Developmental Increase in Ecto-5′-Nucleotidase ActivityOverlaps with Appearance of Two Immunologically DistinctEnzyme Isoforms in Rat Hippocampal SynapticPlasma Membranes

Ivana Grkovic & Ivana Bjelobaba &Nadezda Nedeljkovic &

Natasa Mitrovic & Dunja Drakulic & Milos Stanojlovic &

Anica Horvat

Received: 30 December 2013 /Accepted: 4 February 2014# Springer Science+Business Media New York 2014

Ab s t r a c t E c t o - 5 ′ - n u c l e o t i d a s e ( e - 5 NT ) , aglycosylphosphatidylinositol-linked membrane protein, cata-lyzes a conversion of AMP to adenosine, which influencesnearly every aspect of brain physiology, including embryonicand postnatal brain development. The present study aimed toinvestigate a pattern of expression, activity and kinetic prop-erties of e-5NT in the hippocampal formation and synapticplasmamembrane (SPM) preparations in rats at postnatal days(PDs) 7, 15, 20, 30 and 90. By combining gene expressionanalysis and enzyme histochemistry, we observed that e-5NTmRNA reached the adult level at PD20, while the enzymeactivity continued to increase beyond this age. Further analy-sis revealed that hippocampal layers rich in synapsesexpressed the highest levels of e-5NT activity, while in layerspopulated with neuronal cell bodies, the enzyme activity wasweak or absent. Therefore, activity and expression of e-5NTwere analyzed in SPM preparations isolated from rats atdifferent ages. The presence of two protein bands of about65 and 68 kDa was determined by immunoblot analysis. The65-kDa band was present at all ages, and its abundance

increased from PD7 to PD20. The 68-kDa band appeared atPD15 and increased until PD30, coinciding with the increaseof e-5NT activity, substrate affinity and enzymatic efficiency.Since distinct e-5NT isoforms may derive from different pat-terns of the enzyme protein N-glycosylation, we speculate thatlong-term regulation of e-5NT activity in adulthood may beeffectuated at posttranslational level and without overallchange in the gene and protein expression.

Keywords Ecto-5′-nucleotidase . Adenosine . Synapticplasmamembranes . Hippocampus . Development . Plasticity

Introduction

Intricate cellular processes during central nervous systemdevelopment are highly regulated by a complex signallingsystems, which include extracellular matrix molecules, multi-ple classes of diffusible ligands and numerous receptors andco-receptor that trigger distinct signalling cascades, as well asan epigenetic gene regulation modulating developmentalswitches (Dickins and Salinas 2013). Adenosine is amongthe most recently identified and least investigated diffusiblesignalling factors that contribute to the structural and func-tional remodelling of the brain, both during embryonic andpostnatal development (Wieraszko 1996; Dias et al. 2013). Byacting at G protein-coupled P1 purinoreceptors that denotedA1, A2A, A2B and A3 (Rivkees 1995; Johansson et al. 1997;Ralevic and Burnstock 1998; Doriat et al. 1999), adenosineparticipates in developmental processes, including cell prolif-eration, immature cell migration, axonal growth, synapseformation and apoptosis (Zimmermann 2006).

I. Grkovic (*) :N. Mitrovic :D. Drakulic :M. Stanojlovic :A. HorvatLaboratory of Molecular Biology and Endocrinology, VINCAInstitute of Nuclear Sciences, University of Belgrade, P.O. Box 522,11001 Belgrade, Serbiae-mail: istanojevic@vinca.rs

I. BjelobabaDepartment of Neurobiology, Institute for Biological Research“Sinisa Stankovic”, University of Belgrade, Blvd Despota Stefana142, 11000 Belgrade, Serbia

N. NedeljkovicInstitute for Physiology and Biochemistry, Faculty of Biology,University of Belgrade, Studentski trg 3, 11000 Belgrade, Serbia

J Mol NeurosciDOI 10.1007/s12031-014-0256-0

Extracellular adenosine originates from two sources; it canbe released directly from neurons and glial cells throughbidirectional equilibrative nucleoside transporters (Latini andPedata 2001), or it can be generated from released ATP, bysequential extracel lular hydrolysis catalyzed byectonucleotidase enzyme pathway (Cunha et al. 1996).Ectonucleotidases include several enzyme families that cata-lyze extracellular degradation of ATP. Ecto-nucleoside tri-phosphate diphosphohydrolases (E-NTPDases) dephosphory-late ATP and ADP to AMP with different substrate specific-ities and affinities (Heine et al. 1999; Zimmermann 2000;Yegutkin 2008). Ecto-5′-nucleotidase (e-5NT; also known asCD73) catalyzes the final and the rate-limiting step of conver-sion of AMP to adenosine (Zimmermann 1996). Therefore, e-5NT critically controls the level of adenosine in the extracel-lular space and duration and extent of P1 receptor activation.

Ecto-5′-nucleotidase is a glycosylphosphatidylinositol(GPI)-linked membrane-bound glycoprotein, with its activesite facing the extracellular compartment. Sequence analysisrevealed that rat e-5NT comprises 576 amino acids (Wadaet al. 1986) and possesses five potential N-linked glycosyla-tion sites (Misumi et al. 1990). Themature form of the enzymeends at Ser-523, which carries the GPI anchor (Ogata et al.1990), and has predicted a molecular mass of 57–59 kDa(Wada et al. 1986). Variations in the enzyme apparent molec-ular mass arise from different patterns of N-linked glycosyla-tion and the nature of glycan chains bound to the matureprotein (Wada et al. 1986; Misumi et al. 1990). Finally, thefunctional membrane-bound enzyme exists as a dimer, withthe two subunits linked by hydrogen bonds and hydrophobicinteractions (Heuts et al. 2012).

