Ca2+/calmodulin-dependent kinase II signalling cascade mediates P2X7 receptor-dependent inhibition of neuritogenesis in neuroblastoma cells ´ ´ ´ Rosa 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 ´ ´ Departamento de Bioquımica y Biologıa Molecular, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain ´ Centro de Biologıa Molecular ‘Severo Ochoa’, CSIC-UAM, Madrid, Spain ´ Departamento de Neurobiologıa Celular Molecular y del Desarrollo, Instituto Cajal, CSIC, Madrid, Spain ´ ´ Centro de Investigacion Biomedica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain ´ ´ 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: mtmiras@vet.ucm.es (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 modulator 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 presence of endogenous P2X1, P2X3, P2X4 and P2X7 subunits in these cells Of these, the P2X7 receptor was the only functional receptor, as its activation induced intracellular calcium increments similar to those observed in primary neuronal cultures, exhibiting pharmacological properties characteristic 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-containing medium, whereas P2X7 overexpression significantly reduced the formation 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-dependent 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+ ⁄ calmodulindependent 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 ´ R Gomez-Villafuertes et al P2X7 negatively controls neuritogenesis via CaMKII 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 transmembranespanning segments separated by an N-glycosylated extracellular loop containing 10 conserved cysteine residues [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 pharmacological tools and gene knockout ⁄ knockdown approaches, it has become clear that P2X receptors are involved in a wide and growing range of physiological processes [10] In the CNS, ATP directly mediates fast excitatory synaptic transmission, acting via P2X receptors in mammalian neurons [11–13] Furthermore, 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 facilitated 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 dorsal 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 signalling in the initiation and direction of cell differentiation [28] A trophic effect of purines in regeneration 5308 and neurite outgrowth has also been reported in various cell systems, such as PC12 phaeochromocytoma cells [29,30], retinal ganglion cells [31] and striatal neurons [32] P2X receptors also participate in the formation 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 neurons [35] Furthermore, ATP, acting via P2X7 receptors, 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 differentiation 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 receptors was further examined in mouse Neuro-2a (N2a) neuroblastoma cells Pharmacological inhibition and interference with P2X7 receptor expression were associated with neuritogenesis in N2a cells cultured in low serum-containing medium, whereas P2X7 overexpression 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 effectors, including focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K), Akt and glycogen synthase kinase (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 purinergic 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 FEBS Journal 276 (2009) 5307–5325 ª 2009 The Authors Journal compilation ª 2009 FEBS ´ R Gomez-Villafuertes et al P2X7 negatively controls neuritogenesis via CaMKII A M H2O N B H2O N B H2O N B H2O N B 60 40 P2X7 P2X6 β-Actin C P2X 80 75 kDa 20 50 kDa 35 kDa α-tubulin P2 X1 P2 X2 P2 X3 P2 X4 P2 X5 P2 X6 P2 X7 Normalized to β-actin (R.U.) P2X5 P2X4 P2X P2X P2X3 P2X P2X P2X2 B P2X P2X P2X1 M H2O N B H2O N B H2O N B H2O N B Fig 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, DirectLoadÔ Wide Range DNA marker (B) Quantitative real-time PCR showing the expression levels of P2X subunits in N2a cells Values were normalized by the content of b-actin transcript (C) Total protein isolated from N2a cells was subjected to SDS-PAGE, transferred onto poly(vinylidene difluoride) membrane and incubated with specific antibodies against P2X1–7 subunits Immunodetection of a-tubulin was used as a loading control and P2X6 was practically undetectable (Fig 1B) Western blot analyses were performed on total protein extracts obtained from N2a cells using commercial subunit-specific antibodies Specific neutralizing peptides were employed to guarantee the specificity of these bands (data not shown) As expected, the expression 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 subunits has been reported in the literature, probably caused by differences in glycosylation and ⁄ or oligomerization The predicted sizes for unglycosylated P2X subunits have