Ontogenetic profile of ecto-5′-nucleotidase in rat brain synaptic plasma membranes

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Int. J. Devl Neuroscience 29 (2011) 397–403

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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu

Ontogenetic profile of ecto-5 -nucleotidase in rat brain synaptic plasma membranes Ivana Stanojevic´ a,∗ , Ivana Bjelobaba b , Nadeˇzda Nedeljkovic´ c , Dunja Drakulic´ a , Snjeˇzana Petrovic´ a , Mirjana Stojiljkovic´ b,c , Anica Horvat a a

Laboratory for Molecular Biology and Endocrinology, Institute of Nuclear Sciences “Vinca”, University of Belgrade, Mike Petrovica 12-14, 11000 Belgrade, Serbia Department of Neurobiology, Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Bulevar Despota Stevana 142, 11000 Belgrade, Serbia c Institute for Physiology and Biochemistry, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia b

a r t i c l e

i n f o

Article history: Received 13 December 2010 Received in revised form 28 February 2011 Accepted 8 March 2011 Keywords: Ecto-5 -nucleotidase AMP hydrolysis Synaptosomes Synaptic plasma membranes Ontogeny profile Rat brain

a b s t r a c t Ecto-5 -nucleotidase (CD73; EC, e-5NT) is regarded as the key enzyme in the extracellular formation of adenosine, which acts as a neuromodulator and important trophic and homeostatic factor in the brain. In the present study, we have investigated e-5NT activity, kinetic properties concerning AMP hydrolysis and the enzyme protein abundance in the purified synaptic plasma membrane (SPM) preparations isolated from whole female rat brain at different ages. We observed pronounced increase in AMP hydrolyzing activity in SPM during maturation, with greatest increment between juvenile (15day-old) and pre-pubertal (30-day-old) rats. Immunodetection of e-5NT protein in the SPM displayed the reverse pattern of expression, with the maximum relative abundance at juvenile and minimum relative abundance in the adult stage. Negative correlation between the enzyme activity and the enzyme protein abundance in the SPM indicates that e-5NT has additional roles in the synaptic compartment during postnatal brain development, other than those related to AMP hydrolysis. Determination of kinetic parameters, Km and Vmax , suggested that the increase in the enzyme activity with maturation was entirely due to the increase in the enzyme catalytic efficiency (Vmax /Km ). Finally, double immunofluorescence staining against e-5NT and presynaptic membrane marker syntaxin provided first direct evidence for the existence of this ecto-enzyme in the presynaptic compartment. The results of the study suggest that e-5NT may be a part of general scheme of brain development and synapse maturation and provide rationale for the previously reported inconsistencies between enzyme immunohistochemical and biochemical studies concerning localization of e-5NT in the brain. © 2011 ISDN. Published by Elsevier Ltd. All rights reserved.

1. Introduction Adenosine is an important neuromodulator and homeostatic regulator in the nervous system (Ribeiro and Sebastiao, 2010), exerting effects in development, cell proliferation, migration, differentiation and synaptic network formation (for review see, Zimmermann, 2006). By acting on its own P1 receptor family coupled to either inhibition (A1 and A3 ) or activation (A2A and A2B ) of adenylate cyclase (Cunha et al., 1996a; Cunha, 2005), adenosine modulates neuronal activity by inhibiting or facilitating synaptic transmission (Stone, 1981; Ribeiro and Sebastiao,

Abbreviations: e-5NT, ecto-5 -nucleotidase; AMP, adenosine monophosphate; SPM, synaptic plasma membrane. ∗ Corresponding author at: Laboratory of Molecular Biology and Endocrinology, Institute of Nuclear Sciences “Vinˇca”, P.O. Box 522, 11001 Belgrade, Serbia. Tel.: +381 113443619, fax: +381 112455561. ´ E-mail address: [email protected] (I. Stanojevic). 0736-5748/$36.00 © 2011 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2011.03.003

