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F Benfenati

Publications and source records attributed to F Benfenati.

At least 37 records · Page 2Linked to original sources
PubMed1997

Kinetic analysis of the phosphorylation-dependent interactions of synapsin I with rat brain synaptic vesicles.

1. Synapsin I, a major synaptic vesicle (SV)-associated phosphoprotein, is involved in the regulation of neurotransmitter release and synapse formation. By binding to both phospholipid and protein components of SV with high affinity and in a phosphorylation-dependent fashion, synapsin I is believed to cluster SV and to attach them to the actin-based cytoskeleton of the nerve terminal. 2. In the present study we have investigated the kinetic aspects of synapsin I-SV interactions and the mechanisms of their modulation by ionic strength and site-specific phosphorylation, using fluorescence resonance energy transfer between suitable fluorophores linked to synapsin I and to the membrane bilayer. 3. The binding of synapsin I to the phospholipid and protein components of SV has fast kinetics: mean time constants ranged between 1 and 4 s for association and 9 and 11's for ionic strength-induced dissociation at 20 degrees C. The interaction with the phospholipid component consists predominantly of a hydrophobic binding with the core of the membrane which may account for the membrane stabilizing effect of synapsin I. 4. Phosphorylation of synapsin I by either SV-associated or purified exogenous Ca2+/calmodulin-dependent protein kinase II (CaMPKII) inhibited the association rate and the binding to SV at steady state by acting on the ionic strength-sensitive component of the binding. When dephosphorylated synapsin I was previously bound to SV, exposure of SV to Ca2+/calmodulin in the presence of ATP triggered a prompt dissociation of synapsin I with a time constant similar to that of ionic strength-induced dissociation. 5. In conclusion, the reversible interactions between synapsin I and SV are highly regulated by site-specific phosphorylation and have kinetics of the same order of magnitude as the kinetics of SV recycling determined in mammalian neurons under comparable temperature conditions. These findings are consistent with the hypothesis that synapsin I associates with, and dissociates from, SV during the exo-endocytotic cycle. The on-vesicle phosphorylation of synapsin I by the SV-associated CaMPKII, and the subsequent dissociation of the protein from the vesicle membrane, though not involved in mediating exocytosis of primed vesicles evoked by a single stimulus, may represent a prompt and efficient mechanism for the modulation of neurotransmitter release and presynaptic plasticity.

Animals↗
PubMed1997

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.

Animals↗
PubMed1997

Tat protein from HIV-1 activates MAP kinase in granular neurons and glial cells from rat cerebellum.

We have investigated the effect of extracellularly applied Tat protein of the human immunodeficiency virus type 1 (HIV-1) on tyrosine phosphorylation processes, which represent a major signal transduction pathway of cells of the central nervous system. Primary cultures of rat cerebellar astrocytes or granule cells were incubated with synthetic Tat (10 ng/ml) for various periods of time and analyzed for their phosphotyrosine content by Western blotting. In both types of cultures Tat was able to induce the phosphorylation of mitogen-activated protein kinase (MAP kinase) on tyrosine residues, although with different kinetics and isoform specificity. In addition, in neuronal cells, but not in astrocytes, Tat increased the phosphotyrosine content of Shc, a protein involved in signal transduction downstream of receptor tyrosine kinase activation. This study shows that Tat applied extracellularly is able to induce the generation of intracellular signals in neuronal as well as glial cells.

Animals↗
PubMed1997

Molecular and functional diversity at synapses of individual neurons in vitro.

We have quantified activity-dependent uptake of the fluorescent dye FM1-43 in combination with immunocytochemistry for synaptic vesicle-associated proteins (SVPs) at individual synapses in primary cultures of rat cortical neurons. We show that expression of synaptic proteins is highly variable and that the levels of synaptophysin (p38), synapsin I and sv2, but not synapsin II, correlate with the extent of FM1-43 labelling at synapses. The data indicate that SVP levels affect the uptake of FM1-43 with different efficacy (p38 > synapsin I > sv2 or synapsin II). We also found that the relative levels of SVPs vary at individual boutons of single neurons grown in isolation, which indicates that differential regulation of specific SVPs may contribute to the selective modulation of activity at synapses of the same neuron.

Animals↗
PubMed1997

Phosphorylation-dependent effects of synapsin IIa on actin polymerization and network formation.