During brain development, e-5NT is associated with thesurface of migrating and immature nerve cells (Fenoglio et al.1995) and with a subset of synapses during synaptogenesisand synaptic remodelling (Schoen et al. 1988; Bailly et al.1995; Fenoglio et al. 1995; Heilbronn et al. 1995; Heilbronnand Zimmermann 1995), while in the adult brain, the enzymeis located at the astrocytes, microglia, myelin and nerve cells(Cammer et al. 1985; Schoen et al. 1988; Fuchs 1991;Zimmermann 1992, 1996; Bjelobaba et al. 2007, 2011;Langer et al. 2008). Importantly, e-5NT activity increases upto fivefold during postnatal development in all the tested brainareas (de Paula Cognato et al. 2005; Mackiewicz et al. 2006).Since similar increase was not observed in tissues other thanthe brain (Fuchs 1991), the finding implies that age-dependentincrease in e-5NT activity represents developmental phenom-enon unique to the brain, rather than some common mecha-nisms of cell aging.

Being the last member of the enzyme cascade which de-grades extracellular ATP, it is thought that the main role of e-5NT is the production of adenosine. However, there are sub-stantial data indicating that functions of e-5NT go beyond theactivity of adenosine-producing enzyme (Zimmermann

1996). For instance, localization of P1 receptors in the ratbrain only partially overlaps with e-5NT (Fastbom et al.1987). Furthermore, e-5NT carries the epitopes implicated inthe cell-cell and cell-matrix interactions (Vogel et al. 1993),and since the enzyme exhibits non-homogeneous and polar-ized distribution in many cell types (Wada et al. 1986;Zimmermann 1992), it was implicated in the processes ofintercellular adhesion, signalling and cell migration.

Since e-5NT displays striking age-dependent increase inthe activity, in the present study, we addressed the question ofdevelopmental changes in the expression and function of e-5NT in the hippocampal area, a brain structure that undergoessubstantial synaptic plasticity and rearrangements during de-velopment. We have analyzed e-5NT expression and functionin whole hippocampal formation and in synaptic plasmamembrane preparations isolated from the hippocampal areaduring postnatal development.

Materials and Methods

Animals

Female rats of Wistar strain from VINCA Institute breedingstock were used in all experiments. The animals were kept ona 12-h light/dark regime at a temperature of 22±2 °C and hadfree access to water and food. Animals (n=15 per age group)were sacrificed at postnatal days 7 (PD7), 15 (PD15), 20(PD20), 30 (PD30) and 90 (PD90). Animals in PD7, PD15and PD20 groups were kept in the litter during the wholeexperimental procedure, while animals in PD30 and PD90groups were grouped at the 20th postnatal day and kept inthree/cage until they were sacrificed. Animals were treated inaccordance with the principles from Guide for Care and Useof Laboratory Animals (NIH publication no. 80-23), and theEthical Committee for the Use of Laboratory Animals ofVINCA Institute of Nuclear Sciences, University ofBelgrade, Belgrade, Serbia, approved the protocols.

Analysis of Gene Expression by Semi-Quantitative RT-PCR

The expression analysis of e-5NT was performed by a semi-quantitative RT-PCR assay. Rats of different age (PD7, PD15,PD20, PD30 and PD90; n=3/age) were sacrificed by decap-itation, the brains were removed and hippocampi were dis-sected and immediately frozen in liquid nitrogen. The totalRNA extraction was performed using TRIzol reagent(Invitrogen, USA), in accordance with the manufacturer’sinstructions. The complementary DNA (cDNA) species weresynthesized using First Strand cDNA Synthesis Kit(Fermentas, Lithuania), according to the manufacturer’s in-structions. Preliminary optimization experiments were carriedout to determine amounts of cDNA for non-saturating

J Mol Neurosci

conditions and number of amplification cycles to achievelinear amplification. The cDNAs were amplified usingprimers designed for the amplification of e-5NT (F: 5′-CCCGGGGGCCACTAGCACCTCA-3′; R: 5′-GCCTGGACCACGGGAACCTT-3′), together with a housekeepinggene, glyceraldehide-3-phosphate dehydrogenase (GAPDH,F: 5′-AAGGTGAAGGTCGGAGTCAACG-3′; R: 5′-GGCAGAGATGATGACCCTTTTGGC-3′). For polymerase chainreaction (PCR), appropriate dilutions of cDNA samplesrepresenting 2.5 μg total RNA were mixed with PCR buffercontaining 10mMdeoxyribonucleoside triphosphate, 2.1 mMMgCl2, 0.25 μM primers for e-5NT, 0.125 μM primers forGAPDH, and 1-U Taq polymerase (Kapa Biosystems), in atotal volume of 25 μl. The cDNAs were amplified for 28cycles (Thermal Cycler, Eppendorf), using the following con-ditions: denaturation 94 °C/45 s, annealing 58 °C/1 min,extension 72 °C/1 min, and final extension 72 °C/7 min. ThePCR products were analyzed on a 2 % agarose gel togetherwith an O’GeneRuler (Fermentas) and visualized under UVlight using ethidium bromide. The intensity of PCR productswas quantified using ImageJ software package. The relativeunits related to the e-5NT were normalized against to respec-tive GAPDH products obtained in the same PCR reaction.

