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Biomedical subjects

J Blasi

Publications and source records attributed to J Blasi.

At least 19 recordsLinked to original sources

Early induction of secretoneurin expression following kainic acid administration at convulsant doses in the rat and gerbil hippocampus.

The expression of secretogranin-II and its major proteolytic product secretoneurin (SN) is under the control of neuronal excitation, as demonstrated by treating rats with the excitotoxic kainic acid (KA). Differences in the structure and function of the hippocampus in rats and gerbils have been described; these suggest possible differential reactive responses to KA. In the present study, the SN immunostaining pattern in relation with cell damage is analyzed from 6 h to 4 days following KA administration in rats and gerbils. Dramatic differences in the expression of SN were found in the hippocampal complex following KA administration in gerbils and rats. A robust increase in SN immunoreactivity was detected in the pyramidal cell layer of the rat hippocampus, especially in the CA1 area. In the gerbil, however, a strong increase in SN immunostaining was detected in interneurons of the hippocampal formation, as shown by double-labeling immunohistochemistry to SN and the calcium-binding proteins parvalbumin, calbindin, and calretinin. In addition, no damage (in the hippocampal formation) or moderate damage (in the entorhinal cortex) was observed in the gerbil, in contrast to the rat. The administration of KA and the GABA-B receptor inhibitors (CGP56999A or CGP36742) to the gerbil resulted in a strong rise in SN immunoreactitivty in the CA1 pyramidal cell layer of the hippocampus, as in the rat. However, no increased cell damage was observed under these conditions. The present data provide evidence of a species-differential reactive response to KA that might be based, in part, on distinct inhibitory intrahippocampal circuitry.

Animals↗

HC fragment (C-terminal portion of the heavy chain) of tetanus toxin activates protein kinase C isoforms and phosphoproteins involved in signal transduction.

A recent report [Gil, Chaib-Oukadour, Pelliccioni and Aguilera (2000) FEBS Lett. 481, 177-182] describes activation of signal transduction pathways by tetanus toxin (TeTx), a Zn(2+)-dependent endopeptidase synthesized by the Clostridium tetani bacillus, which is responsible for tetanus disease. In the present work, specific activation of protein kinase C (PKC) isoforms and of intracellular signal-transduction pathways, which include nerve-growth-factor (NGF) receptor trkA, phospholipase C(PLC)gamma-1 and extracellular regulated kinases (ERKs) 1 and 2, by the recombinant C-terminal portion of the TeTx heavy chain (H(C)-TeTx) is reported. The activation of PKC isoforms was assessed through their translocation from the soluble (cytosolic) compartment to the membranous compartment, showing that clear translocation of PKC-alpha, -beta, -gamma and -delta isoforms exists, whereas PKC-epsilon showed a slight decrease in its soluble fraction immunoreactivity. The PKC-zeta isoform showed no consistent response. Using immunoprecipitation assays against phosphotyrosine residues, time- and dose-dependent increases in tyrosine phosphorylation were observed in the trkA receptor, PLCgamma-1 and ERK-1/2. The effects shown by the H(C)-TeTx fragment on tyrosine phosphorylation were compared with the effects produced by NGF. The trkA and ERK-1/2 activation were corroborated using phospho-specific antibodies against trkA phosphorylated on Tyr(490), and antibodies against Thr/Tyr phosphorylated ERK-1/2. Moreover, PLCgamma-1 phosphorylation was supported by its H(C)-TeTx-induced translocation to the membranous compartment, an event related to PLCgamma-1 activation. Since H(C)-TeTx is the domain responsible for membrane binding and lacks catalytic activity, the activations described here must be exclusively triggered by the interaction of TeTx with a membrane component.

Animals↗

Differential regulation of chromogranin A, chromogranin B and secretoneurin protein expression after transient forebrain ischemia in the gerbil.