The synapsins are a family of synaptic vesicle phosphoproteins which play a key role in the regulation of neurotransmitter release and synapse formation. In the case of synapsin I, these biological properties have been attributed to its ability to interact with both synaptic vesicles and the actin-based cytoskeleton. Although synapsin II shares some of the biological properties of synapsin I, much less is known of its molecular properties. We have investigated the interactions of recombinant rat synapsin Ila with monomeric and filamentous actin and the sensitivity of those interactions to phosphorylation, and found that: i) dephosphorylated synapsin II stimulates actin polymerization by binding to actin monomers and forming actively elongating nuclei and by facilitating the spontaneous nucleation/elongation processes; ii) dephosphorylated synapsin II induces the formation of thick and ordered bundles of actin filaments with greater potency than synapsin I; iii) phosphorylation by protein kinase A markedly inhibits the ability of synapsin II to interact with both actin monomers and filaments. The results indicate that the interactions of synapsin II with actin are similar but not identical to those of synapsin I and suggest that synapsin II may play a major structural role in mature and developing nerve terminals, which is only partially overlapping with the role played by synapsin I.

Actins↗
PubMed1996

Effects of synaptic vesicles on actin polymerization.

We have analyzed the effects of synaptic vesicles on actin polymerization by using a time-resolved spectrofluorometric assay. We have found that synaptic vesicles have complex effects on the kinetics of actin polymerization, which vary depending on whether the synaptic vesicle-specific phosphoprotein synapsin I is absent or present on their membrane. Synapsin I bound either to synaptic vesicles or to pure phospholipid vesicles exhibits phosphorylation-dependent actin-nucleating activity. Synaptic vesicles depleted of endogenous synapsin I decrease the rate and the final extent of actin polymerization, an effect which is not observed with pure phospholipid vesicles. Thus, the state of association of synapsin I with synaptic vesicles, which is modulated by its state of phosphorylation, may affect actin assembly and the physico-chemical characteristics of the synaptic vesicle microenvironment.

Actins↗
PubMed1996

Biochemical and functional characterization of the synaptic vesicle-associated form of CA2+/calmodulin-dependent protein kinase II.

Ca+/calmodulin-dependent protein kinase II (CaMPKII) is a brain-enriched protein kinase that plays important roles in synaptic transmission and plasticity. In nerve terminals, a form of CaMPKII is associated with synaptic vesicles and binds the COOH-terminal region of synapsin I (SYNI). The biochemical properties of the vesicle-associated form of CAMPKII have been investigated and compared with those of the soluble forebrain enzyme. Both the alpha- and beta-subunits of CaMPKII copurifying with synaptic vesicles were tightly associated with the vesicle membrane. The vesicle-associated form of CaMPKII was indistinguishable from the soluble form with respect to sites of autophosphorylation, kinetics of both autophosphorylation and SYNI phosphorylation, and induction of autonomous activity upon autophosphorylation. Although both subunits of the soluble CaMPKII interacted with a photoactivatable SYNI derivative, only the alpha-subunit of the synaptic vesicle-associated CaMPKII bound to the COOH-terminal region of SYNI. The latter interaction was strongly dependent on the phosphorylation state of SYNI and on divalent cations, but appeared to be independent of autophosphorylation. These results demonstrate that, although the vesicle-associated form of CaMPKII is catalytically indistinguishable from the soluble form, it exhibits distinct characteristics concerning its association with the vesicle membrane and with SYNI.

Animals↗
PubMed1996

Neurotrophins stimulate phosphorylation of synapsin I by MAP kinase and regulate synapsin I-actin interactions.

The ability of neurotrophins to modulate the survival and differentiation of neuronal populations involves the Trk/MAP (mitogen-activated protein kinase) kinase signaling pathway. More recently, neurotrophins have also been shown to regulate synaptic transmission. The synapsins are a family of neuron-specific phosphoproteins that play a role in regulation of neurotransmitter release, in axonal elongation, and in formation and maintenance of synaptic contacts. We report here that synapsin I is a downstream effector for the neurotrophin/Trk/MAP kinase cascade. Using purified components, we show that MAP kinase stoichiometrically phosphorylated synapsin I at three sites (Ser-62, Ser-67, and Ser-549). Phosphorylation of these sites was detected in rat brain homogenates, in cultured cerebrocortical neurons, and in isolated presynaptic terminals. Brain-derived neurotrophic factor and nerve growth factor upregulated phosphorylation of synapsin I at MAP kinase-dependent sites in intact cerebrocortical neurons and PC12 cells, respectively, while KCl- induced depolarization of cultured neurons decreased the phosphorylation state at these sites. MAP kinase-dependent phosphorylation of synapsin I significantly reduced its ability to promote G-actin polymerization and to bundle actin filaments. The results suggest that MAP kinase-dependent phosphorylation of synapsin I may contribute to the modulation of synaptic plasticity by neurotrophins and by other signaling pathways that converge at the level of MAP kinase activation.

