated forms are between 50 and 79 kDa (reviewed in

[3]). In this study, the bands obtained for P2X1

(59 kDa), P2X3 (64 kDa), P2X5 (63 kDa) and P2X7

(78 kDa) subunits are consistent with the molecular

masses reported for the glycosylated monomers [6,38–

40]. The low-molecular-mass bands observed for P2X1

(48 kDa) and P2X4 (42 kDa) subunits probably repre-

sent unglycosylated proteins [6,41]. A band detected at

45 kDa with the P2X7 antibody may correspond to a

truncated splice variant of the P2X7 receptor, as

described in humans [42]. High-molecular-mass oligo-

meric forms (dimers or trimers) of P2X subunits were

not observed.

Pharmacological characterization of P2X7


The pharmacological characterization of P2X receptors

was carried out by analysing the change in intracellular

free calcium concentration ([Ca2+]i) elicited by a vari-

ety of purinergic compounds, in combination with spe-

cific P2X7 antagonists, in N2a cells loaded with the

calcium indicator fura-2 acetoxymethyl ester (Fura-2

AM). First, we tested the ability of several purinergic

agonists, including a,b-methylene-ATP (a,b-meATP),

CTP, 2¢,3¢-O-(4-benzoyl)-benzoyl ATP (BzATP) and

ATP, to increase [Ca2+]i in N2a cells (Fig. 2A). Nei-

ther a,b-meATP (100 lm) nor CTP (300 lm) modified

[Ca2+]i, whereas BzATP (100 lm), which is known to

induce Ca2+influx in cells expressing P2X7 receptors,

elicited a discrete [Ca2+]i increase in the presence of

Mg2+ ions in the superfusion medium. Last, ATP

(100 lm) triggered a substantial calcium response in

N2a cells, even in the absence of Ca2+ in the extracel-

lular medium (data not shown), indicating the partici-

pation of functional metabotropic P2Y receptors in

this cell line, as reported previously [43].

Decreases in extracellular Ca2+ and Mg2+ concen-

tration are known to modulate P2X7 receptor-medi-

ated responses [44–46]. Based on these findings, N2a

cells were challenged with BzATP in Locke’s solution

with or without 1.2 mm Mg2+. As shown in Fig. 2B,

the potency of BzATP increased by two-fold when an

Mg2+-free medium was employed. Moreover, BzATP

P2X1

P2X2

P2X3

P2X4

P2X5

P2X6

P2X7

0

20

40

60

80

No

rmal

ized

to

β-a

ctin

(R

.U.)

M H2O H2O H2O H2ON B

P2X1 P2X2 P2X5P2X3 P2X4 P2X7 P2X6 β-Actin

N B N B N B M H2O H2O H2O H2ON B N B N B N B

75 kDa

50 kDa

35 kDa

P2X

1P

2X2

P2X

3P

2X4

P2X

5P

2X6

P2X

7

α-tubulin

A

B C

Fig. 1. Several P2X receptors are simultaneously expressed in nondifferentiated murine N2a neuroblastoma cells. (A) RT-PCR expression of

P2X receptors. The bands for P2X1–7 receptors were around 100 bp and were amplified from both N2a cells (N) and adult whole mouse

brain mRNA extracts (B). No amplification products were observed in parallel assays carried out without template (H2O). M, DirectLoad�Wide Range DNA marker. (B) Quantitative real-time PCR showing the expression levels of P2X subunits in N2a cells. Values were normal-

ized by the content of b-actin transcript. (C) Total protein isolated from N2a cells was subjected to SDS-PAGE, transferred onto poly(vinyli-

dene difluoride) membrane and incubated with specific antibodies against P2X1–7 subunits. Immunodetection of a-tubulin was used as a

loading control.

R. Gomez-Villafuertes et al. P2X7 negatively controls neuritogenesis via CaMKII


dose–response curves evaluated in cellular populations

using Locke’s solution with or without Mg2+ showed

significantly different EC50 values (P < 0.01):

114.02 ± 9.55 lm in the presence of Mg2+ and

68.23 ± 5.714 lm in its absence. No differences were

found in the maximal effect or in the Hill coefficients,

the latter being 2.625 ± 0.596 in the presence of

Mg2+ and 3.737 ± 1.089 in the absence of the cation

(Fig. 2C). It should be noted that the BzATP-induced

[Ca2+]i increase was abolished in Ca2+-free medium

(Locke’s solution supplemented with a 12 mm Tri-

s ⁄EGTA mixture) in most cells analysed, indicating the

ionotropic nature of the response.

We further characterized the presence of functional

P2X7 receptors by testing the effect of the P2X7 antag-

onists KN62, brilliant blue G (BBG) and A438079 on

the calcium response elicited by BzATP in Mg2+-free

conditions. These three compounds have been reported

to inhibit the purinergic-induced increase in [Ca2+]ithrough P2X7 receptors without producing any effect

on their own [47–49]. Administered 5 min before and

during BzATP application, KN62 (50 nm), BBG

(1 lm) and A438079 (1 lm) inhibited by 66.2 ± 5.9%,

75.7 ± 2.3% and 71.7 ± 13.0%, respectively, the

increase in [Ca2+]i elicited by 100 lm BzATP

(Fig. 3A–C). Interestingly, both KN62 and BBG

behaved as slowly reversible antagonists, as the

response to BzATP remained inhibited even with a 25–

30 min washout (Fig. 3D,E). In contrast, the inhibi-

tory effect of A438079 was rapidly reversed after a

5 min washout (Fig. 3F).

Biophysical identification of P2X7 receptors in

N2a cells

The above results, based on fluorescence measurements

of the intracellular calcium changes evoked by puriner-

gic agonists, as well as their modulation by P2X7

receptor-selective antagonists, suggest that N2a cells

express functional P2X receptors most probably

belonging to the P2X7 subtype. To obtain direct evi-

dence supporting this conclusion, we conducted patch-

clamp experiments to detect the ion flux mediated by

this type of ionotropic receptor. In these experiments,

we employed cells seeded at a low density (25 000 cell-

sÆcm)2) and systematically chose cells isolated from

neighbours and devoid of cytoplasmic processes.

Ligand-gated ionic currents were activated by challeng-

ing cells bathed in Mg2+-free saline with BzATP

(100 lm), ATP (100 lm) or a,b-meATP (100 lm).

Agonist application was consistently associated with

the appearance of inwardly directed and nondesensitiz-

ing currents that differed markedly in their respective

0.5

1.0

1.5

2.0

100 s

α, βmeATP CTPF

340/

F38

0F

340/

F38

0BzATP ATP

100 s

0.5

1.0

1.5

2.0

2.5BzATP BzATP BzATP

Mg2+ free EGTA

–6 –5 –4 –30

20

40

60

80

100With Mg2+

Without Mg2+

Log [BzATP]

Δ In

trac

ellu

lar

Ca2+

(% o

f m

axim

al r

esp

on

se)

A

B

C

Fig. 2. BzATP induces intracellular calcium transients in nondifferen-

tiated N2a cells that are potentiated in the absence of Mg2+ ions. (A)

Intracellular calcium increments evoked by different purinergic agon-

ists in N2a cells. Cells were challenged with maximal concentrations

of the agonists a,b-meATP (100 lM), CTP (300 lM), BzATP (100 lM)

and ATP (100 lM), and the calcium transients were calculated. Hori-

zontal bars indicate stimulation periods. Trace represents

mean ± standard error of the mean (SEM) from 157 individual cells.

