Developmental Increase in Ecto-5′-Nucleotidase Activity Overlaps with Appearance of Two Immunologically Distinct Enzyme Isoforms in Rat Hippocampal Synaptic Plasma Membranes

Abstract

Ecto-5'-nucleotidase (e-5NT), a glycosylphosphatidylinositol-linked membrane protein, catalyzes a conversion of AMP to adenosine, which influences nearly every aspect of brain physiology, including embryonic and postnatal brain development. The present study aimed to investigate a pattern of expression, activity and kinetic properties of e-5NT in the hippocampal formation and synaptic plasma membrane (SPM) preparations in rats at postnatal days (PDs) 7, 15, 20, 30 and 90. By combining gene expression analysis and enzyme histochemistry, we observed that e-5NT mRNA reached the adult level at PD20, while the enzyme activity continued to increase beyond this age. Further analysis revealed that hippocampal layers rich in synapses expressed the highest levels of e-5NT activity, while in layers populated with neuronal cell bodies, the enzyme activity was weak or absent. Therefore, activity and expression of e-5NT were analyzed in SPM preparations isolated from rats at different ages. The presence of two protein bands of about 65 and 68 kDa was determined by immunoblot analysis. The 65-kDa band was present at all ages, and its abundance increased from PD7 to PD20. The 68-kDa band appeared at PD15 and increased until PD30, coinciding with the increase of e-5NT activity, substrate affinity and enzymatic efficiency. Since distinct e-5NT isoforms may derive from different patterns of the enzyme protein N-glycosylation, we speculate that long-term regulation of e-5NT activity in adulthood may be effectuated at posttranslational level and without overall change in the gene and protein expression.

Unveiling the Role of Ecto-5′-Nucleotidase/CD73 in Astrocyte Migration by Using Pharmacological Tools

“…Based on the carbohydrate content, CD73 may be classified as a high-mannose type, complex carbohydrates type and hybrid type, which contains both complex carbohydrates and sialic acid residues ( Meflah et al, 1984 ; van den Bosch et al, 1986 , 1988 ; Wada et al, 1986 ; Baron and Luzio, 1987 ). As it is quite clear that the differences in glycosylation may be responsible for the variations found in apparent molecular weight of CD73 isolated from different sources ( Turnay et al, 1989 ; Olmo et al, 1992 ; Zimmermann, 1992 ; Navarro et al, 1998 ; Grkovic et al, 2014 ; Lavrnja et al, 2015 ), it has been increasingly evident that the variations in glycan content may be responsible for the functional differences of CD73 found in different cell types and tissues. Since glycans are charged molecules, changes in the carbohydrate composition of CD73 or incorporation of negatively charged sialic acid molecules ( Meflah et al, 1984 ), may significantly affect membrane charge density and thus cell behavior.…”

“…Since glycans are charged molecules, changes in the carbohydrate composition of CD73 or incorporation of negatively charged sialic acid molecules ( Meflah et al, 1984 ), may significantly affect membrane charge density and thus cell behavior. Indeed, some earlier and recent studies demonstrate that the post-translational modifications of CD73 and alterations in the carbohydrate content may be responsible for a short-term ( Vogel et al, 1991 ; Schoen and Kreutzberg, 1994 ; Lavrnja et al, 2015 ) and long term ( Grkovic et al, 2014 ) regulation of CD73 functions, including its role in cell adhesion and migration. All these data point out that the regulation of CD73 by cells is a very complex mechanism, and include transcriptional and post-translational modification, together with cell-specific and tissue-specific regulators that have yet to be established.…”

Neonatal Seizures and Purinergic Signalling

“…NTPDase 2, which is the dominant ectonucleotidase expressed by progenitors in the late embryonic and adult mouse brain, has been identified from E18 in neurogenic regions [259,260], whereas NTPDases 1, 3, 5 and 6 are detected in later stages of brain development (P7-21) [261]. Concerning ecto-5´-ectonucleotidase, its expression increases during postnatal stages (it has been identified in migrating neuroblasts of the cerebellum and is related with synaptogenesis processes [192,[262][263][264][265]). Certain ectonucleotidases of the E-NNPs family are also expressed at early stages of neural development, such as E-NNP-2, for which the splice variant autotaxin was identified in the floor plate of the neural tube at E9.5 [266].…”

Role of Ectonucleotidases in Synapse Formation During Brain Development: Physiological and Pathological Implications

“…Moreover, it is found that expression and function of eN significantly increases in the hippocampal region from birth to adulthood, whereby eN-mRNA markedly increases from PD7 to PD20, reaching a plateau, while the enzyme activity continued to increase beyond this age. Namely, at PD7, the eN gene is weakly expressed concomitant with poor enzyme activity in most hippocampal fields, while in some subregions the activity was completely absent [114]. Further analysis also reveals that layers rich in synapses generally displays the highest levels of eN activity, while in neuronal cell bodies layers, the enzyme expression is weak or completely absent.…”

“…To date, there is only scarce information concerning the role of ATP, other nucleotides as well as expression pattern of individual ectonucleotidases in the development of synaptic network activity, although it is shown by several studies that synaptic compartments comprise ectonucleotidases, i.e . NTPDase1–3, eN, [68, 87, 100, 114, 115, 126, 132-134]. It is also found that TNAP is selectively expressed in the synaptic cleft of sensory cortical areas in adult primates [69] and in humans [135] and its activity is regulated by sensory experience in the primate visual cortex [82].…”