Actins↗
PubMed1996

Synapsin-like molecules in Aplysia punctata and Helix pomatia: identification and distribution in the nervous system and during the formation of synaptic contacts in vitro.

The distribution and biochemical features of the synapsin-like peptides recognized in Aplysia and Helix by various antibodies directed against mammalian synapsins were studied. The peptides can be extracted at low pH and are digested by collagenase; further, they can be phosphorylated by both protein kinase A and Ca2+/calmodulin-dependent protein kinase II. In the ganglia of both snails, they are associated with the soma of most neurons and with the neuropil; punctate immunostaining is present along the neurites. Using cocultures of a Helix serotoninergic neuron and of its target cell, we analysed the redistribution of the synapsin-like peptides during the formation of active synaptic contacts. When the presynaptic neuron is plated in isolation, both synapsin and serotonin immunoreactivities are restricted to the distal axonal segments and to the growth cones; in the presence of the target, the formation of a chemical connection is accompanied by redistribution of the synapsin and serotonin immunoreactivities that concentrate in highly fluorescent round spots scattered along the newly grown neurites located close to the target cell. Almost every spot that is stained for serotonin is also positive for synapsin. In the presynaptic cell plated alone, the number of these varicosity-like structures is substantially stable throughout the whole period; by contrast, when the presynaptic cell synapses the target, their number increases progressively parallel to the increase in the mean amplitude of cumulative excitatory postsynaptic potentials recorded at the same times. The data indicate that mollusc synapsin-like peptides to some extent resemble their mammalian homologues, although they are not exclusively localized in nerve terminals and their expression strongly correlates with the formation of active synaptic contacts.

Animals↗
PubMed1996

Epidermal growth factor-mediated inhibition of neurotransmitter glutamate release from rat forebrain synaptosomes.

We investigated the possibility that receptor tyrosine kinases are involved in modulating neurotransmitter release from isolated nerve terminals. We examined the effects of epidermal growth factor on the release of neurotransmitter glutamate evoked from rat forebrain synaptosomes by KCI and 4-aminopyridine. We detected a significant inhibition of the Ca2+-dependent component of release. This effect appears to be mediated by a reduction in the depolarization-evoked increase in cytosolic free calcium levels, in the absence of significant effects on the plasma membrane potential. On depolarization, a Ca2+-dependent increase was observed in the phosphotyrosine content of bands at molecular masses of approximately 107 and approximately 40 kDa. The addition of epidermal growth factor before depolarization induced a significant phosphorylation of the growth factor receptor in the absence of detectable changes in the phosphotyrosine pattern of total synaptosomal proteins, suggesting that phosphorylation of a minor protein is responsible for the epidermal growth factor-mediated inhibition of glutamate release.

4-Aminopyridine↗
PubMed1995

Accelerated structural maturation induced by synapsin I at developing neuromuscular synapses of Xenopus laevis.

The role of synapsin I, a synaptic vesicle-associated phosphoprotein, in the maturation of nerve-muscle synapses was investigated in nerve-muscle co-cultures prepared from Xenopus embryos loaded with the protein by the early blastomere injection method. The stage of maturation of the synapses was analysed by electron microscopy as well as by whole-cell patch-clamp recording. The acceleration in the functional maturation of neuromuscular synapses induced by synapsin I was accompanied by a profound rearrangement in the ultrastructure of the nerve terminal. Nerve terminals formed by synapsin I-loaded neurons were characterized by a higher number of small synaptic vesicles organized in clusters and predominantly localized close to the nerve terminal plasma membrane, a smaller number of large dense-core vesicles and no significant change in the number of coated vesicles. Precocious development of active zone-like structures as well as deposition of basal lamina into the synaptic cleft were also observed at these synapses. These results support a role for synapsin I in the architectural changes which occur during synaptogenesis and lead to the maturation of quantal neurotransmitter release mechanisms.

Animals↗
PubMed1995

Neuroexocytosis.

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Animals↗
PubMed1995

Phosphorylation of VAMP/synaptobrevin in synaptic vesicles by endogenous protein kinases.