Enzyme Histochemistry

For the enzyme histochemistry, the brains were quickly andcarefully removed from the skulls and fixed in 4 %paraformaldehyde/0.1 M phosphate buffer (pH 7.4). Aftercryoprotection in graded sucrose (10–30 % in 0.2 M phos-phate buffer) at 4 °C, 25-μm-thick brain sections weremounted on gelatine-coated slides, dried for 2 h at roomtemperature and stored at −20 °C until use. The e-5NT histo-chemistry was performed using Langer’s modification ofWachstein and Meisel protocol (Wachstein and Meisel 1957;Langer et al. 2008). All solutions were phosphate free andcontained 2 mM levamisole to block endogenous alkalinephosphatase activity. Cryosections were preincubated for30 min at room temperature with TRIS-maleate sucrose(TMS) buffer containing 0.25 M sucrose, 50 mM TRIS-maleate, 2 mM levamisole and 2 mM MgCl2 (pH 7.4). Theenzyme reaction was performed at 37 °C in a TMS-bufferedsubstrate solution for 90 min, containing 1 mM AMP assubstrate in addition to 2 mM Pb(NO3)2, 5 mM MnCl2,2 mM MgCl2, 50 mM TRIS-maleate, 3 % dextran T250 and0.25 M sucrose (pH 7.4). TMS-buffered substrate solutionwithout substrate served as a control. After washing andsubsequent colour development in a solution of 1 % (v/v)(NH4)2S, as a result of e-5NT activity, a brown deposit ofthe enzyme reaction became visible. Finally, the sections weredehydrated in graded ethanol (70%EtOH, 80%EtOH, 100%EtOH, 100 % isopropanol, and 100 % xylol) and mountedwith DPX (Fluka). The sections were examined and

photographed with a Zeiss Axiovert microscope. The sectionswere examined by two observers, who scored relative label-ling intensity independently. Five grades have been assignedto the labelling intensity: − indicates no staining, ± very faint,+ faint, ++ moderate and +++ strong. Neuroanatomical struc-tures were identified and labelled according to Swanson(1998).

Synaptic Membrane Preparations

After decapitation with a small animal guillotine (HarvardApparatus, Holliston, MA, USA), hippocampi from the sameage group (three brains/group) were pooled for synaptic plas-ma membrane (SPM) isolation, starting with ice-cold medium(0.32 M sucrose, 5 mM Tris–HCl, pH 7.4) as describedpreviously (Horvat et al. 2010). The protein content wasdetermined according to the modified Lowry method(Markwell et al. 1978), using bovine serum albumin as astandard. Samples of SPM were kept at −70 °C until use.Three independent SPM isolations were made for each agegroup.

Immunoblotting

Immunoblot analysis was performed as previously de-scribed (Stanojevic et al. 2011). Samples were adjustedto a final SPM protein concentration of 4 mg/ml, mixedwith Laemmli’s sample buffer and boiled for 5 min. Thesamples (40 μg of proteins) were subjected to 10 %polyacrylamide gel electrophoresis (PAGE) in the pres-ence of sodium dodecyl sulphate (SDS), electrophoreti-cally transferred to polyvinylidene difluoride (PVDF)membranes (0.45 μm, from Millipore). After blockingin 5 % BSA in Tris-buffered saline (50 mM Tris-HClpH 7.4, 150 mM NaCl and 0.05 % Tween 20; TBST)for 1 h, support membranes were incubated at 4 °Cwith a primary goat polyclonal anti-e-5NT antibody(1:1.000 dilution; Santa Cruz Biotechnology, SantaCruz) and goat polyclonal anti-β-actin antibody(1:2.000 dilution; Santa Cruz Biotechnology), as a load-ing control. Visualization of bands was performed with3,3′-diaminobenzidine (DAB; Sigma), after incubatingsupport membrane in donkey anti-goat IgG horseradishperoxidase-conjugated antibody (1:7.500 dilution; SantaCruz Biotechnology). Densitometric analysis was per-formed using image analysis software ImageJ. Opticaldensities of each band corresponding to e-5NT werenormalized to the optical density obtained for β-actin.Results are expressed as mean optical density relative toβ-actin ± SEM, from five separate experiments per-formed on three different SPM preparations.

J Mol Neurosci

Ecto-5′-Nucleotidase Assay

Ecto-5′-nucleotidase assay was performed in the reaction me-dium containing the following: 5 mMMgCl2 and 50mMTris-HCl buffer, pH 7.4 and 20 μg of SPM proteins (Stanojevicet al. 2011). The reaction was initiated by the addition of1.0 mM AMP (Sigma-Aldrich, Germany). Following 30-min incubation at 37 °C, the reaction was stopped by theaddition of 22 μl of 3 M per chloric acid and by transferringthe test tube on ice. The release of inorganic phosphate (Pi)was measured by Malachite Green Phosphate Assay Kit(BioAssay Systems), according to the manufacturer’s instruc-tions, with KH2PO4 as a Pi standard. Kinetic properties of e-5NT in the presence of increasing AMP concentrations (0.05–1.1 mM) were determined in the same reaction mixture.Incubation time and protein concentration were chosen inorder to ensure the linearity of the reaction. Data are presentedas mean activity (nmol Pi/mg/min) ± SEM from three inde-pendent experiments performed in triplicate.

Data Analysis

Kinetic analysis of e-5NT activity was performed bycomputer-assisted least square fitting of the data to theMichaelis-Menten equation, while kinetic parametersVmax (apparent maximum activity) and Km (apparentMichaelis constant) values were calculated from Eadie-Hofstee semi-reciprocal plot of V versus V/[S], usingOrigin 7.5 software package. A one-way analysis ofvariance (ANOVA) followed by Tukey’s post hoc test(considering p<0.01 as significant) was used to deter-mine the significant changes in relative protein andmessenger RNA (mRNA) abundance, the enzyme activ-ity and kinetic parameters between different age groups.