been reported to range between 43 and 68 kDa, whereas the molecular masses of glycosylated 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 represent 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 oligomeric forms (dimers or trimers) of P2X subunits were not observed Pharmacological characterization of P2X7 receptors in N2a cells The pharmacological characterization of P2X receptors was carried out by analysing the change in intracellular free calcium concentration ([Ca2+]i) elicited by a variety of purinergic compounds, in combination with specific 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) Neither 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 extracellular medium (data not shown), indicating the participation of functional metabotropic P2Y receptors in this cell line, as reported previously [43] Decreases in extracellular Ca2+ and Mg2+ concentration are known to modulate P2X7 receptor-mediated 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 FEBS Journal 276 (2009) 5307–5325 ª 2009 The Authors Journal compilation ª 2009 FEBS 5309 ´ R Gomez-Villafuertes et al P2X7 negatively controls neuritogenesis via CaMKII A α, βmeATP CTP BzATP ATP F340/F380 2.0 1.5 1.0 0.5 100 s B Mg2+ free BzATP EGTA BzATP BzATP 2.5 F340/F380 2.0 1.5 1.0 0.5 100 s Δ Intracellular Ca2+ (% of maximal response) C 100 With Mg2+ Without Mg2+ 80 60 40 20 –6 –5 –4 –3 Log [BzATP] Fig BzATP induces intracellular calcium transients in nondifferentiated N2a cells that are potentiated in the absence of Mg2+ ions (A) Intracellular calcium increments evoked by different purinergic agonists 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 Horizontal 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 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 lM to 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 5310 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 Tris ⁄ 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 antagonists 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+]i through P2X7 receptors without producing any effect on their own [47–49] Administered 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 washout (Fig 3D,E) In contrast, the inhibitory effect of A438079 was rapidly reversed after a 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 purinergic 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 evidence supporting this conclusion, we conducted patchclamp 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 cellsỈcm)2) and systematically chose cells isolated from neighbours and devoid of cytoplasmic processes Ligand-gated ionic currents were activated by challenging 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 nondesensitizing currents that differed markedly in their respective FEBS Journal 276 (2009) 5307–5325 ª 2009 The Authors Journal compilation ª 2009 FEBS ´ R Gomez-Villafuertes et al P2X7 negatively controls neuritogenesis via CaMKII Fig P2X7 antagonists reduce intracellular calcium transients elicited by BzATP in nondifferentiated N2a cells N2a cells were stimulated twice with 100 lM BzATP and subsequently exposed to 50 nM KN62 (A), lM BBG (B) or lM A438079 (C) for 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 individual 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 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 current density of about 14 ppF)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 functionally expressed in N2a cells Current–voltage relationships 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 = cells) The involvement of P2X7 receptors in BzATP-induced currents was confirmed by assaying their sensitivity to extracellular Mg2+, as well as to BBG (1 lm) and A438079 (1 lm) In cells exposed for to a bath solution containing mm Mg2+, a rapid and fully reversible inhibition ( 58%; n = 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 = and n = cells, respec- FEBS Journal 276 (2009) 5307–5325 ª 2009 The Authors Journal compilation ª 2009 FEBS 5311 ´ R Gomez-Villafuertes et al P2X7 negatively controls neuritogenesis via CaMKII A B 30 pA 10 α, β-meATP 20 pA 50 pA 20 ATP 2s –100 –80 –60 –40 –20 20 40 60 mV –10 BzATP 2s BzATP –20 400 –30 30 –40 I (pA) 200 100 20 10 * ** 0 BzATP ATP BzATP C D α, β-meATP BzATP BzATP + A438079 BzATP (2 Mg2+) 20 pA Wash BzATP (0 Mg2+) + BBG Wash 1s Control 2s Control 75 40 30 ** 20 IBzATP (pA) 75 IBzATP (pA) IBzATP (pA) 20 pA I (pA) 300 50 * 25 50 25 ** 10 0 0 Mg2+ Mg2+ Control A438079 Wash Control BBG Wash Fig 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 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–voltage 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 intervals and A438079 or BBG were superfused 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 tively) (Fig 