2010). Extracellular adenosine originates from two sources; it can be released directly through bidirectional nonconcentrative adenosine transporters (Brundege and Dunwiddie, 1996) or it can arise from released ATP and adenine nucleotides, by extracellular catabolism via ecto-nucleotidase enzyme pathway (Cunha et al., 1996b). Several ecto-nucleotidase enzyme families contribute to the extracellular catabolism of adenine nucleotides. Currently known ecto-nucleotidases include ectonucleotide pyrophosphatase/phosphodiesterase family (E-NPP) and ecto-nucleosidetriphosphate diphosphohydrolase family (ENTPDase), that hydrolyze extracellular ATP and ADP to AMP and ecto-5 -nucleotidase (Zimmermann, 2000), that hydrolyzes AMP to adenosine. This catabolic pathway constitutes the predominant way of extracellular adenosine formation at nerve terminals (Zimmermann, 1996; Cunha et al., 1996b). Ecto-5 -nucleotidase is the rate limiting enzyme in this pathway, since its feed-forward inhibition by ATP and ADP controls the timing and extent of adenosine formation (James and Richardson, 1993) and consequently time course of neuromodulatory effects of adenosine.


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Ecto-5 -nucleotidase (CD73; EC, e-5NT) is an ectoenzyme that is anchored to the extracellular surface of cell membrane through a glycosyl phosphatidylinositol (GPI) linkage. There is considerable amount of evidence showing that this enzyme has distinct role during brain development and plasticity. E-5NT is expressed on the surface of migrating nerve cells during postnatal development (Fenoglio et al., 1995; Schoen et al., 1988) and it becomes transiently associated with synapses during synaptogenesis and synapse remodeling (Bailly et al., 1995; Fenoglio et al., 1995; Schoen and Kreutzberg, 1995). Several studies clearly documented that e-5NT activity increase in brain during ontogeny. Thus, up to five-fold increase of activity was observed in the cerebral cortex (de Paula Cognato et al., 2005; Mackiewicz et al., 2006), hippocampus (Cunha, 2001; de Paula Cognato et al., 2005), spinal cord (Torres et al., 2003) and in most brain regions of aged compared to young rats (Fuchs, 1991). Alterations of e-5NT activity were also observed in developing mouse retina (Braun et al., 1995) and rat cerebral cortex (Naidoo and Pratt, 1954). However, although all cited studies measured specific activity of e-5NT in postnatal stages, none has investigated changes in its kinetic properties with ontogeny. Contrary to the unequivocal findings on age-related increase, cellular distribution of e-5NT in the brain is still matter of controversy. Namely, previous studies on the enzyme distribution in brain faced inconsistencies between enzyme histochemical and immunocytochemical staining. Enzyme histochemical staining and biochemical studies revealed broad distribution of e-5NT and its association with myelin, astrocytes, activated microglia and neurons (for review, see Zimmermann, 1992; Zimmermann, 1996; Langer et al., 2008). However, immunohistochemical methods demonstrated more restricted enzyme localization at glial structures, such as perivascular endfeets (Schoen et al., 1987, 1988), oligodendroglia and myelinated fibers (Cammer et al., 1985), whereas neuronal localization was rarely observed (Nacimiento and Kreutzberg, 1990; Bjelobaba et al., 2007). The same paradox exists in respect to the synaptic localization of e-5NT. Although biochemical studies repeatedly demonstrated presence of AMP hydrolyzing activity in the presynaptic elements (Cunha et al., 1992; Zimmermann, 1992; James and Richardson, 1993; Cunha, 2001; de Paula Cognato et al., 2005; Schmatz et al., 2009; Sigueira et al., 2010), immunocytochemical studies have shown only sporadic association of the enzyme with nerve terminals (Schoen et al., 1987; Schoen and Kreutzberg, 1997; Zimmermann et al., 1993). Therefore, in the present study, we have readdressed the question of the developmental changes in the activity, kinetic properties and the expression of e-5NT in the synaptic plasma membranes isolated from whole brain of female rats. As syntaxin is presynaptic membrane-bound protein involved in synaptic anchoring and exocytosis, also considered a reliable marker of synaptogenesis (Bennett et al., 1992, 1993; Calakos et al., 1994; Sudhof, 1995), we provided evidence of pre-synaptic localization of e-5NT by means of double immunofluorescence staining for e-5NT and syntaxin in purified synaptosomes isolated from female rat brain at different postnatal stages.