Results

e-5NT Gene Expression in Developing Hippocampus

Figure 1 presents a profile of e-5NT-mRNA expression in thehippocampal region during postnatal development. More thantwofold increase in e-5NT-mRNA abundance between PD7and PD15 (p<0.001) and prominent significant increase be-tween PD15 and PD20 (p<0.01) were evidenced, with theexpression plateau that reached at PD20 and remained un-changed through the adulthood.

e-5NT Histochemistry in Developing Hippocampus

Figure 2a–e shows e-5NT histochemistry in the hippocampalsections at different ages. Since the method is based on adeposition of the enzyme activity product at the site of the

reaction, this approach gives both information on the activityand topographical localization of the enzyme. We observedstrong overall age-dependent increase in e-5NT activity in thehippocampal formation. However, closer inspection revealedthat the expression pattern differed in distinct hippocampalsubregions (Table 1). In cornu ammonis (CA) regions, stain-ing intensity increased with age in the stratum oriens andstratum radiatum of the CA1, while in the correspondinglayers of the CA3, the staining intensity increased only slight-ly with age. In the stratum lucidum of CA3, staining was firstdetected at PD15, and afterwards, it gradually increased withage. Staining was very faint in the pyramidal cell layer at PD7(Fig. 2a) and PD15 (Fig. 2b), whereas at later stages, it almostdisappeared. Age-dependent increase in staining intensity wasobserved in dentate gyrus. Specifically, in the inner and outermolecular layers, staining was observed at P20 and afterwards(Fig. 2c–e). Staining intensity increased strongly in thesubgranular cell layer, which generally expressed the mostintense labelling. Although granule cell layer remainedcompletely devoid of staining, fibres traversing this layercould be discerned at PD30 (Fig. 2d) and PD90 (Fig. 2e). At

Fig. 1 e-5NT gene expression in the hippocampal formation at differentages. a Representative agarose gel photographs showing ethidiumbromide-stained RT-PCR products of the e-5NT and GAPDH gene dur-ing postnatal development (PD). b e-5NT-mRNA abundance was obtain-ed by a densitometric analysis of the target mRNA, expressed relative tothe expression of GADPH mRNA. Bars represent mean relative e-5NT-mRNA abundance ± SEM from two independent determinations per-formed in triplicate. Letters indicate significance level of p<0.01 com-pared with PD7 (a) and PD15 (b)

J Mol Neurosci

PD90, the staining was present in all hippocampal regions,more intensive in CA1 and CA2 than in CA3. Exceptionswere the pyramidal and granule cell layers, which were la-belled faintly. In contrast, the heaviest staining was observedin the stratum lucidum, stratum radiatum, outer and inner

molecular layers of dentate gyrus and stratum lacunosum-moleculare which mainly contain fibres and synapses.

Developmental Profile of e-5NT Protein Expressionin the Hippocampal SPM Preparation

Next, we examined the pattern of e-5NT expression and theactivity in SPM preparations at different ages. Figure 3bshows representative support membrane probed with anti-e-5NT antibody after resolving SPM proteins by SDS-PAGEelectrophoresis in non-reducing conditions. In all groups,antibody against e-5NT recognized a prominent band at about65 kDa. Starting from PD15, the antibody recognized a sec-ond band at about 68 kDa. Densitometric analysis revealedthat the abundance of 65-kDa band gradually increased fromPD7 to PD20, when it reached the level attained at later ages(Fig. 3a). The abundance of 68-kDa band also increased withage, reaching the stable level at PD30 (with statistical differ-ence between successive age groups being p<0.01). By cal-culating the ratios between 68- and 65-kDa abundances ateach age, it was concluded that the abundances of 68-kDaband relative to 65-kDa band increased from about 10 % atPD7 to about 80 % at PD90 (Fig. 3c).

Developmental Profile of e-5NTActivity in HippocampalSPM Preparation

e-5NT activity profile was determined by assaying AMPhydrolysis in hippocampal SPM preparations isolated fromrats at different ages (Fig. 4). An almost threefold increase ine-5NT activity was observed between PD7 (13.2±0.7 nmolPi/mg/min) and PD90 (34.5±1.0 nmol Pi/mg/min). Thehighest increment in e-5NT activity was observed between

Fig. 2 Enzyme histochemistry for e-5NT showing staining intensity andtopographical distribution of e-5NT at different postnatal ages. Sectionsobtained from the rat brains at PD7 (a), PD15 (b), PD20 (c), PD30 (d) andPD90 (e) were processed as described in Materials and Methods sectionand analyzed under Zeiss Axiovert microscope. SO stratum oriens, SPpyramidal cell layer, SR stratum radiatum, SLM stratum lacunosum-moleculare, SL stratum lucidum, ML molecular layer, GCL granule celllayer, SGl subgranular layer, PoDG polymorphic layer of dentate gyrus.Scale bar represents 200 μm (scale in c applies to d and e)

Table 1 Relative e-5NTstaining intensity in the hippocampal subregionsat different postnatal ages

Hippocampal subregions PD7 PD15 PD20 PD30 PD90

Cornu Ammonis (CA)

Stratum oriens ± + ++ ++ ++

Pyramidal cell layer ± ± − − −Stratum lucidum − ± + ++ ++

Stratum radiatum ± + + ++ ++

Stratum lacunosum-moleculare ± ++ ++ +++ +++

Dentate gyrus

Outer molecular layer − − + ++ ++

Inner molecular layer − − + ++ ++

Granule cell layer − − − − −Subgranular layer ± + ++ +++ +++

Polymorph layer − + + ++ ++

− no staining, ± very faint, + faint, ++ moderate, +++ strong

PD postnatal day

J Mol Neurosci

PD20 (17.1±0.67 nmol Pi/mg/min) and PD30 (27.4±0.7 nmolPi/mg/min).