4D) As already noted in microfluorimetric experiments, the effect of BBG was reversed only in part after a 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 neuro5312 blastoma tumour cells [36] Interestingly, P2X7 receptor 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 serumcontaining medium for 72 h in the presence or absence of BBG (100 nm or 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 FEBS Journal 276 (2009) 5307–5325 ª 2009 The Authors Journal compilation ª 2009 FEBS ´ R Gomez-Villafuertes et al Control BBG μM E 40 α-Tub α-Tub C DiBucAMP B BBG 100 nM D Neur ite length (μm) A P2X7 negatively controls neuritogenesis via CaMKII * 30 20 10 C on tr ol D iB uc A M P B B G μM B B G 10 nM G F α-Tub Number of neurites/cell α-Tub H *** *** *** 10 G B B G B B uc iB D nM M A tr on C μM P ol Fig The P2X7 receptor antagonist BBG promotes neurite formation in N2a cells N2a cells were cultured in 0.5% fetal bovine serum-containing medium for 72 h in the absence (A) or presence of mM DiBucAMP (B), 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 receptor serum (red) determine subcellular P2X7 receptor location Scale bar, 20 lm (H) Detail of a distal region of a neurite with a growth conelike structure, where high levels of P2X7 receptor-positive immunostaining can be observed Scale bar, lm Cellular morphology was defined using a-tubulin antibody (green) and nuclei were labelled with DAPI (blue) in paraformaldehyde (PFA)-fixed cells incubated for 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 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 BBGtreated 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 pSUPER.neo.GFP vector-derived small hairpin RNA (shRNA) strategy was designed to knock down native P2X7 receptor expression Although the usefulness of FEBS Journal 276 (2009) 5307–5325 ª 2009 The Authors Journal compilation ª 2009 FEBS 5313 ´ R Gomez-Villafuertes et al P2X7 negatively controls neuritogenesis via CaMKII 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-P2X7GFP expression vector or with a vector carrying the interference RNA for P2X7 (hereafter termed shRNA- P2X7 α-tubulin P2X7/ α-tubulin (R.U.) A P2X7) Control cells were transfected with nonspecific shRNA-luciferase vector (shRNA-Luc) P2X7 expression 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 2.0 ** 1.5 1.0 0.5 * 2x A -P -G R N R sh sh P2 X7 sh R N A -L uc FP 0.0 B Control A N uc -L X7 P2 Neurite length (μm) DiBucAMP FP R sh N 2X -P A C 30 GFP G * 20 10 FP A -P 2X sh R N -G X7 D P2 iB C uc on A tr M P ol GFP P2X7-GFP GFP shRNA-P2X7 Number of neurites/cell D ** * * 5314 sh R A -P 2X X7 P2 N -G M D iB uc A tr C on GFP FP P ol Fig P2X7 receptor regulates the differentiation of N2a cells (A) Western blotting of N2a cells transfected with either pcDNAP2X7-GFP or shRNA-P2X7 The levels of a-tubulin were used as a loading control and the P2X7 ⁄ a-tubulin ratio was used to estimate 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 transfected with empty GFP vector, P2X7-GFP or shRNA-P2X7 As a positive control, cells transfected with empty GFP vector were treated with mM DiBucAMP After 48 h in culture, cells were fixed and GFP-positive cells were observed in order to study cellular 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 FEBS Journal 276 (2009) 5307–5325 ª 2009 The Authors Journal compilation ª 2009 FEBS ´ R Gomez-Villafuertes et al 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 positive control, cells were transfected with empty GFP vector and treated with 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 receptors 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 plasticity (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 outgrowth, whereas the suppression of Ca2+ transients accelerates axon extension [52] CaMKII is a calciumdependent serine ⁄ threonine kinase that also possesses the ability to become calcium independent through autophosphorylation [53] Moreover, once it is activated, it remains functional for at least several hours [54] Previously, we have reported that P2X7 receptor activation induces CaMKII phosphorylation in cerebellar granule neurons [55], whereas P2X7 receptor inhibition reduces its phosphorylation in cultured hippocampal neurons [35] As CaMKII and its downstream targets could be good effectors in N2a cell differentiation induced by P2X7 antagonists, we analysed the phosphorylation state of CaMKII in N2a cells exposed to 100 nm BBG This antagonist produced a significant decrease in the ratio between phosphoCaMKII (active form of the enzyme) and total CaMKII after 60 of treatment (Fig 7A–D), suggesting that CaMKII participates in the negative regulation of neuritogenesis exerted by P2X7 receptors To corroborate this hypothesis, N2a cells were cultured in 0.5% fetal