2. Materials and methods 2.1. Animals Female rats of the Wistar strain were used in the study: juvenile (15-day-old) (n = 9), pre-pubertal (30-day-old) (n = 9), young adult (60-day-old) (n = 9) and adult (90-day-old) (n = 9). Animals were housed in perspex cages (4–5/cage), with sawdust on the floor, under standard conditions: 12 h light/dark cycle, constant temperature (22 ± 2 ◦ C) and free access to food and water. All animals were treated in accordance with the principles from Guide for Care and Use of Laboratory Animals (NIH Publication No. 80-23) and the Belgrade University Animal Care and Use Committee approved the protocols. Efforts were made to minimize the number of used animals and their suffering.

2.2. Synaptosomes and synaptic plasma membranes preparations After decapitation using a small animal guillotine (Harvard Apparatus, Holliston, MA, USA), whole brains from the same age group (3 brains/group/isolation) were rapidly removed for immediate synaptosome and synaptic plasma membrane (SPM) isolation, starting with ice-cold medium (0.32 mol/l sucrose, 5 mmol/l Tris–HCl, pH 7.4). Synaptosomes from pooled brain homogenates of same group were purified according to modified method of Cotman and Matthews (1971), as described previously (Horvat et al., 2010). Parts of purified synaptosomes from all ages were resuspended in medium (in mmol/l): 140 NaCl, 5 KCl, 1.2 NaH2 PO4 , 5 NaHCO3 , 1 MgCl2 , 10 glucose, 10 Tris–HCl, pH 7.4. The contamination of SPM fractions, based on morphological and biochemical markers, were less than 7% (Horvat et al., 1995). Another part of synaptosomal fraction was proceeded for SPM preparation, as described previously (Horvat et al., 2010). Synaptosomes and SPM protein levels at different ages were determined according to Markwell et al. (1978) using bovine serum albumin as a standard. Samples of SPM were kept at −70 ◦ C until use. Three independent isolations were made for each age group. 2.3. Enzyme assays Ecto-5 -nucleotidase assay was described previously (Horvat et al., 2010). Briefly, reaction mixture contained 50 mmol/l Tris–HCl buffer, pH 7.4, 5 mmol/l MgCl2 , 1.0 mmol/l AMP and 80 ␮g SPM. The reaction mixture was pre-incubated for 10 min at 37 ◦ C. The reaction was started by the addition of AMP and stopped after 30 min by the addition of 22 ␮l 3 mol/l perchloric acid. The samples were chilled on ice 10 min and taken for the assay of released inorganic phosphate (Pi) (Pennial, 1966), using KH2 PO4 as a reference standard. Incubation time and protein concentration were chosen in order to ensure the linearity of the reaction. Activation of e-5NT in the presence of increasing AMP concentrations (0.05–2.5 mmol/l) was determined in the same reaction mixture without varying other conditions. All age samples were run in triplicate in 6 independent determinations from three independent SPM isolations. 