(B) Intracellular calcium increments elicited by BzATP (100 lM) in

N2a cells are potentiated in the absence of extracellular Mg2+ ions

and require the presence of extracellular calcium in the superfusion

medium. Horizontal bars indicate stimulation periods. Both Mg2+

washout and Tris ⁄ EGTA addition were performed 5 min before and

during BzATP application. Trace represents mean ± SEM from 108

individual cells. (C) Concentration–response curves for intracellular

calcium mobilization induced by BzATP in N2a cell suspensions in

the presence or absence of extracellular Mg2+ ions. Cell suspensions

were stimulated with graded doses of BzATP ranging from 5 lM to

1 mM prepared in Locke’s buffer or in Mg2+ -free medium. Results

were normalized with respect to the maximal response elicited by

BzATP, and the logistic curve was fitted by nonlinear regression.

Data are the mean ± SEM of at least five different experiments.



amplitudes (Fig. 4A). Thus, peak currents evoked by

BzATP were of 162.8 ± 53.19 pA (n = 25 cells)

which, considering an average membrane capacitance

of 11.62 ± 1.09 pF (n = 27 cells), implies a mean cur-

rent density of about 14 pAÆpF)1. The currents

induced by ATP or a,b-meATP were much smaller

than those elicited by BzATP (10% and 13% smaller,

respectively), thus pointing to P2X7 receptors as the

main ionotropic purinergic receptor subtype function-

ally expressed in N2a cells. Current–voltage relation-

ships for 100 lm BzATP-activated responses were

approximately linear between )90 and +50 mV

(Fig. 4B) and reversed at )3.94 mV (value corrected

for the liquid junction potential; n = 2 cells). The

involvement of P2X7 receptors in BzATP-induced cur-

rents was confirmed by assaying their sensitivity to

extracellular Mg2+, as well as to BBG (1 lm) and

A438079 (1 lm). In cells exposed for 2 min to a bath

solution containing 2 mm Mg2+, a rapid and fully

reversible inhibition (� 58%; n = 6 cells) of current

responses to BzATP was observed (Fig. 4C). Likewise,

following a similar preincubation time, both BBG and

A438079 considerably reduced BzATP-evoked currents

(� 80% and � 62%; n = 7 and n = 4 cells, respec-

Fig. 3. P2X7 antagonists reduce intracellular

calcium transients elicited by BzATP in non-

differentiated N2a cells. N2a cells were

stimulated twice with 100 lM BzATP and

subsequently exposed to 50 nM KN62 (A),

1 lM BBG (B) or 1 lM A438079 (C) for

5 min before and during 100 lM BzATP

stimulation. The recovery of BzATP-induced

calcium responses was then followed for

15–30 min, depending on the antagonist

assayed. Traces represent mean ± standard

error of the mean from at least 150 individ-

ual cells. The inhibition of BzATP responses

by KN62 (D), BBG (E) and A438079 (F) is

expressed as a percentage of the average

response to the initial two BzATP stimuli

(Control). Recovery was evaluated at the

indicated times (Wash). **P < 0.01;

***P < 0.001.



tively) (Fig. 4D). As already noted in microfluorimetric

experiments, the effect of BBG was reversed only in

part after a 5 min washout. These results corroborate

that ionotropic P2X7 receptors mediating nonselective

cation currents are expressed in N2a cells.

Pharmacological block of P2X7 receptors

facilitates N2a cell differentiation and the

formation of neurites

Previous studies have reported that P2X7 receptor

activation induces the proliferation of several human

neuroblastoma cell lines, as well as primary neuro-

blastoma tumour cells [36]. Interestingly, P2X7 recep-

tor inhibition promotes axonal growth and branching

in cultured hippocampal neurons [35]. Based on these

findings, we set out to explore the effect of BBG

on the morphological features of N2a cells. Thus,

N2a cells were cultured in 0.5% fetal bovine serum-

containing medium for 72 h in the presence or

absence of BBG (100 nm or 1 lm). As a positive

control, cells were treated for the same time period

with dibutyryl-cAMP (DiBucAMP), a well-established

inductor of N2a differentiation that produces the

outgrowth of long bipolar or monopolar axon-like

processes [50]. Immunocytochemistry was conducted

0

10

20

30

40

0 Mg2+ 2 Mg2+

**

I BzA

TP

(pA

)

–40

–30

–20

–10

10

20

30

–100 –80 –60 –40 –20 20 40 60

mV

pA

BzATP

20 p

A

1 s

BzATP

I BzA

TP

(pA

)Control BBG Wash

0

25

50

75

**

I BzA

TP

(pA

)

*

Control A438079 Wash0

25

50

75

0

100

200

300

400

I (p

A)

BzATP ATP

**

Control

Wash

+ A438079

Control

Wash

+ BBG

50 p

A

2 s

BzATP

ATP

BzATP (0 Mg2+)20 p

A

2 s

α, β-meA

BA

C D

TP

BzATP

20 p

A

2 s

BzATP (2 Mg2+)

0

10

20

30

BzATP

*

I (p

A)

α, β-meATP

Fig. 4. Biophysical identification of P2X7 receptors in N2a cells. (A) Effect of BzATP (100 lM), ATP (100 lM) or a,b-meATP (100 lM) on

whole-cell currents from N2a cells. Top panels: current responses to BzATP or ATP (left part) and to BzATP or a,b-meATP (right part) applied

at 5 min intervals. Note that a,b-meATP was applied between two pulses of BzATP. Bottom panels: peak current amplitudes evoked by

paired applications of BzATP and ATP (left graph) or BzATP and a,b-meATP (right graph) in four and three cells, respectively. (B) Current–volt-

age relationship for BzATP current responses. (C) Effect of extracellular Mg2+ on current responses to BzATP (100 lM). Top panel: current

responses to BzATP in the absence (0 Mg2+) and presence (2 mM Mg2+) of extracellular Mg2+. Bottom panel: peak current amplitudes

evoked by successive applications of BzATP in the absence and presence of extracellular Mg2+ in six cells. (D) Effect of A438079 (1 lM) or

BBG (1 lM) on current responses to BzATP (100 lM). Top panels: current responses to BzATP in the absence (Control; Wash) and presence

of A438079 (+A438079) (left part) or BBG (+BBG) (right part). BzATP was applied at 5 min intervals and A438079 or BBG were superfused