bovine serum-containing medium for 72 h in the presence or absence of lm KN93, a well-known P2X7 negatively controls neuritogenesis via CaMKII CaMKII inhibitor As positive controls, cells were treated for the same time period with either mm DiBucAMP or lm BBG Immunocytochemistry was performed in PFA-fixed cells incubated for 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, pointing 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 hippocampal neurons [35] FAK is an actin-interacting protein 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 receptors in N2a cells Previous studies have reported that one of the downstream targets of FAK is PI3K [58,59] It is generally accepted that PI3K plays a central role in cell signalling, mediating neurite extension [60,61], cell proliferation, 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-containing medium were treated with 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 mm DiBucAMP as a positive control Immunocytochemical labelling was performed in PFA-fixed cells incubated for h with a-tubulin FEBS Journal 276 (2009) 5307–5325 ª 2009 The Authors Journal compilation ª 2009 FEBS 5315 ´ R Gomez-Villafuertes et al P2X7 negatively controls neuritogenesis via CaMKII BBG 100 nM A E Number of neurites/cell pCaMKII CaMKII -actin ** 93 G N B K iB D Time (min) pCaMKII/Actin (R.U.) B uc on 60 C 30 A tr M P ol 0 B ** ** F 1.5 BBG 100 nM 1.0 pFAK Y397 *** 0.5 FAK 0.0 30 30 60 Time (min) 60 Time (min) G 1.5 1.0 *** 0.5 pFAKY397/FAK (R.U.) 0.0 2.0 * * 1.5 1.0 0.5 0.0 30 60 30 Time (min) H 1.5 1.0 ** 0.5 Number of neurites/cell ** ### ** ** 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 neuritogenesis, such as Akt [64] and GSK3 [65,66] In order to estimate PI3K function, we studied whether B LY + G B B G LY P M uc A iB D C Time (min) 60 ol 30 tr -2 94 00 0.0 5316 ** on pCaMKII/CaMKII (R.U.) D 60 Time (min) B CaMKII/Actin (R.U.) C Fig P2X7 receptor inhibition modifies CaMKII, FAK and PI3K activity in N2a cells (A–D) Western blots of pT286 ⁄ 287CaMKII a ⁄ b and CaMKII a ⁄ b in N2a cells treated with 100 nM BBG for 30 and 60 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 cultured in 0.5% fetal bovine serum-containing medium were treated with either lM BBG or 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) Western blots of pY397FAK and FAK in N2a cells treated with 100 nM BBG for 30 and 60 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 serumcontaining medium were treated with lM BBG for 72 h in the absence or presence of 50 lM LY-294002 Bar graphs show quantitatively the number of neurites per cell Data are the mean ± SEM of 80 cells (**P < 0.01 versus control; ###P < 0.001 versus BBG) 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 (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 FEBS Journal 276 (2009) 5307–5325 ª 2009 The Authors Journal compilation ª 2009 FEBS ´ R Gomez-Villafuertes et al A BBG 100 nM pAKT B 2.5 pAkt/Akt (R.U.) P2X7 negatively controls neuritogenesis via CaMKII 2.0 AKT 30 * 1.5 1.0 0.5 0.0 60 BBG 100 nM pGSK3 GSK3 60 3.0 * 2.5 ** 2.0 1.5 1.0 0.5 0.0 30 60 Time (min) E F BBG 100 nM Tau-1 PHF-1 30 60 30 60 Time (min) 2.5 ** 2.0 Tau-1/PHF-1 Fig 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 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 Actin was used as a loading control and the graph represents the mean ± standard 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 Bar graph represents the mean ± SEM of the tau-1 ⁄ PHF-1 ratio from three different experiments (*P < 0.05; **P < 0.01) D pGSK3/Actin (R.U.) C 30 Time (min) Time (min) * 1.5 1.0 0.5 Time (min) dephosphorylated tau (tau-1 epitope) and hyperphosphorylated 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 heterogeneity of primary neuronal cultures frequently represent a disadvantage in the unequivocal characterization of signalling pathways and molecular cascades For this reason, clonal neuroblastoma cell lines are commonly used as models to study neuronal differentiation, 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 0.0 30 60 Time (min) molecular, immunocytochemical, pharmacological and biophysical techniques in nondifferentiated murine N2a neuroblastoma cells Pharmacological inhibition and the downregulation of P2X7 receptors were associated 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 correspond to a truncated splice variant of the P2X7 receptor, as described in humans [42] The functionality of P2X7 receptors in N2a cells was defined on pharmacological and biophysical grounds Currently, no specific FEBS Journal 276 (2009) 5307–5325 ª 2009 The Authors Journal compilation ª 2009 FEBS 5317 ´ R Gomez-Villafuertes et al P2X7 negatively controls neuritogenesis via CaMKII agonists or antagonists exist for all of the P2X