The chromogranin/secretogranin family of proteins is widely distributed in the central nervous system, where they are stored in large dense-core vesicles. These proproteins are actively processed into small neuroactive peptides, which influence neurotransmitter release, microglial activation and monocyte migration. These properties suggest a possible role of chromogranins/secretogranins in the response that follows central nervous system injury. In the present study, the temporal pattern of expression and the distribution of chromogranin A, chromogranin B and secretoneurin, the major proteolytic product of secretogranin-II, have been studied by immunohistochemistry after 5 min of transient forebrain ischemia in the Mongolian gerbil. A strong increase in the immunoreactivity for chromogranin A and secretoneurin was found in the CA3 pyramidal cell layer of the hippocampus, starting at 12 h, with a peak at 24 h and decrease at 48 h after transient forebrain ischemia. In the hippocampal formation, a rise in chromogranin A immunoreactivity was detected in neurons of the subiculum and the granule cell layer of the dentate gyrus. In addition, increase in the immunoreactivity for chromogranin A and secretoneurin was found in selected neurons of the neocortex. Chromogranin A and secretoneurin immunostaining patterns were similar in ischemic and control gerbils at 4 and 7 days following the ischemic insult. Chromogranin A and secretoneurin immunoreactivity in consecutive sections showed co-localization of both antigens but also selective overexpression of chromogranin A or secretoneurin in various neurons. No changes in chromogranin B immunoreactivity were detected across the time course following transient forebrain ischemia. These data indicate that changes in the expression of the chromogranin family of proteins after ischemia are selective for chromogranin A and secretoneurin. To our knowledge, this is the first study showing that the expression of the chromogranin family of proteins is differentially regulated after an ischemic insult in selected neuronal populations of the hippocampal formation and the cerebral cortex. Furthermore, the present data suggest a possible implication of chromogranin A and secretoneurin in the pathophysiology of transient forebrain ischemia.

Animals↗

Involvement of SNAP-23 and syntaxin 6 in human neutrophil exocytosis.

To understand the molecular basis of exocytosis in human neutrophils, the role of syntaxin 6 and SNAP-23 in neutrophil degranulation was examined. Human syntaxin 6 was cloned and identified as a 255-amino acid protein with a carboxy-terminal transmembrane region and two coiled-coil domains. Syntaxin 6 was localized mainly in the plasma membrane of human resting neutrophils, whereas SNAP-23 was located primarily in the mobilizable tertiary and specific granules. SNAP-23 was translocated to the cell surface, colocalizing with syntaxin 6, on neutrophil activation. In vitro binding studies established that SNAP-23 binds to syntaxin 6. Coimmunoprecipitation assays indicated that SNAP-23 interacts with syntaxin 6 in vivo, and this interaction was dramatically increased on neutrophil activation. Antibodies against SNAP-23 inhibited Ca(++) and GTP-gamma-S-induced exocytosis of CD67-enriched specific granules, but they hardly affected exocytosis of the CD63-enriched azurophilic granules, when introduced into electropermeabilized neutrophils. Anti-syntaxin 6 antibodies prevented exocytosis of both CD67- and CD63-enriched granules in electropermeabilized neutrophils. These data show that syntaxin 6 and SNAP-23 are involved in human neutrophil exocytosis, demonstrating that vesicle SNAP receptor-target SNAP receptor (v-SNARE- t-SNARE) interactions modulate neutrophil secretion. Syntaxin 6 acts as a target for secretion of specific and azurophilic granules, whereas SNAP-23 mediates specific granule secretion.

Amino Acid Sequence↗

Cellubrevin is present in the basolateral endocytic compartment of hepatocytes and follows the transcytotic pathway after IgA internalization.

The endocytic compartment of polarized cells is organized in basolateral and apical endosomes plus those endocytic structures specialized in recycling and transcytosis, which are still poorly characterized. The complexity of the various populations of endosomes has been demonstrated by the exquisite repertoire of endogenous proteins. In this study we examined the distribution of cellubrevin in the endocytic compartment of hepatocytes, since its intracellular location and function in polarized cells are largely unknown. Highly purified rat liver endosomes were isolated from estradiol-treated rats, and the early/sorting endosomal fraction was further subfractionated in a multistep sucrose density gradient, and studied. Analysis of dissected endosomal fractions showed that cellubrevin was located in early/sorting endosomes, with Rab4, annexins II and VI, and transferrin receptor, but in a specific subpopulation of these early endosomes with the same density range as pIgA and Raf-1. Interestingly, only in those isolated endosomal fractions, endosomes enriched in transcytotic structures (of livers loaded with IgA), the polymeric immunoglobulin receptor specifically co-immunoprecipitated with cellubrevin. In addition, confocal and immuno-electron microscopy identification of cellubrevin in tubular structures underneath the sinusoidal plasma membrane together with the re-organization of cellubrevin, in the endocytic compartment, after the IgA loading, strongly suggest the involvement of cellubrevin in the transcytosis of pIgA.

Animals↗

Differential interaction patterns in binding assays between recombinant syntaxin 1 and synaptobrevin isoforms.