2 min before and during the second BzATP application. Bottom panel: peak current amplitudes evoked by successive applications of BzATP

in the absence and presence of A438079 (four cells) or BBG (seven cells). In the top panels of (A), (C) and (D), drugs were administered

during the time indicated by horizontal bars. Broken lines denote the zero current level. Vh = )70 mV. *P < 0.05; **P < 0.01.



in paraformaldehyde (PFA)-fixed cells incubated for

1 h with antibodies raised against P2X7 receptor and

a-tubulin. For morphological quantification, only

processes equal to or greater than one cell diameter

in length were considered as neurites. DiBucAMP

(1 mm) produced a significant increase in both the

average neurite length and number of neurites per cell

compared with control untreated cells (Fig. 5A,B). In

contrast, cells exposed to either 100 nm or 1 lm BBG

showed more neurites per cell, although the average

length of the neurites was not significantly different

from that observed in control cells (Fig. 5A,C,D). It

should be noted that some DiBucAMP- and BBG-

treated cells developed axon-like processes ending in

growth cone-like structures that showed high levels

of P2X7-positive immunostaining, suggesting the

implication of P2X7 receptors in the control of

neuritogenesis (Fig. 5G,H).

Knockdown of the P2X7 receptor induces

neurite formation and P2X7 overexpression

blocks neuritogenesis in N2a cells

In order to corroborate the effect of P2X7 receptor

antagonism in N2a cell differentiation, a pSU-

PER.neo.GFP vector-derived small hairpin RNA

(shRNA) strategy was designed to knock down native

P2X7 receptor expression. Although the usefulness of

Control

BBG 1 μμM

α -Tub

0

10

20

30

40

* ( h

t g

n

e l e t i r u

e N

μ

) m

Control

DiBucA

MP

BBG 1 μM

BBG 100 n

M

Control

DiBucA

MP

BBG 1 μM

BBG 100 n

M

0

1

2

3

4

*** ***

***

l l e c / s e t i r u

e n

f o

r e

b

m

u

N

α -Tub

DiBucAMP

BBG 100 nM

α -Tub

α -Tub

A B E

F

C D

G H

Fig. 5. The P2X7 receptor antagonist BBG promotes neurite formation in N2a cells. N2a cells were cultured in 0.5% fetal bovine serum-con-

taining medium for 72 h in the absence (A) or presence of 1 mM DiBucAMP (B), 1 lM BBG (C) or 100 nM BBG (D). Afterwards, cells were

fixed and immunostained with a-tubulin antibody (green). Scale bar, 50 lm. Bar graphs show quantitatively the neurite length (E) and the

number of neurites per cell (F) found in N2a cells cultured under the four different conditions. Data are the mean ± standard error of the

mean of 60 cells from three different experiments. *P < 0.05; ***P < 0.001. (G) DiBucAMP-treated N2a cells stained with anti-P2X7 recep-

tor serum (red) determine subcellular P2X7 receptor location. Scale bar, 20 lm. (H) Detail of a distal region of a neurite with a growth cone-

like structure, where high levels of P2X7 receptor-positive immunostaining can be observed. Scale bar, 5 lm. Cellular morphology was

defined using a-tubulin antibody (green) and nuclei were labelled with DAPI (blue).



this shRNA has been demonstrated previously in the

HEK293T cell line [35], we confirmed its effectiveness

in the N2a cell line, which expresses native P2X7. N2a

cells were transfected with either a pcDNA3-P2X7-

GFP expression vector or with a vector carrying the

interference RNA for P2X7 (hereafter termed shRNA-

P2X7). Control cells were transfected with nonspecific

shRNA-luciferase vector (shRNA-Luc). P2X7 expres-

sion increased by two-fold in cells transfected with

P2X7-GFP, whereas the expression of native P2X7

was specifically reduced by 82% in cells transfected

with shRNA-P2X7 (Fig. 6A). Once the efficiency and

Control

DiBucAMP

shRNA-P2X7

GFP

GFP

P2X7-GFP

0

10

20

30

Neu

rite

len

gth

(μm

)

P2X7-

GFP

DiBucA

MP

Control

A N

R h

s

7 X 2 P

-

P2X7-

GFP

DiBucA

MP

Control

A N

R h

s

7 X 2 P

-

0

1

2

3

*

*

**

Nu

mb

er o

f n

euri

tes/

cell

GFP

GFP

*

P2X7

α -tubulin /7

X2P

α).

U.R(

nilubut-

c u L - A

N R

h s

P F

G - 7 X

2 P

7 x 2 P - A

N R

h s

c u L - A

N R

h s

P F

G - 7 X

2 P

7 X 2 P

- A N

R h

s

0.0

0.5

1.0

1.5

2.0 **

*

A

B C

D

Fig. 6. P2X7 receptor regulates the differen-

tiation of N2a cells. (A) Western blotting of

N2a cells transfected with either pcDNA-

P2X7-GFP or shRNA-P2X7. The levels of

a-tubulin were used as a loading control and

the P2X7 ⁄ a-tubulin ratio was used to esti-

mate the efficiency of both vectors (n = 3;

*P < 0.05; **P < 0.01). Control cells were

transfected with nonspecific shRNA-Luc

vector. (B) Representative GFP fluorescence

images of N2a cells cultured in 0.5% fetal

bovine serum-containing medium transfect-

ed with empty GFP vector, P2X7-GFP or

shRNA-P2X7. As a positive control, cells

transfected with empty GFP vector were

treated with 1 mM DiBucAMP. After 48 h in

culture, cells were fixed and GFP-positive

cells were observed in order to study cellu-

lar morphology and differentiation degree.

Scale bar, 50 lm. Bar graphs summarize

the results on neurite length (C) and number

of neurites per cell (D) from transfected

cells. Data are the mean ± standard error of

the mean of 60 cells from three different

experiments. *P < 0.05; **P < 0.01.



specificity of both vectors had been confirmed, N2a

cells cultured in 0.5% fetal bovine serum-containing

medium were transfected with either P2X7-GFP or

shRNA-P2X7 and, 48 h later, were PFA-fixed and

analysed. Control cells were transfected with empty

green fluorescent protein (GFP) vector and, as a posi-

tive control, cells were transfected with empty GFP

vector and treated with 1 mm DiBucAMP for 48 h.

N2a cells that overexpressed the P2X7 receptor showed

a rounded morphology and were practically devoid of

visible neurites, hence indicating that P2X7 receptor

activation is involved in the maintenance of N2a cells

in a nondifferentiated state (Fig. 6B–D). As occurred

after BBG treatment, P2X7 receptor knockdown

promoted neurite formation in GFP-positive cells,

although, once again, differences in neurite average

length were only found in DiBucAMP-treated cells

(Fig. 6B–D). Interestingly, the silencing of P2X7 recep-

tors produced alterations in N2a cell morphology,

showing a broad increase in the number of neurites

that displayed lamellipodia-like morphology.

