Heterogeneity of glutamatergic and GABAergic release machinery in cerebral cortex.

We investigated whether cortical glutamatergic and GABAergic release machineries can be differentiated on the basis of the proteins they express, by studying the degree of co-localization of synapsin (SYN) I and II, synaptophysin (SYP) I and II, synaptosomal-associated protein (SNAP)-25 and SNAP-23 in vesicular glutamate transporter (VGLUT) 1-, VGLUT2- and vesicular GABA transporter (VGAT)-positive (+) puncta in the rat cerebral cortex. Co-localization studies showed that SYNI and II were expressed in approximately 90% of VGLUT1+, approximately 30% of VGLUT2+ and 30-50% of VGAT+ puncta; SYPI was expressed in approximately 95% of VGLUT1+, 30% of VGLUT2+, and 45% of VGAT+ puncta; SYPII in approximately 7% of VGLUT1+, 3% of VGLUT2+, and 20% of VGAT+ puncta; SNAP-25 in approximately 94% of VGLUT1+, 5% of VGLUT2+, and 1% of VGAT+ puncta, and SNAP-23 in approximately 3% of VGLUT1+, 86% of VGLUT2+, and 22% of VGAT+ puncta. Since SYPI, which is considered ubiquitous, was expressed in about half of GABAergic axon terminals, we studied its localization electron microscopically and in immunoisolated synaptic vesicles: these studies showed that approximately 30% of axon terminals forming symmetric synapses were SYPI-negative, and that immunoisolated VGAT-positive synaptic vesicles were relatively depleted of SYPI as compared with VGLUT1+ vesicles. Overall, the present investigation shows that in the cerebral cortex of rats distinct presynaptic proteins involved in neurotransmitter release are differentially expressed in GABAergic and in the two major types of glutamatergic axon terminals in the cerebral cortex of rats.

Cellular mechanisms of the acute increase of glutamate release induced by nerve growth factor in rat cerebral cortex.

Nerve growth factor (NGF) was found to increase glutamate release in the developing visual cortex. We investigated the cellular mechanisms of this effect and its dependence on extracellular and intracellular Ca2+. The NGF-induced enhancement of glutamate release from superfused rat visual cortex synaptosomes required mild depolarization. Removal of external Ca2+ during depolarization with 15 mM K+ only halved the effect of NGF on glutamate release. NGF increased [Ca2+]i in K+-depolarized synaptosomes preloaded with fura-2AM both in the presence and in the absence of external Ca2+. The effects of NGF on glutamate release and [Ca2+]i elevation were prevented by an anti-TrkA receptor monoclonal antibody. NGF increased synaptosomal inositol (1,4,5)-triphosphate (InsP3) during depolarization and the InsP3 receptor antagonist heparin abolished the effect of NGF on evoked glutamate release both in the presence and in the absence of external Ca2+. The effect of NGF on the evoked glutamate release in Ca2+-free medium was abolished by dantrolene, a ryanodine receptor blocker, by CGP 37157, a blocker of the mitochondrial Na+/Ca2+ exchanger and by pretreatment of synaptosomes with caffeine. NGF significantly increased the depolarization-induced activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the subsequent phosphorylation of synapsin I in the absence of external Ca2+ and the NGF effect on evoked glutamate release was inhibited by the CaMKII inhibitors KN-93 and CaMKII 281-309 peptide but not by the MAP kinase inhibitor PD 98059. Thus, the effect of NGF on evoked glutamate release is linked to an increase in [Ca2+]i contributed by both Ca2+ entry and mobilization from InsP3-sensitive, ryanodine-sensitive and mitochondrial stores and to the subsequent activation of CaMKII.

Heterogeneity of glutamatergic and GABAergic release machinery in cerebral cortex: analysis of synaptogyrin, vesicle-associated membrane protein, and syntaxin.

To define whether cortical glutamatergic and GABAergic release machineries can be differentiated on the basis of the nature and amount of proteins they express, we studied the degree of co-localization of synaptogyrin (SGYR) 1 and 3, vesicle-associated membrane protein (VAMP) 1 and 2, syntaxin (STX) 1A and 1B in vesicular glutamate transporter (VGLUT)1-, VGLUT2- and vesicular GABA transporter (VGAT)-positive (+) puncta and synaptic vesicles in the rat cerebral cortex. Co-localization studies showed that SGYR1 and 3 were expressed in about 90% of VGLUT1+, 70% of VGLUT2+ and 80% of VGAT+ puncta; VAMP1 was expressed in approximately 45% of VGLUT1+, 55% of VGLUT2+, and 80% of VGAT+ puncta; VAMP2 in about 95% of VGLUT1+, 75% of VGLUT2+, and 80% of VGAT+ puncta; STX1A in about 65% of VGLUT1+, 30% of VGLUT2+, and 3% of VGAT+ puncta, and STX1B in approximately 45% of VGLUT1+, 35% of VGLUT2+, and 70% of VGAT+ puncta. Immunoisolation studies showed that while STX1A was completely segregated and virtually absent from VGAT synaptic vesicles, STX1B, VAMP1/VAMP2, SGYR1/SGYR3 showed a similar pattern with the highest expression in VGLUT1 immunoisolated vesicles and the lowest in VGAT immunoisolated vesicles. Moreover, we studied the localization of STX1B at the electron microscope and found that a population of axon terminals forming symmetric synapses were STX1B-positive.These results extend our previous observations on the differential expression of presynaptic proteins involved in neurotransmitter release in GABAergic and glutamatergic terminals and indicate that heterogeneity of glutamatergic and GABAergic release machinery can be contributed by both the presence or absence of a given protein in a nerve terminal and the amount of protein expressed by synaptic vesicles.