Syntaxin 1 and synaptobrevin play an essential role in synaptic vesicle exocytosis. Two isoforms for each of these proteins, syntaxin 1A and 1B and synaptobrevin 1 and 2, have been found in nerve endings. Previous morphological studies have revealed a characteristic co-localization of syntaxin 1 and synaptobrevin isoforms in nervous and endocrine systems; however, the physiological significance of differential distribution is not known. In the present study an in vitro assay has been used to study a possible isoform specific interaction between syntaxin and synaptobrevin isoforms. The results show that although both syntaxin 1A and 1B may interact with synaptobrevin 1 and 2, this interaction is not uniform, showing different affinity patterns depending on the syntaxin 1/synaptobrevin isoform combination. The addition of SNAP-25 increased the binding capacity of syntaxin and synaptobrevin isoforms without affecting specific interactions.

Animals↗

Transient increase of synapsin-I immunoreactivity in the mossy fiber layer of the hippocampus after transient forebrain ischemia in the mongolian gerbil.

Synapsin-I is a vesicular phosphoprotein, which regulates neurotransmitter release, neurite development, and maturation of synaptic contacts during normal development and following various brain lesions in adulthood. In the present study, we have examined by immunohistochemistry possible modifications in the expression of synapsin-I in the hippocampus of Mongolian gerbils after transient forebrain ischemia. The animals were subjected to 5 min of transient forebrain ischemia through bilateral common carotid occlusion, and were examined at different time-points post-ischemia. Transient forebrain ischemia produces cell death of the majority of CA1 pyramidal neurons of the hippocampus and polymorphic hilar neurons of the dentate gyrus. This is followed by reactive changes, including synaptic reorganization and modifications in the expression of synaptic proteins, which provide the molecular bases of synaptic plasticity. Transient decrease of synapsin-I immunoreactivity was observed in the inner zone of the molecular layer of the dentate gyrus, thus suggesting denervation and posterior reinervation in this area. In addition, a strong increase in synapsin-I immunoreactivity was observed in the hilus of the dentate gyrus and in the mossy fiber layer of the hippocampus at 2, 4 and 7 days after ischemia. Parallel increases in synaptophysin immunoreactivity were not observed, thus suggesting a selective induction of synapsin-I after ischemia. The present results indicate that synapsin-I participates in the reactive response of granule cells to transient forebrain ischemia in the hippocampus of the gerbil, and suggest a role for this protein in the plastic adaptations of the hippocampus following injury.

Animals↗

Syntaxin 1A and 1B display distinct distribution patterns in the rat peripheral nervous system.

Syntaxin 1 has been shown to play an outstanding role in synaptic vesicle exocytosis. Two isoforms of this protein are expressed in neurons, syntaxin 1A and 1B. However, the physiological significance of the occurrence of such closely related isoforms is not still understood. Here, by means of isoform-specific immunocytochemistry, we show that syntaxin 1A and 1B display different patterns of expression in the rat peripheral nervous system. Nerve terminals of sensory neurons reaching the spinal cord were clearly enriched in immunoreactive syntaxin 1A. Both isoforms were detected in cell bodies of sensory neurons at the dorsal root ganglia, although specific immunolabelling displayed very different patterns at the cellular level. Motor endplates and muscle spindles were only immunostained for syntaxin 1B. Syntaxin 1A was mainly associated with nerve fibres reaching small blood vessels. In addition, nerve plexuses of the enteric nervous system showed immunostaining for both syntaxin isoforms. The different distribution pattern of the two neuronal syntaxin isoforms in the rat peripheral nervous system could be related to isoform-specific biochemical properties involved in the exocytotic process.

Animals↗

Selective early induction of synaptosomal-associated protein (molecular weight 25,000) following systemic administration of kainate at convulsant doses in the rat.