CaMKII-dependent signalling is modulated by

P2X7 receptors in N2a cells

Intracellular calcium is a critical regulator of neuronal

differentiation, neurite outgrowth and synaptic plastic-

ity (for a review, see [51]). It has been reported that

Ca2+ influx and the resulting transient elevation of

[Ca2+]i in growth cones decrease the rate of axon out-

growth, whereas the suppression of Ca2+ transients

accelerates axon extension [52]. CaMKII is a calcium-

dependent serine ⁄ threonine kinase that also possesses

the ability to become calcium independent through

autophosphorylation [53]. Moreover, once it is acti-

vated, it remains functional for at least several hours

[54]. Previously, we have reported that P2X7 receptor

activation induces CaMKII phosphorylation in cere-

bellar granule neurons [55], whereas P2X7 receptor

inhibition reduces its phosphorylation in cultured hip-

pocampal neurons [35]. As CaMKII and its down-

stream targets could be good effectors in N2a cell

differentiation induced by P2X7 antagonists, we analy-

sed the phosphorylation state of CaMKII in N2a cells

exposed to 100 nm BBG. This antagonist produced a

significant decrease in the ratio between phospho-

CaMKII (active form of the enzyme) and total CaM-

KII after 60 min of treatment (Fig. 7A–D), suggesting

that CaMKII participates in the negative regulation of

neuritogenesis exerted by P2X7 receptors. To corro-

borate this hypothesis, N2a cells were cultured in 0.5%

fetal bovine serum-containing medium for 72 h in the

presence or absence of 1 lm KN93, a well-known

CaMKII inhibitor. As positive controls, cells were

treated for the same time period with either 1 mm

DiBucAMP or 1 lm BBG. Immunocytochemistry was

performed in PFA-fixed cells incubated for 1 h with

a-tubulin antibodies. As reported previously, both

DiBucAMP and BBG produced a significant increase

in the number of neurites per cell compared with

untreated cells. As expected, cells exposed to KN93

also developed more neurites than control cells, point-

ing to a key role of this kinase in the control of

neuritogenesis in N2a cells (Fig. 5E).

Involvement of FAK in BBG-mediated neurite

formation in N2a cells

As shown previously, silencing of P2X7 receptors with

shRNAs produces a significant increase in the number

of neurites exhibiting a lamellipodia-like morphology.

This phenomenon may be explained by alterations in

the actin cytoskeleton, as reported previously in hippo-

campal neurons [35]. FAK is an actin-interacting pro-

tein that regulates focal adhesion contact formation

and lamellipodia stability [56]. It has been reported

that phospho-CaMKIIa is able to phosphorylate FAK

at Ser843 (pFAKS846), which results in a decrease in

FAK activity. In contrast, when the Ser843 residue is

dephosphorylated, FAK is autophosphorylated at

Tyr397 (pFAKT397) and becomes active [57]. In this

line, we examined whether the relative amount of

active pFAKT397 was modified in N2a cells following

BBG treatment. In agreement with a decrease in

CaMKII activity, Tyr397 phosphorylation of FAK

was significantly higher in N2a cells exposed to BBG,

indicating that P2X7 receptor inhibition produces an

increase in the activity of FAK (Fig. 7F,G).

PI3K-dependent signalling is modulated by P2X7


Previous studies have reported that one of the down-

stream targets of FAK is PI3K [58,59]. It is generally

accepted that PI3K plays a central role in cell signal-

ling, mediating neurite extension [60,61], cell prolifera-

tion, survival and motility [62,63]. Based on these

findings, we studied whether PI3K could participate in

the signalling cascade regulated by P2X7 receptors.

N2a cells cultured in 0.5% fetal bovine serum-contain-

ing medium were treated with 1 lm BBG for 72 h in

the presence or absence of the PI3K inhibitor

LY-294002 (50 lm). Cells were treated for the same

time period with 1 mm DiBucAMP as a positive

control. Immunocytochemical labelling was performed

in PFA-fixed cells incubated for 1 h with a-tubulin



antibodies. Interestingly, the BBG-mediated increase in

the number of neurites was completely blocked by

LY-294002 treatment, hence indicating that PI3K is

crucial in the signalling cascade that couples P2X7

receptor inhibition with neurite formation in N2a cells

(Fig. 7H).

Several PI3K targets have also been identified and

characterized, including other kinases involved in neu-

ritogenesis, such as Akt [64] and GSK3 [65,66]. In

order to estimate PI3K function, we studied whether

the activity of Akt and GSK3 was modulated by P2X7

inhibition. Akt phosphorylation was transiently

increased in N2a cells treated with 100 nm BBG for

30 min (Fig. 8A,B). When GSK3 phosphorylation was

analysed, a significant increase in phospho-GSK3

levels was observed in N2a cells exposed to 100 nm

BBG, which resulted in a reduction in GSK3 activity

after treatment (Fig. 8C,D). To support this finding,

we studied the phosphorylation state of tau protein, a

well-known GSK3 substrate [67]. The ratio between

0.0

0.5

1.0

1.5

0.0

0.5

1.0

1.5

**

0 30 60Time (min)

0 30 60Time (min)

0 30 60Time (min)

pC

aMK

II/C

aMK

II (R

.U.)

0.0

0.5

1.0

1.5

2.0

**

Time (min)

pF

AK

Y39

7/F

AK

(R

.U.)

***

CaM

KII/

Act

in (

R.U

.)

0 30 60

0.0

0.5

1.0

1.5

***

pC

aMK

II/A

ctin

(R

.U.)

BBG 100 nMA E

B F

C G

D H

pCaMKII

CaMKII

-actin

0 30 60Time (min)

pFAK Y397

FAK

BBG 100 nM

0 30 60Time (min)

Control

DiBucA

MPBBG

KN930

1

2

3

** ****

Nu

mb

er o

f n

euri

tes/

cell

Control

DiBucA

MPBBG

LY-294

002

BBG + L

Y0

1

2

3

**

**###

**

**

Nu

mb

er o

f n

euri

tes/

cell

Fig. 7. P2X7 receptor inhibition modifies

CaMKII, FAK and PI3K activity in N2a cells.

(A–D) Western blots of pT286 ⁄ 287CaMKII a ⁄ band CaMKII a ⁄ b in N2a cells treated with

100 nM BBG for 30 and 60 min. Actin was

used as a loading control and the graphs

represent the mean ± standard error of the

mean (SEM) of pCaMKII, CaMKII and the

pCaMKII ⁄ CaMKII ratio from three different

experiments (**P < 0.01). (E) N2a cells cul-

tured in 0.5% fetal bovine serum-containing

medium were treated with either 1 lM BBG

or 1 lM KN93 for 72 h. Bar graphs show

quantitatively the number of neurites per

cell. Data represent the mean ± SEM of 80

cells (*P < 0.05; ***P < 0.001). (F, G) Wes-

tern blots of pY397FAK and FAK in N2a cells

treated with 100 nM BBG for 30 and

60 min. Bar graph represents the mean ±

SEM of the pFAKY397 ⁄ FAK ratio from three

different experiments (*P < 0.05). (H) N2a

cells cultured in 0.5% fetal bovine serum-

containing medium were treated with 1 lM

BBG for 72 h in the absence or presence of

50 lM LY-294002. Bar graphs show quanti-

tatively the number of neurites per cell.