receptors, 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 receptors 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 concentration 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 demonstrating 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, voltageclamp studies showed that BzATP elicits nondesensitizing inward currents in N2a cells, with current–voltage relationships that not display rectification Moreover, the fact that BzATP-activated currents reversed at about mV and were sensitive to BBG and A438079 indicates that they are mediated by genuine P2X7 receptors Together, these data point to the existence 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 plasticity 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, respectively) 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, independent 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 sub5318 stance P-dependent mechanism [36], and that a functional 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 immunolabelling 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 procedures) must be derived from the removal of an extracellular nucleotidic tone Interestingly, it has been reported that neuroblastoma cells maintain a steady ATP concentration in the incubation medium close to 100–200 nmolỈL)1 [36] Moreover, in some physiological fluids, such as aqueous humour, an ATP concentration close to 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 vesicular or granule secretion In neuronal cells, ATP is packaged within vesicles close to the presynaptic membrane, and the stimulation of exocytosis leads to the release of ATP into the extracellular medium [72,73] Concerning other mechanisms implicated in nonvesicular 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+]i with microtubule depolymerization [75] Indeed, the transient receptor potential vanilloid receptor 1, a nonselective cation channel, plays an inhibitory role in sensory neuronal extension and motility by regulating the disassembly of microtubules [76] In a similar way, P2X7 receptors could be mediating cytoskeleton reorganization in a Ca2+ -dependent manner and, consequently, the repression of neurite outgrowth It should be noted that P2X7 receptors can interact directly with structural proteins such as b-actin, a-actinin, laminin 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 FEBS Journal 276 (2009) 5307–5325 ª 2009 The Authors Journal compilation ª 2009 FEBS ´ R Gomez-Villafuertes et al 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 neuronal differentiation through the activation of FAK [79] FAK is an integrin adaptor protein crucial for focal adhesion formation, tumour migration [80], axonal branching and synapse configuration [81] FAK activity is reduced by CaMKII-mediated phosphorylation 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 pathway regulated by P2X7 receptors in N2a cells, we evaluated 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 downstream target of pFAKY397 [58,59] In a previous study, we characterized the participation of PI3K in the control of axonal growth and branching in hippocampal neurons [35] The present study also demonstrates that PI3K plays a key role in the formation of neurites induced by P2X7 receptor inhibition Moreover, 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 activity can also induce the inhibition of GSK3, which has been proposed to promote axonal growth and branching 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 mechanistic insight into the P2X7-triggered signalling pathways 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, P2X7 negatively controls neuritogenesis via 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 characterization 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, DiBucAMP and DirectLoadÔ Wide Range DNA marker were purchased from Sigma (St Louis, MO, USA), Fura-2 AM was obtained from Molecular Probes (Leiden, the Netherlands), and KN62, KN93, LY-294002 and A438079 were obtained from Tocris Bioscience (Bristol, UK) The commercial 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-CaMKII a ⁄ b (pT286 ⁄ 287) antibody was obtained from Upstate Cell Signalling Solutions (Lake Placid, NY, USA); CaMKII a ⁄ b, phospho-FAK (pY397), FAK and GSK3 a ⁄ b antibodies 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 glutamaxÒ (Invitrogen), penicillin ⁄ streptomycin (Invitrogen) and 10% heat-inactivated fetal bovine serum (EuroClone, Padova, Italy) Cells were grown at 37 °C in a humidified atmosphere containing 5% CO2 Transfections N2a cells were plated at · 104 cellsỈcm)2 and transiently transfected using Lipofectamin 2000 (Life Technologies, Milan, Italy) following the manufacturer’s instructions FEBS Journal 276 (2009) 5307–5325 ª 2009 The Authors Journal