2.4. Western blot analysis Samples were adjusted to a final SPM protein concentration of 4 mg/ml, mixed with Laemmli’s sample buffer and boiled for 5 min. Samples (50 ␮g of proteins) were subjected to 10% polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulphate (SDS), electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (0.45 ␮m, from Millipore) for 1 h at 100 V. After blocking in 5% bovine serum albumin in Tris-buffered saline (50 mmol/l Tris–HCl pH 7.4, 150 mmol/l NaCl, 0.05% Tween-20, TBS-T) for 1 h at room temperature, the membranes were incubated overnight at 4 ◦ C with primary antibody against e-5NT (1:1000 dilution, anti-CD73 goat polyclonal, Santa Cruz Biotechnology, Inc., SCB). After four washing periods for 5–10 min with TBS-T, the membranes were incubated with secondary anti-goat horseradish peroxidase (HRP)-conjugated antibody (SCB) (1:7500 dilution) for 2 h at room temperature. The goat polyclonal anti-␤-actin antibody (SCB) was used (1:2000 dilution) as an equal loading control, on the same membrane as e-5NT. Negative control with omitted primary antibody was done. Finally, immunopositive bands were visualized with 3,3 -diaminobenzidine (DAB; Sigma). Image J (image analysis software) was used for semi-quantitative analysis. The finale value of e-5NT relative protein abundance was normalized based on the respective ␤-actin. Values were expressed as mean relative intensity ± SEM. 2.5. Synaptosome immunofluorescence Double labeling of e-5NT and syntaxin was performed as follows. Synaptosomal pellet, concentrations of 1 mg/ml, was resuspended in 1 ml of phosphate buffered saline (PBS) (in mmol/l): 137 NaCl, 2.6 KCl, 1.5 KH2 PO4 , 8.1 Na2 HPO4 , pH 7.4 and was allowed to attach to poly-l-lysine-coated cover slips for 30 min. Then, the synaptosomes were fixed for 15 min with 4% paraformaldehyde in PBS. After several washes in PBS, synaptosomes were incubated for 1 h in PBS containing 5% normal donkey serum, 3% BSA, and 0.1% Triton X-100. Incubation with the appropriate primary antibody was carried overnight at 4 ◦ C. Anti-ecto-5 -nucleotidase antibody was applied at 1:50 dilution, and anti-syntaxin antibody (anti-syntaxin rabbit polyclonal, Santa Cruz Biotechnology, Inc., SCB) was applied in 1:500 dilution. After washing in PBS, synaptosomes were incubated for 2 h at room temperature with the appropriate fluorescent secondary antibodies (donkey anti-rabbit Alexa Fluor 488, donkey anti-goat Alexa Fluor 555; dilution 1:200; Invitrogen, Carlsbad, CA,). Finally, synaptosomes were washed in PBS and mounted on glass slides with Mowiol (Calbiochem). Controls were performed following the same procedure, with the omission of the primary antibodies. Synaptosomes were viewed under a Zeiss Axiovert microscope equipped with camera and EC Plan-Neofluor 100× objective and using an Apotome system for obtaining optical sections. 2.6. Data analysis Data obtained for the enzyme activities are presented as mean activity (nmol Pi/mg/min) ± SEM from three independent SPM preparations (n = 9 rats/developmental stage) performed in triplicate. Kinetic analysis of AMP hydrolyz-

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3. Results 3.1. Developmental profile of e-5NT activity in synaptic plasma membranes AMP hydrolyzing activity showed gradual and significant increase in rats at different ages (Fig. 1). Low level of the enzyme activity was found in the SPM preparation isolated from juvenile rats (7.57 ± 0.40 nmol Pi/mg/min), but increased more than 2-fold in pre-pubertal rats (17.54 ± 3.12 nmol Pi/mg/min) and was 3-fold higher in young adults (21.70 ± 5.51 nmol Pi/mg/min) compared to juvenile animals (F[3,74] = 116.4, p < 0.001). Adult rats had nearly the same level of e-5NT activity (27.62 ± 0.60 nmol Pi/mg/min) as young adults, but significantly higher compared to juvenile animals (F[1,38] = 19.1, p < 0.001).