Data are the mean ± SEM of 80 cells

(**P < 0.01 versus control; ###P < 0.001

versus BBG).



dephosphorylated tau (tau-1 epitope) and hyper-

phosphorylated tau (PHF-1 epitope) was increased

almost two-fold after BBG treatment (Fig. 8E,F), thus

corroborating that BBG inhibits GSK3 activity.

Discussion

The limited biological material and the cellular hetero-

geneity of primary neuronal cultures frequently

represent a disadvantage in the unequivocal character-

ization of signalling pathways and molecular cascades.

For this reason, clonal neuroblastoma cell lines are

commonly used as models to study neuronal differenti-

ation, as they retain the capacity to differentiate into a

neuronal-like phenotype, whilst expressing a rich

repertoire of membrane receptors coupled to the most

well-known second messenger systems. In this study,

we characterized the presence of P2X7 receptors by

molecular, immunocytochemical, pharmacological and

biophysical techniques in nondifferentiated murine

N2a neuroblastoma cells. Pharmacological inhibition

and the downregulation of P2X7 receptors were associ-

ated with neuritogenesis in N2a cells. Moreover, we

found that the inhibition of P2X7 receptors decreased

CaMKII activity, enhanced FAK activation and

increased the phosphorylation of Akt and GSK3.

The presence of P2X7 receptors was studied directly

by both PCR and western blotting techniques. In

agreement with the molecular masses reported for the

glycosylated monomers, we found a molecular mass of

78 kDa for the P2X7 protein [68]. A small band

detected at 45 kDa with the P2X7 antibody may corre-

spond to a truncated splice variant of the P2X7 recep-

tor, as described in humans [42]. The functionality of

P2X7 receptors in N2a cells was defined on pharmaco-

logical and biophysical grounds. Currently, no specific

GSK3

pGSK3

0 30 60Time (min)

0 30 60Time (min)

Tau-1

PHF-1

BBG 100 nM

BBG 100 nM

0 30 60

***

Time (min)

pG

SK

3/A

ctin

(R

.U.)

0 30 600.0

0.5

1.0

1.5

2.0

2.5

0.0

0.5

1.0

1.5

2.0

2.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

** *

Time (min)

Tau

-1/P

HF

-1

0 30 60

*

Time (min)

pA

kt/A

kt (

R.U

.)

AKT

pAKT

0 30 60Time (min)

BBG 100 nMA B

C D

E F

Fig. 8. P2X7 receptor inhibition increases

Akt and GSK3 phosphorylation in N2a cells.

(A, B) Western blots of pS473Akt and Akt in

N2a cells treated with 100 nM BBG for 30

and 60 min. Bar graph represents the

mean ± standard error of the mean (SEM)

of the pAkt ⁄ Akt ratio from three different

experiments (*P < 0.05). (C, D) Western

blots of pS9 ⁄ 21GSK3 and GSK3 a ⁄ b in N2a

cells treated with 100 nM BBG for 30 and

60 min. Actin was used as a loading control

and the graph represents the mean ± stan-

dard deviation of the pGSK3 ⁄ actin ratio from

three different experiments (*P < 0.05;

**P < 0.01). (E, F) Western blots of tau-1

and PHF-1 in N2a cells treated with 100 nM

BBG for 30 and 60 min. Bar graph repre-

sents the mean ± SEM of the tau-1 ⁄ PHF-1

ratio from three different experiments

(*P < 0.05; **P < 0.01).



agonists or antagonists exist for all of the P2X recep-

tors, and therefore their pharmacological identification

relies on the effects of a number of compounds (for a

review, see [69]). ATP and BzATP were able to induce

an increase in [Ca2+]i, whereas a,b-meATP and CTP

were ineffective. The potency for BzATP was increased

by two-fold following the omission of Mg2+ from the

extracellular medium, suggesting that ATP4) is the

active ligand. Likewise, the fact that an increase in

[Ca2+]i could not be evoked by a,b-meATP clearly

demonstrated that, if present, P2X1 and P2X3 recep-

tors are not functionally prominent in N2a cells [70].

The lack of CTP-mediated calcium responses also

excludes the presence of functional P2X4 receptors in

these cells [41]. In contrast, both the substantial

increase in [Ca2+]i and the small ionic current evoked

by micromolar concentrations of ATP indicate that, in

this cell model, the response to micromolar ATP con-

centration is mainly mediated by metabotropic P2Y

receptor activation, as reported previously [43]. The

P2X7 receptor antagonists KN62, BBG and A438079

inhibited BzATP-evoked calcium responses, thus dem-

onstrating the presence of P2X7 receptors [47–49].

Interestingly, both KN62 and BBG behaved as slowly

reversible antagonists, whereas the inhibitory effect of

A438079 was rapidly reversed. In addition, voltage-

clamp studies showed that BzATP elicits nondesensitiz-

ing inward currents in N2a cells, with current–voltage

relationships that do not display rectification. More-

over, the fact that BzATP-activated currents reversed

at about 0 mV and were sensitive to BBG and

A438079 indicates that they are mediated by genuine

P2X7 receptors. Together, these data point to the exis-

tence of native functional P2X7 receptors in N2a cells.