SNAP-25 (synaptosomal-associated protein of mol. wt 25,000) is an essential component for neurotransmitter release, and its expression has been related to the plastic responses that follow CNS injury. In the present study, transient induction of SNAP-25 in selected brain areas is shown by immunohistochemistry at short times after a single intraperitoneal injection of kainate at convulsant doses. Six hours after kainate injection, SNAP-25 immunoreactivity was noticed in the perikarya of certain neurons of the perirhinal and lateral cortices, polymorphic layer of the dentate gyrus, CA3 pyramidal area of the hippocampus, and thalamus. In the same areas, a strong increase in SNAP-25 immunorectivity was detected at 12 and 24 h after kainate injection in cell bodies and fibers. Four days after kainate administration, the immunostaining pattern was similar to that observed in control animals. Intraperitoneal injection of cycloheximide blocked the expression of SNAP-25, thus suggesting de novo SNAP-25 protein synthesis following kainate administration. Kainate-dependent induction of SNAP-25a messenger RNA synthesis was observed by in situ hybridization in the mentioned brain areas. Heat shock protein of mol. wt 72,000 (HSP70/72) is a chaperone whose expression is induced early under stress conditions. Its expression and distribution were compared to that of SNAP-25 after the excitotoxic insult. Brain areas overexpressing SNAP-25 and HSP70/72 overlapped. In addition, partial co-localization of both antigens was observed by double-labeling immunohistochemistry. These results provide evidence of an involvement of SNAP-25 in the reactive response that follows kainate administration, and support the role of this protein in the plastic events that take place after kainate excitotoxicity.

Animals↗

Axonal transport of ribonucleoprotein particles (vaults).

RNA was previously shown to be transported into both dendritic and axonal compartments of nerve cells, presumably involving a ribonucleoprotein particle. In order to reveal potential mechanisms of transport we investigated the axonal transport of the major vault protein of the electric ray Torpedo marmorata. This protein is the major protein component of a ribonucleoprotein particle (vault) carrying a non-translatable RNA and has a wide distribution in the animal kingdom. It is highly enriched in the cholinergic electromotor neurons and similar in size to synaptic vesicles. The axonal transport of vaults was investigated by immunofluorescence, using the anti-vault protein antibody as marker, and cytofluorimetric scanning, and was compared to that of the synaptic vesicle membrane protein SV2 and of the beta-subunit of the F1-ATPase as a marker for mitochondria. Following a crush significant axonal accumulation of SV2 proximal to the crush could first be observed after 1 h, that of mitochondria after 3 h and that of vaults after 6 h, although weekly fluorescent traces of accumulations of vault protein were observed in the confocal microscope as early as 3 h. Within the time-period investigated (up to 72 h) the accumulation of all markers increased continuously. Retrograde accumulations also occurred, and the immunofluorescence for the retrograde component, indicating recycling, was weaker than that for the anterograde component, suggesting that more than half of the vaults are degraded within the nerve terminal. High resolution immunofluorescence revealed a granular structure-in accordance with the biochemical characteristics of vaults. Of interest was the observation that the increase of vault immunoreactivity proximal to the crush accelerated with time after crushing, while that of SV2-containing particles appeared to decelerate, indicating that the crush procedure with time may have induced perikaryal alterations in the production and subsequent export to the axon of synaptic vesicles and vault protein. Our data show that ribonucleoprotein-immunoreactive particles can be actively transported within axons in situ from the soma to the nerve terminal and back. The results suggest that the transport of vaults is driven by fast axonal transport motors like the SV2-containing vesicles and mitochondria. Vaults exhibit an anterograde and a retrograde transport component, similar to that observed for the vesicular organelles carrying SV2 and for mitochondria. Although the function of vaults is still unknown studies of the axonal transport of this organelle may reveal insights into the mechanisms of cellular transport of ribonucleoprotein particles in general.

Animals↗

Colchicine administration in the rat central nervous system induces SNAP-25 expression.

he arrest of axonal transport by colchicine administration has been extensively used in immunocytochemical studies to increase the levels of neuroactive compounds in neuronal somata. In order to study the accumulation rates of a variety of proteins with location and physiological action at the synaptic terminal, we analysed, by immunocytochemical methods, the neuronal cell body content of these synaptic proteins in colchicine-injected rats. In sham-injected animals, all synaptic proteins tested were essentially observed in nerve fibres and terminal boutons. After colchicine administration, intense SNAP-25 immunoreactivity was found in many neuronal cell bodies throughout the CNS. In contrast, immunostaining for the rest of the synaptic proteins analysed (syntaxin 1A and 1B, synaptobrevin I and II, Rab3A, synaptophysin, synapsin I, synaptotagmin I and GAP-43) was virtually absent in neuronal cell bodies in treated animals. Furthermore, northern blot and in situ hybridization analysis revealed an increase in SNAP-25a and SNAP-25b messenger RNA isoforms in the brains of adult colchicine-administered animals. In addition, colchicine administration in five-day-old rat pups induced a notable increase in both SNAP-25 transcript isoforms. The present results indicate that in vivo colchicine administration, under conditions known to inhibit axoplasmic transport, upregulates SNAP-25 expression in the rat brain.