VAMP/synaptobrevin (SYB), an integral membrane protein of small synaptic vesicles, is specifically cleaved by tetanus neurotoxin and botulinum neurotoxins B, D, F, and G is thought to play an important role in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. Potential phosphorylation sites for various kinases are present in SYB sequence. We have studied whether SYB is a substrate for protein kinases that are present in nerve terminals and known to modulate neurotransmitter release. SYB can be phosphorylated within the same vesicle by endogenous Ca2+/calmodulin-dependent protein kinase II (CaMKII) associated with synaptic vesicles. This phosphorylation reaction occurs rapidly and involves serine and threonine residues in the cytoplasmic region of SYB. Similarly to CaMKII, a casein kinase II (CasKII) activity copurifying with synaptic vesicles is able to phosphorylate SYB selectively on serine residues of the cytoplasmic region. This phosphorylation reaction is markedly stimulated by sphingosine, a sphingolipid known to activate CasKII and to inhibit CaMKII and protein kinase C. The results show that SYB is a potential substrate for protein kinases involved in the regulation of neurotransmitter release and open the possibility that phosphorylation of SYB plays a role in modulating the molecular interactions between synaptic vesicles and the presynaptic membrane.

Amino Acid Sequence↗
PubMed1995

Dephosphorylated synapsin I anchors synaptic vesicles to actin cytoskeleton: an analysis by videomicroscopy.

Synapsin I is a synaptic vesicle-associated protein which inhibits neurotransmitter release, an effect which is abolished upon its phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Based on indirect evidence, it was suggested that this effect on neurotransmitter release may be achieved by the reversible anchoring of synaptic vesicles to the actin cytoskeleton of the nerve terminal. Using video-enhanced microscopy, we have now obtained experimental evidence in support of this model: the presence of dephosphorylated synapsin I is necessary for synaptic vesicles to bind actin; synapsin I is able to promote actin polymerization and bundling of actin filaments in the presence of synaptic vesicles; the ability to cross-link synaptic vesicles and actin is specific for synapsin I and is not shared by other basic proteins; the cross-linking between synaptic vesicles and actin is specific for the membrane of synaptic vesicles and does not reflect either a non-specific binding of membranes to the highly surface active synapsin I molecule or trapping of vesicles within the thick bundles of actin filaments; the formation of the ternary complex is virtually abolished when synapsin I is phosphorylated by CaM kinase II. The data indicate that synapsin I markedly affects synaptic vesicle traffic and cytoskeleton assembly in the nerve terminal and provide a molecular basis for the ability of synapsin I to regulate the availability of synaptic vesicles for exocytosis and thereby the efficiency of neurotransmitter release.

Actins↗
PubMed1994

Interaction of Grb2 via its Src homology 3 domains with synaptic proteins including synapsin I.

Grb2 is a 25-kDa adaptor protein composed of a Src homology 2 (SH2) domain and two flanking Src homology 3 (SH3) domains. One function of Grb2 is to couple tyrosine-phosphorylated proteins (through its SH2 domain) to downstream effectors (through its SH3 domains). Using an overlay assay, we have identified four major Grb2-binding proteins in synaptic fractions. These proteins interact with wild-type Grb2 but not with Grb2 containing point mutations in each of its two SH3 domains corresponding to the loss of function mutants in the Caenorhabditis elegans Grb2 homologue sem-5. Two of the proteins, mSos and dynamin, were previously shown to bind Grb2. The third protein of 145 kDa is brain specific and to our knowledge has not been previously described. The fourth protein is synapsin I. Dynamin is required for synaptic vesicle endocytosis and synapsin I is thought to mediate the interaction of synaptic vesicles with the presynaptic cytomatrix. These data suggest that Grb2, or other proteins containing SH3 domains, may play a role in the regulation of the exo/endocytotic cycle of synaptic vesicles and therefore of neurotransmitter release.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Tetanus and botulinum neurotoxins are zinc proteases specific for components of the neuroexocytosis apparatus.

Tetanus and botulinum neurotoxins bind to nerve cells, penetrate the cytosol, and block neurotransmitter release. Comparison of their amino-acid sequences shows the presence of the highly conserved His-Glu-x-x-His zinc-binding motif of zinc-endopeptidases (HExxH). Atomic absorption measurements of clostridial neurotoxins show the presence of one atom of zinc/toxin molecule bound to the light chain. The toxin-bound zinc ion is essential for the neurotoxins inhibition of neurotransmitter release in Aplysia neurons injected with the toxins. Phosphoramidon, a very specific inhibitor of zinc-endopeptidases, blocks the intracellular activity of the clostridial neurotoxins. Highly purified preparations of the light chain of tetanus and botulinum B and F neurotoxins cleaved specifically VAMP/synaptobrevin, an integral membrane protein of small synaptic vesicles, both in vivo and in vitro. From these studies, it can be concluded that the clostridial neurotoxins responsible for tetanus and botulism block neuroexocytosis via the proteolytic cleavage of specific components of the neuroexocytotic machinery.

Amino Acid Sequence↗