Calcium is an essential second messenger involved in

neuronal remodelling, this term referring to neuronal

differentiation, neurite outgrowth and synaptic plastic-

ity. Our results demonstrate that BzATP induces an

increase in [Ca2+]i in N2a cells that can be prevented

by the addition of specific P2X7 receptor antagonists

(BBG, KN62 and A438079) and is associated with

opposite changes (inhibition or facilitation, respec-

tively) in neuritogenesis. A trophic effect of ATP in

regeneration and neurite development has been

reported previously in various cell systems, such as

phaeochromocytoma cells (PC12), retinal ganglion cells

and striatal neurons [29–32]. It is worth mentioning

that ATP, acting via metabotropic P2Y receptors, can

also stimulate neurite outgrowth in N2a cells, indepen-

dent of calcium entry and other neurotrophic factors

[43]. With regard to P2X7 receptors, previous studies

have shown that ATP, via P2X7 receptors, sustains the

growth of human neuroblastoma cells through a sub-

stance P-dependent mechanism [36], and that a func-

tional decrease in P2X7 receptors seems to be

associated with retinoic acid-induced differentiation of

N2a cells [37]. In this context, the current study shows

that pharmacological inhibition of P2X7 receptors and

their downregulation with shRNAs promote neurite

formation in N2a cells, whereas P2X7 overexpression

significantly reduces neuritogenesis. Furthermore, a

local control of neurite formation by P2X7 receptors is

supported by the strong P2X7-positive immunolabel-

ling detected at the axon-like processes and growth

cone-like structures observed in a fraction of N2a cells

treated with DiBucAMP or BBG. It is obvious that

the effects caused by the functional inhibition of P2X7

receptors (either by pharmacological or molecular pro-

cedures) must be derived from the removal of an extra-

cellular nucleotidic tone. Interestingly, it has been

reported that neuroblastoma cells maintain a steady

ATP concentration in the incubation medium close to

100–200 nmolÆL)1 [36]. Moreover, in some physiologi-

cal fluids, such as aqueous humour, an ATP concen-

tration close to 1 lm can be reached [71]. An open

question is the source of extracellular ATP. It is well

known that ATP does not cross the plasma membrane

because of its negative charge, and cells release ATP

either alone or together with other mediators by vesic-

ular or granule secretion. In neuronal cells, ATP is

packaged within vesicles close to the presynaptic mem-

brane, and the stimulation of exocytosis leads to the

release of ATP into the extracellular medium [72,73].

Concerning other mechanisms implicated in nonvesicu-

lar ATP secretion, there is clear evidence to support

the release of ATP in a regulated manner through

connexons, also known as hemichannels [74].

Previous studies have linked increases in [Ca2+]iwith microtubule depolymerization [75]. Indeed, the

transient receptor potential vanilloid receptor 1, a non-

selective cation channel, plays an inhibitory role in sen-

sory neuronal extension and motility by regulating the

disassembly of microtubules [76]. In a similar way,

P2X7 receptors could be mediating cytoskeleton reor-

ganization in a Ca2+ -dependent manner and, conse-

quently, the repression of neurite outgrowth. It should

be noted that P2X7 receptors can interact directly with

structural proteins such as b-actin, a-actinin, lami-

nin a3 and integrin b2 in HEK cells, some of which

might initiate cytoskeletal rearrangements following

receptor activation [77]. We have reported previously

that P2X7 receptor activation is coupled to CaMKII

phosphorylation in cerebellar granule neurons [55] and

hippocampal neurons in culture [35]. In neurons,

CaMKII inhibition induces the reorganization of

F-actin and the formation of growth cones with a



filopodial structure [78]. Interestingly, our data show

that P2X7 receptor inhibition by BBG reduces the

amount of phospho-CaMKII, the active form of the

enzyme, thus pointing towards a common biochemical

mechanism in the control of neuritogenesis by P2X7

receptors in both primary neurons and neuron-like

tumour cells.

In human neuroblastoma cells, insulin-like growth

factor-I produces morphological changes, accompanied

by actin cytoskeletal rearrangement, followed by neu-

ronal differentiation through the activation of FAK

[79]. FAK is an integrin adaptor protein crucial for

focal adhesion formation, tumour migration [80], axo-

nal branching and synapse configuration [81]. FAK

activity is reduced by CaMKII-mediated phosphoryla-

tion at Ser843, whereas, in the absence of this process,

FAK is autophosphorylated at Tyr397, thereby

increasing its activity [57]. This mechanism underlies

the ability of local [Ca2+]i transients to reduce

pFAKY397, and thus to promote focal complex

removal and deadhesion of neuronal growth cones and

fibroblasts [82]. In our studies, phosphorylation of

Tyr397 is augmented after P2X7 receptor inhibition,

indicating that neurite formation induced by BBG is

probably a result of a decrease in calcium influx

through P2X7 receptors.

In an attempt to go further in the signalling path-

way regulated by P2X7 receptors in N2a cells, we eval-

uated the activity of PI3K, Akt and GSK3, all protein

kinases involved in neuritogenesis [60,61,64–66]. Both

Akt and GSK3 are phosphorylated by PI3K, a down-

stream target of pFAKY397 [58,59]. In a previous

study, we characterized the participation of PI3K in

the control of axonal growth and branching in hippo-

campal neurons [35]. The present study also demon-

strates that PI3K plays a key role in the formation of

neurites induced by P2X7 receptor inhibition. More-

over, our results show that P2X7 receptor inhibition

increases the phosphorylation of two PI3K targets,

Akt and GSK3, resulting in Akt activation and GSK3

inhibition. It should be noted that increased Akt activ-

ity can also induce the inhibition of GSK3, which has

been proposed to promote axonal growth and branch-

ing in neurons [83,84] and neurite formation in N2a

cells [85]. The inhibition of GSK3 activity by BBG was

confirmed by the augmentation of dephosphorylated

tau, a well-established GSK3 substrate [67].

Taken together, our results provide the first mecha-

nistic insight into the P2X7-triggered signalling path-

ways regulating neurite formation in N2a cells. P2X7

receptor inhibition leads to increased neurite formation

in N2a cells, this effect occurring in parallel with a

modification of the phosphorylation state of CaMKII,

FAK, Akt and GSK3, all kinases related to axonal

growth and cellular differentiation. In addition, our

results indicate that N2a cells may be considered to be

a reliable and convenient model for the initial charac-

terization of the signalling cascades coupled to P2X

receptors in the nervous system. This cellular model

may also be an excellent system to assay the effect of a

large number of compounds that may be potential

modulators of axonal growth and regeneration via

P2X7 receptors. Moreover, our data support the

involvement of P2X7 receptors in the maintenance of

neuroblastoma cells in a nondifferentiated state, and

point to P2X7 receptor antagonists as future potential

therapeutic agents in the treatment of neuroblastoma

tumour cells.

Experimental procedures

Chemicals and antibodies

ATP, UTP, CTP, a,b-meATP, BzATP, BBG, BSA, DiBu-

cAMP and DirectLoad� Wide Range DNA marker were

purchased from Sigma (St Louis, MO, USA), Fura-2 AM

was obtained from Molecular Probes (Leiden, the Nether-

lands), and KN62, KN93, LY-294002 and A438079 were

obtained from Tocris Bioscience (Bristol, UK). The com-

mercial antibodies used in this study were raised against

mouse P2X1, P2X2, P2X3, P2X4, P2X5, P2X6 and P2X7

(intracellular epitope) receptors, and all were purchased

from Alomone Labs (Jerusalem, Israel); phospho-CaM-

KII a ⁄ b (pT286 ⁄ 287) antibody was obtained from Upstate

Cell Signalling Solutions (Lake Placid, NY, USA); CaM-

KII a ⁄ b, phospho-FAK (pY397), FAK and GSK3 a ⁄ bantibodies were purchased from Invitrogen (San Francisco,

CA, USA); phospho-GSK3 (pS9 ⁄ 21) and phospho-Akt

(pS473) were obtained from Cell Signalling (Beverly, MA,

USA); Akt was obtained from Santa Cruz Biotechnology

(Santa Cruz, CA, USA); and tau-1 was purchased from

Chemicon (Temecula, CA, USA). PHF-1 antibody was a

kind gift from Dr Jesus Avila (CBM, Madrid, Spain).