Protein-protein interactions and protein modules in the control of neurotransmitter release.

Information transfer among neurons is operated by neurotransmitters stored in synaptic vesicles and released to the extracellular space by an efficient process of regulated exocytosis. Synaptic vesicles are organized into two distinct functional pools, a large reserve pool in which vesicles are restrained by the actin-based cytoskeleton, and a quantitatively smaller releasable pool in which vesicles approach the presynaptic membrane and eventually fuse with it on stimulation. Both synaptic vesicle trafficking and neurotransmitter release depend on a precise sequence of events that include release from the reserve pool, targeting to the active zone, docking, priming, fusion and endocytotic retrieval of synaptic vesicles. These steps are mediated by a series of specific interactions among cytoskeletal, synaptic vesicle, presynaptic membrane and cytosolic proteins that, by acting in concert, promote the spatial and temporal regulation of the exocytotic machinery. The majority of these interactions are mediated by specific protein modules and domains that are found in many proteins and are involved in numerous intracellular processes. In this paper, the possible physiological role of these multiple protein-protein interactions is analysed, with ensuing updating and clarification of the present molecular model of the process of neurotransmitter release.

Interleukin-6 inhibits neurotransmitter release and the spread of excitation in the rat cerebral cortex.

Cytokines are extracellular mediators that have been reported to affect neurotransmitter release and synaptic plasticity phenomena when applied in vitro. Most of these effects occur rapidly after the application of the cytokines and are presumably mediated through the activation of protein phosphorylation processes. While many cytokines have an inflammatory action, interleukin-6 (IL-6) has been found to have a neuroprotective effect against ischaemia lesions and glutamate excitotoxicity, and to increase neuronal survival in a variety of experimental conditions. In this paper, the functional effects of IL-6 on the spread of excitation visualized by dark-field/infrared videomicroscopy in rat cortical slices and on glutamate release from cortical synaptosomes were analysed and correlated with the activation of the STAT3, mitogen-activated protein kinase ERK (MAPK/ERK) and stress-activated protein kinase/cJun NH2-terminal kinase (SAPK/JNK) pathways. We have found that IL-6 depresses the spread of excitation and evoked glutamate release in the cerebral cortex, and that these effects are accompanied by a stimulation of STAT3 tyrosine phosphorylation, an inhibition of MAPK/ERK activity, a decreased phosphorylation of the presynaptic MAPK/ERK substrate synapsin I and no detectable effects on SAPK/JNK. The effects of IL-6 were effectively counteracted by treatment of the cortical slices with the tyrosine kinase inhibitor lavendustin A. The inhibitory effects of IL-6 on glutamate release and on the spread of excitation in the rat cerebral cortex indicate that the protective effect of IL-6 on neuronal survival could be mediated by a downregulation of neuronal activity, release of excitatory neurotransmitters and MAPK/ERK activity.

Analysis of the Bak protein expression in human polymorphonuclear neutrophils.

In this study, we investigated the expression of Bak, a member of the Bcl-2 protein family, in human polymorphonuclear neutrophils. Northern blot and Western blot analyses revealed that Bak messenger RNA and protein were constitutively expressed in peripheral polymorphonuclear neutrophils and mononuclear cells, as well as in several hematopoietic cell lines. Remarkably, culturing neutrophils for 24 h in the presence or absence of interferon-gamma or tumor necrosis factor-alpha, which have been described to modulate the survival rate of these cells, did not influence the expression of antigenic Bak. Taken together, our data indicate that the expression of the pro-apoptotic protein Bak in polymorphonuclear neutrophils is constitutive, is not subject to modulation, and does not correlate with the neutrophil life span in culture.

Synapsin I interacts with c-Src and stimulates its tyrosine kinase activity.