Fig. 1. Developmental profile of e-5NT activity in SPM preparations isolated from female rat brain at different ages. Enzyme activity was assayed as described in Section 2 in a presence of 1.0 mmol/l AMP. Bars represent mean activity (nnmol Pi/mg/min) ± SEM. from six different determinations performed in triplicate. (#) Indicates significant difference from juvenile (15-day-old) rats (ANOVA followed by Tukey’s test, F = 116.4, p < 0.001). ing activity in each membrane preparation was performed by computer-assisted least square fitting of the data to the Michaelis–Menten equation, while kinetic parameters Vmax (apparent maximum activity) and Km (apparent Michaelis constant) values were calculated from Eadie–Hofstee semi-reciprocal plot of V vs. V/[S], using Origin 7.5 software package. A one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (considering p < 0.05 as significant) was used to determine the significant changes in enzyme activities, kinetic parameters and relative protein abundance between animal groups.

3.2. Kinetic analysis of e-5NT in SPM preparation at different ages We further assessed kinetic properties of the e-5NT during maturation (Fig. 2). Michaelis–Menten plots of initial velocities vs. AMP concentrations at different ages showed similar patterns of enzyme activation with raising AMP concentrations (0.05–2.5 mmol/l). As expected, maximum enzyme activity at the plateau increased consistently with postnatal development. The kinetic parameters Vmax and Km were calculated from the Eadie–Hofstee transformation (insets in Fig. 2A–D) of the data from Michaelis–Menten plots at different ages (Table 1). Analysis of variance showed significant effect of maturation for Vmax (F[3.8] = 124.3, p < 0.001), whereas Km values did not differ

Fig. 2. Michaelis–Menten plots of initial velocities vs. raising AMP concentrations at day 15 (A), day 30 (B), day 60 (C) and day 90 (D). The enzyme assay was performed as described in Section 2 in presence of 0.05–2.5 mmol/l AMP. Bars represent mean activity (nmol Pi/mg/min) ± SEM. from six independent experiments performed in triplicate. Solid lines represent best fit obtained by using Origin 7.5 software package. Inset (A–D): linear semi-reciprocal Eadie–Hofstee plots of V vs. V/[S] from data presented in the figure.


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Table 1 Kinetic parameters Km , Vmax and Km /Vmax for ecto-5 -nucleotidase activity at different ages. Age (days)

Km (mmol/l)

Juvenile (15) Pre-pubertal (30) Young adults (60) Adults (90)

0.32 0.29 0.30 0.49

± ± ± ±

0.02 0.02 0.09 0.07*

Vmax (nmol Pi/mg/min) 8.70 19.15 26.98 37.51

± ± ± ±

0.24 0.43# 2.70# 2.61#

Vmax /Km 27.3 66.0 89.9 76.5

± ± ± ±

0.1 0.4# 2.0# 6.0#

Kinetic parameters Km and Vmax were obtained by Eadie–Hofstee transformation of the data presented in Fig. 2A–D, whereas Vmax /Km values were obtained from the basic constants. * p < 0.05 – indicates significant difference from juvenile rats. # p < 0.001 – indicates significant difference from juvenile rats.

Fig. 4. Western blot of e-5NT (A) and relative protein abundance (B) in the SPM preparations isolated from whole brains of rats at different ages. The finale value of e-5NT relative protein abundance was normalized based on the respective ␤-actin. Bars represent mean ± SEM. from six independent experiments. (**) Indicates difference from juvenile animals (ANOVA followed by Tukey’s test, F = 5.728, p < 0.01).

between juvenile, pre-pubertal and young adults, but significantly increased in adult animals (F[3,8] = 7.71, p < 0.05), compared to all age groups. Vmax /Km ratios showed that enzyme physiological efficiency increased 2–3-fold in adult compared to juvenile rats (F[3,8] = 217.1, p < 0.05).

brane marker syntaxin at different ages. Our Western blot results indicated that the level of syntaxin in SPM isolated from juvenile rat brains was lower compared to SPM from pre-pubertal, young adult and adult rats (data not shown). As shown in Fig. 5, presence of double-labeled structures suggests that e-5NT was indeed present in the presynaptic membrane compartment. However, we have also observed single labeled, e-5NT – or syntaxin-positive structures, indicating that at part of the e-5NT was not associated with presynaptic membranes. The relative number of double labeled, e5NT-syntaxin-positive synaptosomes was the highest in juvenile rats and declines with the maturation.