Cell culture

N2a cells were cultured in DMEM (Sigma) supplemented

with glutamax� (Invitrogen), penicillin ⁄ streptomycin (Invi-

trogen) and 10% heat-inactivated fetal bovine serum (Euro-

Clone, Padova, Italy). Cells were grown at 37 �C in a

humidified atmosphere containing 5% CO2.

Transfections

N2a cells were plated at 4 · 104 cellsÆcm)2 and transiently

transfected using Lipofectamine� 2000 (Life Technologies,

Milan, Italy) following the manufacturer’s instructions.



After 6 h, the medium was removed and the cells were fur-

ther incubated for the indicated time periods in culture

medium. Expression vectors and shRNAs were obtained as

reported previously [35]. Briefly, human P2X7 full-length

cDNA was cloned into the pd2EGFP-N1 vector (Clontech,

Mountain View, CA, USA), and the ligation product was

confirmed by sequencing. P2X7 knockdown was achieved

by RNA interference (RNAi) using a vector-based shRNA

approach (pSUPER.neo.GFP vector, OligoEngine, Seattle,

WA, USA). The concomitant expression of GFP allowed

transfected cells to be identified by fluorescence microscopy.

RT-PCR and quantitative real-time PCR

Total RNA was extracted from cultured N2a cells and from

whole adult mice brain (strain C57BL ⁄ 6J) using an

Rneasy� plus mini kit (Qiagen, Hilden, Germany), follow-

ing the manufacturer’s instructions. After digestion with

TURBO DNase (Ambion, Austin, TX, USA), total RNA

was quantified and reversed transcribed using M-MLV

reverse transcriptase, 6 lg of random primers and 350 lm

dNTPs (all from Invitrogen). PCRs were carried out using

AmpliTaq Gold� PCR Master Mix (Applied Biosystems,

Foster City, CA, USA), 5 lL of the RT product and spe-

cific commercial oligonucleotide primers for mouse P2X1–5

and P2X7 receptors, as well as for b-actin (Applied Biosys-

tems). P2X6-specific primers were designed using Primer

Express 3.0 Software (Applied Biosystems): forward primer,

5¢-AGGGCAGATGTCCAGAGCAT-3¢; reverse primer,

5¢-GTCTTCATCAGCCCAGCAGTT-3¢. PCRs involved an

initial denaturation step at 94 �C for 5 min, followed by 40

cycles of amplification (94 �C for 30 s, 60 �C for 30 s and

72 �C for 30 s) conducted with a thermocycler GeneAmp

PCR System 2400 (Applied Biosystems). Control reactions

were carried out in the absence of RT product (template)

to avoid cross-contamination. Amplified PCR products

were electrophoresed on a 1% agarose gel and visualized

by SYBR� Safe DNA gel stain (Invitrogen).

Quantitative real-time PCRs were performed using gene-

specific primers and TaqMan MGB probes for mouse

P2X1–5 and P2X7 receptors, and b-actin (all from Applied

Biosystems). For the P2X6 receptor, the primers used have

been described previously and the probe design was FAM-

5¢-CTTCCGTTCCTCTGGC-3¢-MGB. Fast thermal cycling

was performed using a StepOnePlus� Real-Time PCR Sys-

tem (Applied Biosystems) as follows: denaturation, one

cycle of 95 �C for 20 s, followed by 40 cycles each of 95 �Cfor 1 s and 60 �C for 20 s. The results were normalized as

indicated by parallel amplification of the endogenous

control b-actin.

Western blotting

N2a cells were lysed and homogenized for 1 h at 4 �C in

lysis buffer containing 50 mm Tris ⁄HCl, 150 mm NaCl,

1% Nonidet P40 and Complete� Protease Inhibitor Cock-

tail Tablets (Roche Diagnostics GmbH, Mannheim,

Germany), pH 7.4. Separation of the proteins was per-

formed on 10% SDS-PAGE gels. Proteins were transferred

to poly(vinylidene difluoride) (Hybond�-P, Amersham GE

Healthcare, Madrid, Spain) or nitrocellulose membranes,

saturated for 1 h at room temperature with 5% nonfat

dried milk in Tris-buffered saline (10 mm Tris ⁄HCl,

100 mm NaCl and 0.1% Tween; pH 7.5) (TBS-T) and

incubated overnight at 4 �C with the following antisera at

the dilutions specified in parentheses: P2X1 (1 : 200), P2X2

(1 : 200), P2X3 (1 : 500), P2X4 (1 : 500), P2X5 (1 : 400),

P2X6 (1 : 200), P2X7 (1 : 250), pT286 ⁄ 287CaMKII a ⁄ b(1 : 1000), Akt (1 : 1000), pS473Akt (1 : 1000), pS9 ⁄ 21GSK3

(1 : 1000), pY397FAK (1 : 1000) and FAK (1 : 1000). The

monoclonal antibodies used in this study were as follows

(dilution specified in parentheses): a-tubulin (1 : 10 000),

CaMKII a ⁄ b (1 : 1000), GSK3 a ⁄ b (1 : 1000), tau-1

(1 : 5000) and PHF-1 (1 : 100). Blots were then washed in

TBS-T, and incubated for 1 h at room temperature with

goat anti-rabbit or goat anti-mouse IgGs coupled to horse-

radish peroxidase (HRP, Amersham GE Healthcare), both

used at 1 : 5000 dilution. Protein bands were detected by

ECL chemiluminescence (Amersham GE Healthcare).

Calcium microfluorimetric analysis in single cells

N2a cells cultured on coverslips placed in 35 mm dishes

(250 000 cells per well) were washed with Locke’s solution

(composition in mm: NaCl, 140; KCl, 4.5; CaCl2, 2.5;

KH2PO4, 1.2; MgSO4, 1.2; glucose, 5.5; Hepes, 10; pH 7.4),

and loaded with the calcium dye Fura-2 AM (5 lm) for

45 min at 37 �C. Subsequently, the coverslips were washed

and placed in a superfusion chamber on a Nikon Eclipse

TE-2000 microscope (Nikon, Barcelona, Spain), where the

cells were continuously superfused with Locke’s medium at

a rate of 1.5 mLÆmin)1. N2a cells were stimulated for 40 s

with a variety of purinergic receptor agonists at near-maxi-

mal effective concentration: 100 lm a,b-meATP, 300 lm

CTP, 100 lm BzATP and 100 lm ATP. In other studies,

the P2X7 antagonists KN62 (50 nm), BBG (1 lm) and

A438079 (1 lm) were preincubated for 5 min before BzATP

(100 lm) superfusion. In some experiments, an Mg2+-free

Locke’s solution was used, replacing MgSO4 by glucose at

a concentration that conserved the solution osmolarity.