Synapsin I is a synaptic vesicle-associated phosphoprotein that has been implicated in the formation of presynaptic specializations and in the regulation of neurotransmitter release. The nonreceptor tyrosine kinase c-Src is enriched on synaptic vesicles, where it accounts for most of the vesicle-associated tyrosine kinase activity. Using overlay, affinity chromatography, and coprecipitation assays, we have now shown that synapsin I is the major binding protein for the Src homology 3 (SH3) domain of c-Src in highly purified synaptic vesicle preparations. The interaction was mediated by the proline-rich domain D of synapsin I and was not significantly affected by stoichiometric phosphorylation of synapsin I at any of the known regulatory sites. The interaction of purified c-Src and synapsin I resulted in a severalfold stimulation of tyrosine kinase activity and was antagonized by the purified c-Src-SH3 domain. Depletion of synapsin I from purified synaptic vesicles resulted in a decrease of endogenous tyrosine kinase activity. Portions of the total cellular pools of synapsin I and Src were coprecipitated from detergent extracts of rat brain synaptosomal fractions using antibodies to either protein species. The interaction between synapsin I and c-Src, as well as the synapsin I-induced stimulation of tyrosine kinase activity, may be physiologically important in signal transduction and in the modulation of the function of axon terminals, both during synaptogenesis and at mature synapses.

The inhibitory effects of interleukin-6 on synaptic plasticity in the rat hippocampus are associated with an inhibition of mitogen-activated protein kinase ERK.

Several cytokines have short-term effects on synaptic transmission and plasticity that are thought to be mediated by the activation of intracellular protein kinases. We have studied the effects of interleukin-6 (IL-6) on the expression of paired pulse facilitation (PPF), posttetanic potentiation (PTP), and long-term potentiation (LTP) in the CA1 region of the hippocampus as well as on the activation of the signal transducer and activator of transcription-3 (STAT3), the mitogen-activated protein kinase ERK (MAPK/ERK), and the stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK). IL-6 induced a marked and dose-dependent decrease in the expression of PTP and LTP that could be counteracted by the simultaneous treatment with the tyrosine kinase inhibitor lavendustin A (LavA) but did not significantly affect PPF. The IL-6-induced inhibition of PTP and LTP was accompanied by a simulation of STAT3 tyrosine phosphorylation and an inhibition of MAPK/ERK dual phosphorylation, in the absence of changes in the state of activation of SAPK/JNK. Both effects of IL-6 on STAT3 and MAPK/ERK activation were effectively counteracted by LavA treatment. The results indicate the tyrosine kinases and MAPK/ERK are involved in hippocampal synaptic plasticity and may represent preferential intracellular targets for the actions of IL-6 in the adult nervous system.

Specificity of the binding of synapsin I to Src homology 3 domains.

Synapsins are synaptic vesicle-associated phosphoproteins involved in synapse formation and regulation of neurotransmitter release. Recently, synapsin I has been found to bind the Src homology 3 (SH3) domains of Grb2 and c-Src. In this work we have analyzed the interactions between synapsins and an array of SH3 domains belonging to proteins involved in signal transduction, cytoskeleton assembly, or endocytosis. The binding of synapsin I was specific for a subset of SH3 domains. The highest binding was observed with SH3 domains of c-Src, phospholipase C-gamma, p85 subunit of phosphatidylinositol 3-kinase, full-length and NH(2)-terminal Grb2, whereas binding was moderate with the SH3 domains of amphiphysins I/II, Crk, alpha-spectrin, and NADPH oxidase factor p47(phox) and negligible with the SH3 domains of p21(ras) GTPase-activating protein and COOH-terminal Grb2. Distinct sites in the proline-rich COOH-terminal region of synapsin I were found to be involved in binding to the various SH3 domains. Synapsin II also interacted with SH3 domains with a partly distinct binding pattern. Phosphorylation of synapsin I in the COOH-terminal region by Ca(2+)/calmodulin-dependent protein kinase II or mitogen-activated protein kinase modulated the binding to the SH3 domains of amphiphysins I/II, Crk, and alpha-spectrin without affecting the high affinity interactions. The SH3-mediated interaction of synapsin I with amphiphysins affected the ability of synapsin I to interact with actin and synaptic vesicles, and pools of synapsin I and amphiphysin I were shown to associate in isolated nerve terminals. The ability to bind multiple SH3 domains further implicates the synapsins in signal transduction and protein-protein interactions at the nerve terminal level.

Structural determinants of the specificity for synaptic vesicle-associated membrane protein/synaptobrevin of tetanus and botulinum type B and G neurotoxins.

Tetanus and botulinum neurotoxins type B and G are zinc-endopeptidases of remarkable specificity. They recognize and cleave a synaptic vesicle-associated membrane protein (VAMP)/synaptobrevin, an essential protein component of the vesicle docking and fusion apparatus. VAMP contains two copies of a nine-residue motif, also present in SNAP-25 (synaptosomal-associated protein of 25 kDa) and syntaxin, the two other substrates of clostridial neurotoxins. This motif was suggested to be a determinant of the target specificity of neurotoxins. Antibodies raised against this motif cross-react among VAMP, SNAP-25, and syntaxin and inhibit the proteolytic activity of the neurotoxins. Moreover, the various neurotoxins cross-inhibit each other's proteolytic action. The role of the three negatively charged residues of the motif in neurotoxin recognition was probed by site-directed mutagenesis. Substitution of acidic residues in both copies of the VAMP motif indicate that the first one is involved in tetanus neurotoxin recognition, whereas the second one is implicated in binding botulinum B and G neurotoxins. These results suggest that the two copies of the motif have a tandem association in the VAMP molecule.