3.3. E-5NT immunodetection

4. Discussion

The specificity of the antibody used for immunodetection of e5NT was tested using SPM fraction isolated from adult rat brain. As shown in Fig. 3, the antibody recognizes one prominent band at 68 kDa corresponding closely to the molecular weight previously shown for e-5NT from mammalian brain (Vogel et al., 1992). Fig. 4 presents representative Western blot membrane (Fig. 4A) and relative protein abundance of e-5NT in SPM preparations isolated at different postnatal stages (Fig. 4B). Analysis of the data revealed significant negative effect of maturation, in young adult and adult (F[3,40] = 5.728, p < 0.01), for the abundance of e-5NT in the whole brain SPM compared to juvenile rats. Namely, the enzyme protein was more abundant in the SPM preparation isolated from juvenile rats and its abundance consistently decreased with age, being the lowest in adult animals.

The present study confirmed what was previously shown (Fuchs, 1991; Torres et al., 2003; Mackiewicz et al., 2006), that AMP hydrolyzing activity consistently and significantly increases in the synaptic plasma membranes (SPM) isolated from whole female rat brain during ontogeny. As extracellular AMP hydrolysis in synaptic cleft occurs through the action of extracellular surface-located e-5NT, increase in this enzyme activity indicates efficient production of adenosine. However, only when extracellular ATP and ADP levels decrease below the threshold of inhibition of e-5NT, adenosine will be formed in a considerable amount (James and Richardson, 1993). Since the production of AMP is possibly altered with maturation, it is difficult to conclude whether the increased e-5NT activity, which was measured in vitro, will or will not result in increased extracellular adenosine in vivo. Since the most pronounced increase in e-5NT activity coincides with the onset of gonadal function in female rats (Baker, 1979), it is possible that e-5NT is under the regulation of gonadal steroids. Since the study of Torres et al. (2003) reported similar prominent increase in the enzyme activity in the spinal cord synaptosomes of both male and female rats, and unpublished data from our labora-

Fig. 3. Western blot analysis of SPM preparations isolated from whole adult brains. Proteins (50 ␮g per lane) were resolved on 10% gel, transferred to PVDF membranes and probed with anti-e-5NT antibody. E-5NT antibody specifically stained one band at about 68 kDa. Lane 1 shows molecular weight markers, and both lanes 2 and 3 are SPM from 90 days old rat brains.

3.4. Inmunocytochemical localization of e-5NT in purified synaptosomes In order to verify whether e-5NT was indeed associated with SPM, we analyzed the distribution of e-5NT and presynaptic mem-

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Fig. 5. Co-localization of syntaxin and e-5NT in synaptosomes. Double labeling of purified synaptosomes from 15-, 30-, 60- and 90-day-old rats with e-5NT (red) and syntaxin (green) antibodies, and the merged (yellow) of two images. C – control, the background fluorescence in the absence of the primary antibodies; T – phase contrast image of synaptosomes. Scale bar = 3 ␮m in all images. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

tory have shown that level of AMP hydrolyzing activity did not vary with the phase of oestrus cycle, we concluded that female gonadal steroids were not related to the observed alterations. On the other hand, given that similar prominent increase in e-5NT activity in the same developmental period was observed in almost all brain areas (Fuchs, 1991; Torres et al., 2003; Mackiewicz et al., 2006), but not in the other body tissues or cell types (Fuchs, 1991), it was reasonable

to conclude that such phenomena did not reflect a general mechanism of cellular aging, but rather some developmental processes, which are unique to the brain. The novel finding of our study was a direct negative correlation between e-5NT activity and its protein abundance in the SPM at different ages. In other words, e-5NT protein was the most abundant in SPM at juvenile stage when its specific activity was the