Cells were visualized using a Plan Fluor 20· ⁄ 0.5 lens on a

Nikon Eclipse TE-2000-E microscope. The wavelength of

the incoming light (340 or 380 nm) was selected with the

aid of an Optoscan monochromator (10 nm bandwidth),

from Cairn Research (Faversham, UK); 12-bit images were

acquired with an ORCA-ER C 47 42–98 CCD camera

(Hamamatsu, Barcelona, Spain) controlled by Metafluor

6.3r6 PC software (Universal Imaging Corp., Cambridge,

UK). The exposure time was 250 ms for each wavelength

and the changing time was less than 5 ms. The images were



acquired continuously and buffered in a fast SCSI disk.

The time course data represent the average light intensity in

a small elliptical region within each cell. The background

was subtracted at each wavelength and the 340 ⁄ 380 ratio

was calculated. The data are represented as the normalized

F340 ⁄F380 fluorescence ratio, which increases as [Ca2+]iincreases.

Intracellular calcium fluorimetric analysis in

cellular suspensions

Population-averaged intracellular Ca2+ changes were also

determined with the fluorescent indicator Fura-2 AM. N2a

cells were collected by trypsinization from confluent cul-

tures in 75 cm2 flasks, washed and resuspended in Locke’s

solution. The cells were then loaded by incubation with

5 lm Fura-2 AM for 45 min at 37 �C in Locke’s solution

containing 1 mgÆmL)1 BSA. After the loading period, the

cells were washed in fresh Locke’s solution and resuspended

at a density of 106 cellsÆmL)1. Recordings were made on

1.5 mL samples in thermostatically controlled, stirred

cuvettes in a LS-50B fluorometer (Perkin Elmer, Waltham,

MA, USA). The fluorescence intensity was determined with

an excitation wavelength of 340 nm and an emission wave-

length of 510 nm. The results were normalized with respect

to the maximal response elicited by BzATP. Agonists and

other compounds were added to the cuvettes from at least

100-fold concentrated stock solutions to avoid large volume

variations. Fluorescence traces were calibrated individually

by releasing intracellular dye contents with Triton X-100

(0.3%) and determining dye fluorescence in the presence of

a mixture of EGTA ⁄Tris ([Ca2+] < 0.2 nm) and 2.5 mm

Ca2+ to calculate Fmin and Fmax, respectively. [Ca2+]i was

calculated from the fluorescence traces using the equation

of Grynkiewicz [86].

Electrophysiological recordings

Electrophysiological recordings were performed with an

EPC9 patch-clamp amplifier using pulse software (HEKA

Electronic, Lambrecht, Germany). Pipettes were pulled

from Kimax borosilicate glass (Witz Scientific, Holland,

OH, USA) and subsequently wax-coated and fire-polished

to obtain a final resistance of 2–3 MX when filled with

standard solutions. The standard extracellular solution

(bath) contained (in mm): NaCl, 140; KCl, 2.8; CaCl2, 2;

Hepes, 10; glucose, 10 (pH 7.2, adjusted with NaOH;

298 mOsm). Recording pipettes were filled with a solution

containing (in mm): KCl, 140; MgCl2, 1; Hepes, 10

(pH 7.2, adjusted with KOH; 292 mOsm). Cells attached to

glass coverslips were transferred to a recording chamber

placed in the stage of an inverted Zeiss Axiovert 100 micro-

scope (Zeiss, Barcelona, Spain) and continuously super-

fused with bath solution (perfusion rate of 1 mLÆmin)1).

Membrane currents were measured in the whole-cell config-

uration of the patch-clamp technique, filtered at 3 kHz and

sampled at 10 kHz. Once electrical access to the cytoplasm

had been established, cells were held at a voltage (Vh) of

)70 mV and those with holding currents larger than 20 pA

were rejected. Series resistance was compensated by 80%

and monitored throughout the experiment, together with

the cell membrane capacitance.

Ligand-gated currents were activated by P2X receptor

agonists applied onto the cell under investigation by means

of a gravity-driven perfusion system with five independent

lines controlled by electronic valves (The Lee Company,

Westbrook, CO, USA). This system allowed the exchange

of the medium surrounding a cell in < 200 ms. Stock solu-

tions of drugs were diluted daily in extracellular saline and

incorporated into the perfusion system a few minutes before

starting the experiments. Current–voltage relations for

BzATP-evoked responses were obtained by stepping the

holding voltage to the indicated values, 2 s prior to and

during agonist application. Reversal potential values from

individual cells were corrected for the junction potential

between the pipette’s solution and the extracellular solution,

which was calculated to be +4.42 mV using the Patcher’s

Tools module included in igor-pro software (WaveMetrics,

Inc., Lake Oswego, OR, USA). All recordings were

obtained at room temperature (21–24 �C).

Immunocytochemistry

N2a cells cultured on coverslips placed in 35 mm dishes

(250 000 cells per well) were washed with NaCl ⁄Pi and fixed

with 4% PFA for 15 min. After washing with NaCl ⁄Pi, the

cells were permeabilized with 0.1% Triton X-100 and

blocked with 5% goat serum and 10% fetal bovine serum

for 1 h at room temperature. After washing with 3% BSA

in NaCl ⁄Pi, the cells were incubated for 1 h with primary

antibodies against P2X7 (1 : 100) and a-tubulin (1 : 1000).

Subsequently, cells were washed with NaCl ⁄Pi and incu-

bated for 1 h with Cy3�-conjugated donkey anti-rabbit

IgG (Jackson InmunoResearch, Westgrove, PA, USA) or

fluorescein isothiocyanate-goat anti-mouse IgG (Sigma).

Nuclei were counterstained with 4¢,6-diamidino-2-phenylin-

dole (DAPI, from Invitrogen), a fluorescent stain that binds

strongly to DNA. Coverslips were mounted on glass slides

using FluoroSave� Reagent (Calbiochem, Nottingham,

UK). Images were acquired using a Nikon Eclipse TE-200

microscope coupled to a CCD camera (Kappa ACC-1).

The analysis of neurite length and ramifications was carried

out using Neuron J freeware.

Statistical analysis

Results were analysed by unpaired t-test using graph pad

prism 5 (Graph Pad Software Inc., San Diego, CA, USA)

and expressed as the mean ± standard error of the mean.

Differences were considered to be significant at P £ 0.05.



Acknowledgements

PHF-1 antibody was a generous gift from Dr Jesus

Avila (CBM, Madrid, Spain). This work was sup-

ported by research grants from MICINN (BFU2008-

02699, BFU2005-06034 and SAF2006-00906), CAM

(S-SAL-0253-2006), ‘The Spanish Ion Channel Initia-

tive (SICI)’ (CSD2008-00005) and Fundacion Marceli-

no Botın. RGV was supported by SICI. AP was

supported by CIBERNED. MDH and JIDH were sup-

ported by Juan de la Cierva and Ramon y Cajal

Programs, respectively. DB was a research fellow of

the Basque Government.

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Ca2+/calmodulin-dependent kinase II signalling cascade mediates P2X7 receptor-dependent inhibition...

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