compilation ª 2009 FEBS 5319 ´ R Gomez-Villafuertes et al P2X7 negatively controls neuritogenesis via CaMKII After h, the medium was removed and the cells were further 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 RneasyÒ plus mini kit (Qiagen, Hilden, Germany), following 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, lg of random primers and 350 lm dNTPs (all from Invitrogen) PCRs were carried out using AmpliTaq GoldÒ PCR Master Mix (Applied Biosystems, Foster City, CA, USA), lL of the RT product and specific commercial oligonucleotide primers for mouse P2X1–5 and P2X7 receptors, as well as for b-actin (Applied Biosystems) 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 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 SYBRÒ Safe DNA gel stain (Invitrogen) Quantitative real-time PCRs were performed using genespecific 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 FAM5¢-CTTCCGTTCCTCTGGC-3¢-MGB Fast thermal cycling was performed using a StepOnePlusÔ Real-Time PCR System (Applied Biosystems) as follows: denaturation, one cycle of 95 °C for 20 s, followed by 40 cycles each of 95 °C for 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 h at °C in lysis buffer containing 50 mm Tris ⁄ HCl, 150 mm NaCl, 5320 1% Nonidet P40 and CompleteÔ Protease Inhibitor Cocktail Tablets (Roche Diagnostics GmbH, Mannheim, Germany), pH 7.4 Separation of the proteins was performed on 10% SDS-PAGE gels Proteins were transferred to poly(vinylidene difluoride) (HybondÔ-P, Amersham GE Healthcare, Madrid, Spain) or nitrocellulose membranes, saturated for 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 °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 h at room temperature with goat anti-rabbit or goat anti-mouse IgGs coupled to horseradish peroxidase (HRP, Amersham GE Healthcare), both used at : 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 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 mLỈmin)1 N2a cells were stimulated for 40 s with a variety of purinergic receptor agonists at near-maximal 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 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 ms The images were FEBS Journal 276 (2009) 5307–5325 ª 2009 The Authors Journal compilation ª 2009 FEBS ´ R Gomez-Villafuertes et al 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+]i increases 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 cultures in 75 cm2 flasks, washed and resuspended in Locke’s solution The cells were then loaded by incubation with lm Fura-2 AM for 45 at 37 °C in Locke’s solution containing mgỈmL)1 BSA After the loading period, the cells were washed in fresh Locke’s solution and resuspended at a density of 106 cellsỈmL)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 wavelength 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 microscope (Zeiss, Barcelona, Spain) and continuously superfused with bath solution (perfusion rate of mLỈmin)1) Membrane currents were measured in the whole-cell config- P2X7 negatively controls neuritogenesis via CaMKII uration of the patch-clamp technique, filtered at 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 solutions 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, 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 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 h at room temperature After washing with 3% BSA in NaCl ⁄ Pi, the cells were incubated for h with primary antibodies against P2X7 (1 : 100) and a-tubulin (1 : 1000) Subsequently, cells were washed with NaCl ⁄ Pi and incubated for h with Cy3Ô-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-phenylindole (DAPI, from Invitrogen), a fluorescent stain that binds strongly to DNA Coverslips were mounted on glass slides using FluoroSaveÔ 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 (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 FEBS Journal 276 (2009) 5307–5325 ª 2009 The Authors Journal compilation ª 2009 FEBS 5321 ´ R Gomez-Villafuertes et al P2X7 negatively controls neuritogenesis via CaMKII Acknowledgements PHF-1 antibody was a generous gift from Dr Jesus ´ ´ Avila (CBM, Madrid, Spain) This work was supported by research grants from MICINN (BFU200802699, BFU2005-06034 and SAF2006-00906), CAM (S-SAL-0253-2006), ‘The Spanish Ion Channel Initia´ tive (SICI)’ (CSD2008-00005) and Fundacion Marcelino Botı´ n 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 References Burnstock G & Kennedy C (1985) Is there a basis for distinguishing two types of P2-purinoceptor? 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(PI3K), Akt... should be noted that P2X7 receptors can interact directly with structural proteins such as b-actin, a-actinin, laminin a3 and integrin b2 in HEK cells, some of which might initiate cytoskeletal