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lowest, and the least abundant in the adult stage when the enzyme specific activity was the highest. What could be the physiological explanation of the observed discrepancy? Like many GPI-anchored proteins, e-5NT may have functions other than its catalytic activity, such as cell adhesion. In the developing cerebral cortex, between 3 and 9 weeks of age, e-5NT carries HNK-1 epitope, which is implicated in cell-to-cell and cell-to-matrix interactions (Dieckhoff et al., 1986; Vogel et al., 1991; Olmo et al., 1992; Sadej et al., 2006). However, the epitope disappears from the enzyme at 12 weeks of age and afterwards (Vogel et al., 1993). The transient appearance of the HNK-1 epitope in e-5NT protein coincides with the juvenile to pre-pubertal periods in our study, when the enzyme displays the highest abundance and the lowest activity. Thus, the high abundance of e-5NT during juvenile period may be connected with its roles other than catalytic activity, such as synaptogenesis and refinement of synaptic connections during this earliest stage of postnatal development (Vogel et al., 1993) and these interactions may modulate the enzyme catalytic activity (Dieckhoff et al., 1986; Olmo et al., 1992; Sadej et al., 2006). Consecutively, decrease in the enzyme relative abundance may be the results of developmental down-regulation during later stages of ontogeny, when e-5NT functions solely as a final member of the ectonucleotidase pathway. Our finding raises yet another question: if e-5NT expression in SPM progressively decline during the ontogeny, what could be the mechanism of the increase in its catalytic activity with aging? To address this question we performed kinetic analysis of e-5NT activity and calculated kinetic constants, Km and Vmax at different ages. Kinetic analysis showed significant progressive increase in Vmax between juvenile, pre-pubertal and young adults without changes in apparent Km and further increase of Vmax in adult age with only slight increase in Km value. Therefore, since immunodetection showed significant decline in abundance of the enzyme protein with maturation, we concluded that increase in e-5NT activity in SPM with development was entirely due to the increase in the enzyme catalytic efficiency (Vmax /Km ). The changes we have observed in the enzyme kinetic properties during maturation might be caused by changes in the enzyme protein conformation and topography that has been found during the brain development (Schoen et al., 1988; Zimmermann, 1992; Vogel et al., 1993). Namely, functional e-5NTs are a homodimers with highly diverse pattern of glycosylation (Fini et al., 2003; Strater, 2006), that could influence the enzyme kinetic properties. In addition, e-5NT dimers associate with each other and with other ectonucleotidases, P1 and P2 receptors and nucleoside transporters to form hetero-oligomeric complexes (Schicker et al., 2009) that contribute to e-5NT finetuning. Thus, topology of purinergic components at the cell surface, that could be a subject of developmental changes, might be responsible for changes in the kinetics we observed during the ontogeny (Kenworthy and Edinin, 1998).E-5NT enzyme assay and the Western blot analysis in SPM fraction suggest that the enzyme is associated with the presynaptic membrane compartment. Yet, while the SPM fraction was of satisfactory purity, we could not exclude that the fraction contains some glial or post-synaptic “contamination” that could contribute to the enzyme assay and immunodetection of e-5NT. We have found co-localization of e-5NT protein with individual syntaxin-positive synaptosomes at all ages and thus verify unambiguously that the enzyme was associated with the presynaptic plasma membrane compartment. Using an enzyme cytochemical method, Schoen and Kreutzberg (1997) were localized e-5NT activity on the glial cells and at the main types of asymmetric synapses; the percentage of labeled synapse increased until adulthood (Bailly et al., 1995). In addition, neuronal syntaxin was reported to be limited in distribution to asymmetric (type I) terminals (but not at all) in rat brain, indicating that protein is localized primarily to a subpopulation of synapses that use excitatory neurotransmitter systems (Sesack and Snyder, 1995). We have also found