To further elaborate observed changes in e-5NT activity,we performed kinetic analysis of the enzyme activity at dif-ferent ages (Fig. 5). Michaelis-Menten plots of initial enzymevelocities versus AMP concentrations showed similar patternsof enzyme activation at all ages. Kinetic parameters, Vmax andKm, were calculated from the Eadie-Hofstee transformation ofthe data from the Michaelis-Menten plots at different ages(insets in Fig. 5a–e) and are presented in Table 2. ANOVAshowed significant effect of age on Vmax and Km values. Asexpected, maximum enzyme velocity, Vmax, increased withage, while Km value, which is the measure of the enzymeapparent affinity for AMP, decreased from PD7 to PD20,

when it reached the level attained at later ages. Table 2 alsodepicts Vmax/Km ratio, which represents enzymatic efficiencyof the enzyme. The e-5NT efficiency increased up to fivefoldfrom PD7 to PD90.

Discussion

The present study shows that expression and function of e-5NT significantly increase in the hippocampal region frombirth to adulthood. By combining gene expression analysisand enzyme histochemistry, we observed that e-5NT geneexpression hit the highest level at P20, while the enzymeactivity continued to increase beyond this age. Namely, atPD7 at the level of hippocampal formation, the e-5NTgene was weakly expressed as well as the enzyme activityin most hippocampal fields, while in some subregions,such as dentate gyrus and stratum lucidum of the CA3,the activity was completely absent. e-5NT gene expressionmarkedly increased from PD7 to PD20, reaching a pla-teau, while the enzyme activity continued to increasebeyond this age, from faint to moderate and strong, de-pending on the hippocampal field. Further analysis re-vealed that layers rich in synapses generally expressedthe highest levels of e-5NT activity, while in layers pop-ulated with neuronal cell bodies, the enzyme expressionwas weak or completely absent.

Since enzyme histochemistry revealed that e-5NT activitywas mainly associated with hippocampal layers rich in syn-apses, the study was further carried out to correlate observedchanges with the expression and function of e-5NT associatedwith the presynaptic compartment. Therefore, we monitored

Fig. 3 Immunoblot analysis of e-5NT protein abundance in hippocampalSPM preparations obtained from rats at different ages. a Relative abun-dances of 65- and 68-kDa bands, relative to the abundance of β-actin.Bars represent mean relative abundance ± SEM, from five immunoblotexperiments performed on three separately isolated SPM preparations. bRepresentative immunoblot obtained by probing the support membrane

with anti-e-5NT antibody and anti-β-actin antibody. Analysis revealedthe presence of two bands corresponding to 65 and 68 kDa. c 68/65-kDaabundance ratio at different ages. PD postnatal day. Letters indicatesignificance level of p<0.01 compared with PD7 (a), PD15 (b) andPD20 (c)

Fig. 4 e-5NT activity in hippocampal SPM preparations isolated fromrats at different ages. Values represent mean activity (nmol Pi/mg/min) ±SEM from three independent experiments performed in triplicate. Lettersindicate significance level of p<0.01 compared with PD7 (a), PD15 (b),PD20 (c) and PD30 (d)

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protein expression and the enzyme kinetics in purified pre-synaptic plasma membrane (SPM) preparations isolatedfrom hippocampi at different ages. At SPM level, e-5NTactivity consistently increased during the ontogeny, whilee-5NT protein abundance reached a plateau at PD20. Thelack of correlation between e-5NT protein expression and

the enzyme activity after PD20 indicates that the modu-lation of enzyme kinetics in adulthood occurs at post-translational level. In fact, in three independently isolatedSPM preparations, we consistently observed two proteinbands of about 65- and 68-kDa molecular weight. The65-kDa band was present at all ages, and its relative

Fig. 5 Michaelis-Menten plots ofinitial velocities versus raisingAMP concentrations (0.05–1.1 mM) in the hippocampal SPMpreparations isolated from PD7(a), PD15 (b), PD20 (c), PD30 (d)and PD90 (e). Symbols representmean activity (nmol Pi/mg/min) ±SEM from three independent ex-periments performed in triplicate.Solid line in each graph representsthe best fit of the experimentaldata obtained by using Origin 7.5software package. Inset to a–eLinear semi-reciprocal Eadie-Hofstee plots of V versus V/[S]from the data presented in thecorresponding figure

J Mol Neurosci

abundance increased and reached a plateau at PD20,while the 68-kDa band first appeared at PD15 and itsrelative abundance increased until PD30. It is knownthat isoforms of e-5NT derive from different patternsof protein N-glycosylation (Zimmermann 1992), whereastwo immunologically distinct e-5NT isoforms had beenpreviously observed in the hippocampal nerve terminals(Cunha et al. 2000). In the current study, the appearanceof 68-kDa band and its continually growing abundance,both absolute and relative to 65-kDa band, overlappedwith significant increment in e-5NT activity and enzy-matic efficiency. Specifically, the abundance of 68-kDaband during the ontogeny increased from about 10 % atPD7 to 80 % at PD90 relative to 65-kDa band. Duringthe same period, the enzyme substrate affinity and max-imum velocity increased twofold, while enzymatic effi-ciency, a kinetic parameter which incorporates bothsubstrate affinity and catalytic ability of an enzyme,increased more than fivefold. Based on the aforemen-tioned results, we conclude that the appearance andincreasing expression of the 68-kDa isoform duringontogeny overlapped with prominent age-dependent in-crease in e-5NT enzyme activity in hippocampal SPMpreparation. The findings may present a ground for theobserved continual increase in the enzyme activity dur-ing hippocampal maturation, without a correspondingincrease in e-5NT protein expression.