Animals↗

Exocytotic protein components in rat pituitary gland after long-term estrogen administration.

Recently, a set of proteins involved in the docking and fusion machinery of secretory organelles has been identified in anterior pituitary cells. In this study we analyzed, by Western blotting and immunocytochemistry, the expression of several proteins involved in exocytosis after long-term administration of 17beta-estradiol (E2) in Fischer 344 rats. No differences were observed in the amount of synaptosomal-associated protein of 25 kDa, synaptobrevin 2, syntaxin 1, synaptotagmin I and Rab3a in total brain homogenates from treated rats after E2 administration. In striking contrast, the levels of all of these exocytotic proteins, including cellubrevin, were notably decreased in pituitary glands of E2-treated rats. In addition, no differences were observed in the in vitro basal and 8-Br-cAMP-induced prolactin (PRL) release between pituitary cells from control and E2-treated rats, whereas TRH-induced PRL release in anterior pituitary cells from E2-treated animals was higher than in control donors. In conclusion, this study shows that protein components of the exocytotic machinery are specifically down-regulated in the pituitary gland of E2-treated Fischer 344 rats.

Animals↗

TrkB and TrkC signaling are required for maturation and synaptogenesis of hippocampal connections.

Recent studies have suggested a role for neurotrophins in the growth and refinement of neural connections, in dendritic growth, and in activity-dependent adult plasticity. To unravel the role of endogenous neurotrophins in the development of neural connections in the CNS, we studied the ontogeny of hippocampal afferents in trkB (-/-) and trkC (-/-) mice. Injections of lipophilic tracers in the entorhinal cortex and hippocampus of newborn mutant mice showed that the ingrowth of entorhinal and commissural/associational afferents to the hippocampus was not affected by these mutations. Similarly, injections of biocytin in postnatal mutant mice (P10-P16) did not reveal major differences in the topographic patterns of hippocampal connections. In contrast, quantification of biocytin-filled axons showed that commissural and entorhinal afferents have a reduced number of axon collaterals (21-49%) and decreased densities of axonal varicosities (8-17%) in both trkB (-/-) and trkC (-/-) mice. In addition, electron microscopic analyses showed that trkB (-/-) and trkC (-/-) mice have lower densities of synaptic contacts and important structural alterations of presynaptic boutons, such as decreased density of synaptic vesicles. Finally, immunocytochemical studies revealed a reduced expression of the synaptic-associated proteins responsible for synaptic vesicle exocytosis and neurotransmitter release (v-SNAREs and t-SNAREs), especially in trkB (-/-) mice. We conclude that neither trkB nor trkC genes are essential for the ingrowth or layer-specific targeting of hippocampal connections, although the lack of these receptors results in reduced axonal arborization and synaptic density, which indicates a role for TrkB and TrkC receptors in the developmental regulation of synaptic inputs in the CNS in vivo. The data also suggest that the genes encoding for synaptic proteins may be targets of TrkB and TrkC signaling pathways.

Animals↗

Increase in SNAP-25 immunoreactivity in the mossy fibers following transient forebrain ischemia in the gerbil.

SNAP-25 (a synaptosomal-associated protein of 25 kDa) has been shown to be involved both in synaptic vesicle exocytosis and in axonal outgrowth. In the present study, we investigated the changes in SNAP-25 immunoreactivity in the hippocampus of the Mongolian gerbil (Meriones unguiculatus) at different time points after transient forebrain ischemia insult. In parallel, immunostaining for GAP-43, a protein involved in axonal outgrowth, and for syntaxin-1 (stx1A and stx1B), another protein implicated in neurotransmitter release, was also analyzed. The animals were subjected to 2.5 or 5 min of transient forebrain ischemia through bilateral common carotid occlusion, and examined at different intervals after ischemia. SNAP-25 immunoreactivity was increased in the mossy fiber layer as early as 2 days after 5 min of ischemia. Increased SNAP-25 immunoreactivity in mossy fibers was also detected at days 4 and 7 after ischemia. On day 15, SNAP-25 staining was similar to that observed in control non-ischemic animals. In contrast, no changes in GAP-43 and syntaxin-1 immunoreactivity were observed in the mossy fiber layer following 5 min of ischemia. No modifications in SNAP-25, syntaxin-1 or GAP-43 immunoreactivity were observed following 2.5 min of ischemia, the longest period for which no neuronal damage is observed. These results provide evidence of a specific involvement of SNAP-25 in the reactive changes associated with transient forebrain ischemia.