syntaxin positive synaptosomes without e-5NT signal and in much lesser extent, e-5NT immunosignal associated with unidentifiable syntaxin-negative structures. Therefore, the immunofluorescence microscopic study confirmed that at least part of e-5NT was located in the part of excitatory synapses at all ages. The e-5NT might regulate local levels of its product adenosine, that acts as a potent trophic factor regulating development, growth, cell proliferation and differentiation (Neary and Burnstock, 1996), neurite extension (Abbracchio et al., 1989), neurogenesis (Weaver, 1996) and apoptosis (Abbracchio et al., 1995; Bronte et al., 1996), as well as a neuromodulator (Zimmermann, 1996) affecting the level of neuronal activity. Furthermore, there is considerable evidence that the e-5NT is associated with the neural surface during synaptic plasticity and remodeling (review in Zimmermann, 2006) and plays a significant role in cellular contacts during synaptic formation. Low enzyme activity and high expression in early postnatal development, indicated e-5NT adhesion role in synaptogenesis and possibly participation in the substrate selection of the developing synapses. Its function may thus go beyond its activity as an adenosine-producing enzyme. When the intensive formation of synapses is completed, then the e-5NT has mainly hydrolyzing activity that is seen in adulthood. In this respect, changes in the e-5NT activity and abundance during ontogeny suggest that the enzyme may be part of general scheme of brain development and maturation. In summary, the results presented in this study provide direct evidence for the existence of e-5NT in the presynaptic compartment. The e-5NT protein is enriched in the presynaptic compartment in early postnatal development and decreases with maturation, whereas its activity follows the reverse pattern of expression. Such changes could have broad influence on the process of brain development and maturation and additionally provide rationale for the inconsistency between enzyme immunohistochemical and biochemical studies concerning the cellular and subcellular localization of e-5NT in the nervous system. Conflict of interest The authors declare no conflict of interest. Acknowledgments This study was supported by Serbian Ministry of Science and Technology projects nos. 173044 and 41014. References Abbracchio, M.P., Cattabeni, F., Clementi, F., Sher, E., 1989. Adenosine receptors likened to adenylate cyclase activity in human neuroblastoma cells: modulation during cell differentiation. Neuroscience 30, 819–825. Abbracchio, M.P., Ceruti, S., Barbieri, D., Franceschi, C., Malorni, W., Biondo, L., Burnstock, G., 1995. A novel action for adenosine: apoptosis of astroglial cells in rat brain primary cultures. Biochem. Biophys. Res. Commun. 213, 908–915. Bailly, Y., Schoen, S.W., Delhaye-Bouchaud, N., Kreutzberg, G.W., Mariani, J., 1995. 5 -Nucleotidase activity as a synaptic marker of parasagittal compartmentation in the mouse cerebellum. J. Neurocytol. 24, 879–890. Baker, D.J., 1979. Reproduction and breeding. In: Baker, D.J., Lindsey, J.R., Wisbroth, S.H. (Eds.), The Laboratory Rat Biology and Diseases, vol. 1. Academic Press, New York, pp. 154–167. Bennett, M.K., Calakos, N., Scheller, R.H., 1992. Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science 275, 255–259. Bennett, M.K., Garcia-Araras, J.E., Elferink, L.A., Peterson, K., Fleming, A.M., Hazuka, C.D., Scheller, R.H., 1993. The syntaxin family of vesicular transport receptors. Cell 74, 863–873. Bjelobaba, I., Stojiljkovic, M., Pekovic, S., Dacic, S., Lavrnja, I., Stojkov, D., Rakic, Lj., Nedeljkovic, N., 2007. Immunohistological determination of ecto-nucleoside triphosphate diphosphohydrolase1 (NTPDase1) and 5 -nucleotidase in rat hippocampus reveals overlapping distribution. Cell. Mol. Neurobiol. 27, 731–743.

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