As adenosine in the extracellular space arises fromthe action of e-5NT, increase in the enzyme activitysuggests increased production of adenosine during post-natal development. In parallel to e-5NT activity increaseobserved in this study, there are data indicating gradualup-regulation of A1 receptor expression in the hippo-campus (Rivkees 1995). Thus, low e-5NT activity andlow A1 receptor expression early in the postnatal devel-opment could provide conditions for undisturbed nervegrowth. The most prominent rise of e-5NT activitycorresponds to the end of synaptogenesis, suggesting

that the adenosine-mediated signalling may have animportant role at this developmental stage. This typeof signalling and the fact that adenosine receptors andthe enzyme involved in adenosine production are all inclose proximity at synaptic sites would guarantee a highspecificity of action of adenosine at the synaptic level.

Various aspects of e-5NT molecular properties andfunctions have been reported and reviewed in a numberof papers (Zimmermann 1992, 2006; Colgan et al. 2006;Langer et al. 2008). Cumulative evidence shows that e-5NT exhibits highly variable expression and activity indifferent tissues and cell types, whereas alterations ofthe enzyme activity have been associated with consid-erable number of human disorders (Schetinger et al.2007; Deaglio and Robson 2011; Bonan 2012;Ghiringhelli et al. 2012). Additional unique property ofe-5NT activity in the brain is striking the age-dependentup-regulation in all the tested brain areas (de PaulaCognato et al. 2005; Mackiewicz et al. 2006). Thehighly variable levels of e-5NT activity in health anddisease raise an important question of the regulatorymechanism(s) controlling the enzyme expression at dif-ferent levels. Among the several regulatory elementspresent in the promoter region of e-5NT gene (Spychalaet al. 1999), there is a binding site for TCF1/LEF transcriptionfactor, which is the nuclear target of Wnt signalling (Behrenset al. 1996; Molenaar et al. 1996), crucially involved indendritogenesis, axon guidance and synaptogenesis (Ille andSommer 2005; Salinas 2012). Thus, developmental expres-sion of e-5NT is under direct transcriptional control by num-ber of transcription factors (Spychala et al. 1999), growthfactors (Kohring and Zimmermann 1998) and hormones(Bavaresco et al. 2007). However, the results obtained in thisstudy show for the first time that long-term regulation of e-5NT activity in adulthood may be effectuated at the posttrans-lational level, i.e. without overall change in the gene and/orprotein expression. The most ubiquitous posttranslational pro-tein modification is N-glycosylation, a complex series ofreactions catalyzed in the lumen of the endoplasmic reticulumby oligosaccharyltransferase enzymes. Demonstration that in-crease in e-5NTactivity during ontogeny may be regulated ona longer timescale possibly by different pattern of N-glycosyl-ation suggests that this might also be the case in situations inwhich lack of correlation between e-5NT gene/protein expres-sion and enzyme activity was observed (Bavaresco et al. 2007;Brisevac et al. 2012; Da Silva et al. 2012).More generally, thisopens a question of developmental regulation of extremelycomplex enzymatic machinery that executes glycosylation,particularly when defects in the attachment of carbohydrateto protein have been implicated in many inheritable andcongenital developmental disorders (Freeze 2006; Hewitt2009; Hennet 2012), many of which affect nervous systemdevelopment.

Table 2 Kinetic parameters Km, Vmax and Km/Vmax for e-5NT activity atdifferent ages

PD Km (mmol/l) Vmax (nmol Pi/mg/min) Vmax/Km

7 0.25±0.02 16.47±0.50 65.9±3.6

15 0.15±0.01a 17.43±0.44 116.8±5.5a

20 0.09±0.01a,b 18.58±0.34a 201.7±11.9a,b

30 0.10±0.01a,b 30.20±0.71a,b,c 298.7±22.6a,b,c

90 0.11±0.01a,b 35.03±0.78a,b,c,d 324.8±23.4a,b,c,d

Letters in superscripts indicate significance level of p<0.01 or less,compared with a PD7, b PD15, c PD20 and d PD30

PD postnatal day

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Acknowledgments This work was supported by the Ministry of Edu-cation, Science and Technological Development, project nos. 173044 and41014.

References

Bailly Y, Schoen SW, Delhaye-Bouchaud N, Kreutzberg GW, Mariani J(1995) 5′-Nucleotidase activity as a synaptic marker of parasagittalcompartmentation in the mouse cerebellum. J Neurocytol 24:879–890

Bavaresco L, Bernardi A, Braganhol E, Wink MR, Battastini AM (2007)Dexamethasone inhibits proliferation and stimulates ecto-5′-nucleo-tidase/CD73 activity in C6 rat glioma cell line. J Neurooncol 84:1–8

Behrens J, von Kries JP, Kuhl M, Bruhn L, Wedlich D, Grosschedl R,Birchmeier W (1996) Functional interaction of beta-catenin with thetranscription factor LEF-1. Nature 382:638–642