Animals↗

Synaptobrevin isoforms in secretory granules and synaptic-like microvesicles in anterior pituitary cells.

A set of synaptic proteins have been shown to be essential for the life cycle and exocytosis of synaptic vesicles at the nerve terminal. Recently, these proteins have also been identified in certain endocrine cells. Here we analysed the presence and location of some of these synaptic proteins in anterior pituitary cells. Immunoblotting data demonstrated that Rab3a, synaptotagmin, cellubrevin, synaptobrevin 2, syntaxin 1, SNAP-25 and synaptophysin were well represented in anterior pituitary cells as well as in the corticotroph cell line AtT-20. Cellubrevin was the most abundant synaptobrevin isoform present in pituitary cells. Moreover, both cellubrevin and synaptobrevin 2 took part of a protein complex involved in the fusion process in adenohypophyseal cells. Immunocytochemical and subcellular fractionation showed that cellubrevin, synaptobrevin 2, Rab3a and synaptotagmin were located in both secretory granules and synaptic-like microvesicles fractions. In contrast, SNAP-25 and syntaxin 1 were mainly associated with plasma membrane fractions. Therefore, these results suggest similar secretory mechanisms for synaptic vesicles and secretory organelles in both neuronal and endocrine cells.

Animals↗

Dystrophic neurites of senile plaques are defective in proteins involved in exocytosis and neurotransmission.

Dystrophic neurites are major components of neuritic (both immature and mature) senile plaques in Alzheimer disease. Previous studies have shown strong immunoreactivity for different neuropeptides, and chromogranin A, a protein associated with dense-core vesicles, in dystrophic neurites. In the present study, antibodies to synaptophysin, synapsin, Rab3a and synaptotagmin (synaptic vesicle proteins), and SNAP-25 (synaptosomal-associated protein of 25 kD) and syntaxin (presynaptic plasma membrane proteins) have been used to learn about the dystrophic neurite equipment of proteins that are necessary for the docking and fusion of synaptic vesicles, and then for exocytosis and neurotransmission. The present results have shown that, although most neuritic senile plaques have chromogranin A- and SNAP-25-immunoreactive dystrophic neurites, only a percentage of them contain synaptophysin, and a minority contain synaptotagmin and Rab3a. Dystrophic neurites do not contain synapsin and syntaxin. These results show that dystrophic neurites of senile plaques are defective in proteins that control exocytosis and neurotransmission.

Alzheimer Disease↗

Block of transmitter release by botulinum C1 action on syntaxin at the squid giant synapse.

Electrophysiological, morphological, and biochemical approaches were combined to study the effect of the presynaptic injection of the light chain of botulinum toxin C1 into the squid giant synapse. Presynaptic injection was accompanied by synaptic block that occurred progressively as the toxin filled the presynaptic terminal. Neither the presynaptic action potential nor the Ca2+ currents in the presynaptic terminal were affected by the toxin. Biochemical analysis of syntaxin moiety in squid indicates that the light chain of botulinum toxin C1 lyses syntaxin in vitro, suggesting that this was the mechanism responsible for synaptic block. Ultrastructure of the injected synapses demonstrates an enormous increase in the number of presynaptic vesicles, suggesting that the release rather than the docking of vesicles is affected by biochemical lysing of the syntaxin molecule.

Animals↗

Intracellular location of SNAP-25 in human neutrophils.

Exocytosis plays an essential role in the physiological functions of human neutrophils. Although SNAP-25 is considered to play a key role in vesicle-membrane fusion, it has been detected almost exclusively in the neuronal system. Using different specific antibodies to SNAP-25, we have identified in the membrane fraction of resting human neutrophils an immunoreactive band with the same molecular mass observed in brain homogenates. Immunoblot analysis of subcellular fractions of neutrophils revealed that SNAP-25 protein was found in the granule membrane fraction, but not in the cytosolic and plasma membrane fractions. Granule localization for neutrophil SNAP-25 was further demonstrated by confocal and immunoelectron microscopy. Furthermore, SNAP-25 was mainly located in the morphologically defined neutrophil peroxidase-negative granules, which are mobilizable upon cell activation. In addition, the protein was specifically cleaved by botulinal neurotoxin A, as observed in brain homogenate. These findings reveal the presence of SNAP-25 in the granule membranes of human neutrophils.

Botulinum Toxins, Type A↗