Bjelobaba I, Stojiljkovic M, Pekovic S, Dacic S, Lavrnja I, Stojkov D,Rakic L, Nedeljkovic N (2007) Immunohistological determinationof ecto-nucleoside triphosphate diphosphohydrolase1 (NTPDase1)and 5′-nucleotidase in rat hippocampus reveals overlapping distri-bution. Cell Mol Neurobiol 27:731–743

Bjelobaba I, Parabucki A, Lavrnja I, Stojkov D, Dacic S, Pekovic S,Rakic L, Stojiljkovic M, Nedeljkovic N (2011) Dynamic changes inthe expression pattern of ecto-5′-nucleotidase in the rat model ofcortical stab injury. J Neurosci Res 89:862–873

Bonan CD (2012) Ectonucleotidases and nucleotide/nucleoside trans-porters as pharmacological targets for neurological disorders. CNSNeurol Disord Drug Targets 11:739–750

Brisevac D, Bjelobaba I, Bajic A, Clarner T, Stojiljkovic M, Beyer C,Andjus P, Kipp M, Nedeljkovic N (2012) Regulation of ecto-5′-nucleotidase (CD73) in cultured cortical astrocytes by differentinflammatory factors. Neurochem Int 61:681–688

Cammer W, Sacchi R, Kahn S (1985) Immunocytochemical localizationof 5′-nucleotidase in oligodendroglia and myelinated fibers in thecentral nervous system of adult and young rats. Brain Res 352:89–96

Colgan SP, Eltzschig HK, Eckle T, Thompson LF (2006) Physiologicalroles for ecto-5′-nucleotidase (CD73). Purinergic Signal 2:351–360

Cunha RA, Vizi ES, Ribeiro JA, Sebastiao AM (1996) Preferentialrelease of ATP and its extracellular catabolism as a source ofadenosine upon high- but not low-frequency stimulation of rathippocampal slices. J Neurochem 67:2180–2187

Cunha RA, Brendel P, Zimmermann H, Ribeiro JA (2000)Immunologically distinct isoforms of ecto-5′-nucleotidase in nerveterminals of different areas of the rat hippocampus. J Neurochem 74:334–338

Da Silva RS, Richetti SK, Tonial EM, Bogo MR, Bonan CD (2012)Profile of nucleotide catabolism and ectonucleotidase expressionfrom the hippocampi of neonatal rats after caffeine exposure.Neurochem Res 37:23–30

de Paula Cognato G, BrunoAN, Vuaden FC, Sarkis JJ, Bonan CD (2005)Ontogenetic profile of ectonucleotidase activities from brain synap-tosomes of pilocarpine-treated rats. Int J Dev Neurosci 23:703–709

Deaglio S, Robson SC (2011) Ectonucleotidases as regulators ofpurinergic signaling in thrombosis, inflammation, and immunity.Adv Pharmacol 61:301–332

Dias RB, Rombo DM, Ribeiro JA, Henley JM, Sebastiao AM (2013)Adenosine: setting the stage for plasticity. Trends Neurosci 36:248–257

Dickins EM, Salinas PC (2013) Wnts in action: from synapse formationto synaptic maintenance. Front Cell Neurosci 7:162

Doriat JF, Koziel V, Humbert AC, Daval JL (1999) Medium- and long-term alterations of brain A1 and A2A adenosine receptor

characteristics following repeated seizures in developing rats.Epilepsy Res 35:219–228

Fastbom J, Pazos A, Palacios JM (1987) The distribution of adenosine A1receptors and 5′-nucleotidase in the brain of some commonly usedexperimental animals. Neuroscience 22:813–826

Fenoglio C, Scherini E, Vaccarone R, Bernocchi G (1995) A re-evaluation of the ultrastructural localization of 5′-nucleotidase ac-tivity in the developing rat cerebellum, with a cerium-based method.J Neurosci Methods 59:253–263

Freeze HH (2006) Genetic defects in the human glycome. Nat Rev Genet7:537–551

Fuchs JL (1991) 5′-Nucleotidase activity increases in aging rat brain.Neurobiol Aging 12:523–530

Ghiringhelli F, Bruchard M, Chalmin F, Rebe C (2012) Production ofadenosine by ectonucleotidases: a key factor in tumorimmunoescape. J Biomed Biotechnol 2012:473712

Heilbronn A, Zimmermann H (1995) 5′-Nucleotidase activates and aninhibitory antibody prevents neuritic differentiation of PC12 cells.Eur J Neurosci 7:1172–1179

Heilbronn A, Maienschein V, Carstensen K, Gann W, Zimmermann H(1995) Crucial role of ecto-5′-nucleotidase in differentiation andsurvival of developing neural cells. Neuroreport 7:257–261

Heine P, Braun N, Heilbronn A, Zimmermann H (1999) Functionalcha rac t e r i za t ion o f r a t ec to -ATPase and ec to -ATPdiphosphohydrolase after heterologous expression in CHO cells.Eur J Biochem 262:102–107

Hennet T (2012) Diseases of glycosylation beyond classical congenitaldisorders of glycosylation. Biochim Biophys Acta 1820:1306–1317

Heuts DP,WeissenbornMJ, Olkhov RV, Shaw AM, Gummadova J, LevyC, Scrutton NS (2012) Crystal structure of a soluble form of humanCD73 with ecto-5′-nucleotidase activity. Chembiochem 13:2384–2391

Hewitt JE (2009) Abnormal glycosylation of dystroglycan in humangenetic disease. Biochim Biophys Acta 1792:853–861

Horvat A, Stanojevic I, Drakulic D, Velickovic N, Petrovic S, MilosevicM (2010) Effect of acute stress on NTPDase and 5′-nucleotidaseactivities in brain synaptosomes in different stages of development.Int J Dev Neurosci 28:175–182

Ille F, Sommer L (2005) Wnt signaling: multiple functions in neuraldevelopment. Cell Mol Life Sci 62:1100–1108

Johansson B, Georgiev V, Fredholm BB (1997) Distribution and postna-tal ontogeny of adenosine A2A receptors in rat brain: comparisonwith dopamine receptors. Neuroscience 80:1187–1207

Kohring K, Zimmermann H (1998) Upregulation of ecto-5′-nucleotidasein human neuroblastoma SH-SY5Y cells on differentiation byretinoic acid or phorbolester. Neurosci Lett 258:127–130

Langer D, Hammer K, Koszalka P, Schrader J, Robson S, ZimmermannH (2008) Distribution of ectonucleotidases in the rodent brainrevisited. Cell Tissue Res 334:199–217

Latini S, Pedata F (2001) Adenosine in the central nervous system:release mechanisms and extracellular concentrations. J Neurochem79:463–484

Mackiewicz M, Nikonova EV, Zimmermann JE, Romer MA, Cater J,Galante RJ, Pack AI (2006) Age-related changes in adenosinemetabolic enzymes in sleep/wake regulatory areas of the brain.Neurobiol Aging 27:351–360

Markwell MA, Haas SM, Bieber LL, Tolbert NE (1978) A modificationof the Lowry procedure to simplify protein determination in mem-brane and lipoprotein samples. Anal Biochem 87:206–210

Misumi Y, Ogata S, Hirose S, Ikehara Y (1990) Primary structure of ratliver 5′-nucleotidase deduced from the cDNA. Presence of theCOOH-terminal hydrophobic domain for possible post-translational modification by glycophospholipid. J Biol Chem 265:2178–2183

Molenaar M, van de Wetering M, Oosterwegel M, Peterson-Maduro J,Godsave S, Korinek V, Roose J, Destree O, Clevers H (1996) XTcf-

J Mol Neurosci

3 transcription factor mediates beta-catenin-induced axis formationin Xenopus embryos. Cell 86:391–399

Ogata S, Hayashi Y, Misumi Y, Ikehara Y (1990) Membrane-anchoringdomain of rat liver 5′-nucleotidase: identification of the COOH-terminal serine-523 covalently attached with a glycolipid.Biochemistry 29:7923–7927

Ralevic V, Burnstock G (1998) Receptors for purines and pyrimidines.Pharmacol Rev 50:413–492

Rivkees SA (1995) The ontogeny of cardiac and neural A1 adenosinereceptor expression in rats. Brain Res Dev Brain Res 89:202–213

Salinas PC (2012)Wnt signaling in the vertebrate central nervous system:from axon guidance to synaptic function. Cold Spring Harb PerspectBiol 4:a008003. doi:10.1101/cshperspect.a008003

Schetinger MR, Morsch VM, Bonan CD,Wyse AT (2007) NTPDase and5′-nucleotidase activities in physiological and disease conditions:new perspectives for human health. Biofactors 31:77–98

Schoen SW, Graeber MB, Toth L, Kreutzberg GW (1988) 5′-Nucleotidase in postnatal ontogeny of rat cerebellum: a marker formigrating nerve cells? Brain Res 467:125–136

Spychala J, Zimmermann AG, Mitchell BS (1999) Tissue-specific regu-lation of the ecto-5′-nucleotidase promoter. Role of the camp re-sponse element site in mediating repression by the upstream regu-latory region. J Biol Chem 274:22705–22712

Stanojevic I, Bjelobaba I, Nedeljkovic N, Drakulic D, Petrovic S,Stojiljkovic M, Horvat A (2011) Ontogenetic profile of ecto-5′-nucleotidase in rat brain synaptic plasma membranes. Int J DevNeurosci 29:397–403

Swanson LW (1998) Brain maps: structure of the rat brain. A laboratoryguide with printed and electronic templates for data, models andschematics, 2nd edn. Elsevier, Amsterdam

Vogel M, Zimmermann H, Singer W (1993) Transient association of theHNK-1 epitope with 5′-nucleotidase during development of the catvisual cortex. Eur J Neurosci 5:1423–1425

Wachstein M,Meisel E (1957) Histochemistry of hepatic phosphatases ofa physiologic pH; with special reference to the demonstration of bilecanaliculi. Am J Clin Pathol 27:13–23

Wada I, Himeno M, Furuno K, Kato K (1986) Biosynthesis and intracel-lular transport of rat liver 5′-nucleotidase. J Biol Chem 261:2222–2227

Wieraszko A (1996) Extracellular ATP as a neurotransmitter: its role insynaptic plasticity in the hippocampus. Acta Neurobiol Exp (Wars)56:637–648

Yegutkin GG (2008) Nucleotide- and nucleoside-convertingectoenzymes: important modulators of purinergic signalling cas-cade. Biochim Biophys Acta 1783:673–694

Zimmermann H (1992) 5′-Nucleotidase: molecular structure and func-tional aspects. Biochem J 285(Pt 2):345–365

Zimmermann H (1996) Biochemistry, localization and functional roles ofecto-nucleotidases in the nervous system. Prog Neurobiol 49:589–618

Zimmermann H (2000) Extracellular metabolism of ATP and othernucleotides. Naunyn Schmiedebergs Arch Pharmacol 362:299–309

Zimmermann H (2006) Nucleotide signaling in nervous system develop-ment. Pflugers Arch 452:573–588

J Mol Neurosci