Biomedical subjects

Fabio Benfenati

Publications and source records attributed to Fabio Benfenati.

At least 19 recordsLinked to original sources

PubMed2006

Immature neuronal phenotype derived from mouse skin precursor cells differentiated in vitro.

Recent findings indicate that skin-derived precursor cells (SKPs) of mouse dermis can differentiate in cells with neuronal-like morphology. However, direct evidence supporting the establishment of functional phenotype is missing. In the present study, SKP cells were obtained using published in vitro techniques and studied at 14- to 21-day differentiation, when neuronal-like morphology was observed. The experiment was repeated 39 times. Co-cultures with cortical astrocytes were also used to enhance the process of neural differentiation. Expression of GAP43 and light-chain MAP-2(c) but not markers of dendritic and synaptic terminal differentiation such as heavy-chain MAP-2(ab) and synapsin were observed. Voltage-clamp electrophysiology recordings showed potassium currents, but neither action potentials generation nor electrotonic response to exogenous administration of nicotine or kainic acid. These observations suggest that mouse SKPs do not differentiate into mature functional neurons, at least using the published methodologies for in vitro differentiation.

4-Aminopyridine↗

PubMed2005

FAK and PYK2 interact with SAP90/PSD-95-Associated Protein-3.

Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2) are two related non-receptor tyrosine kinases highly expressed in brain. Although they are both involved in synaptic plasticity, little is known about their specific neuronal partners. Using a yeast two-hybrid screen and GST pull-down assays we show that SAPAP3 (SAP90/PSD-95-Associated Protein-3) interacts with FAK (residues 676-840) and PYK2. The three proteins partly co-distribute in the same sucrose gradient fractions as the post-synaptic density protein PSD-95 and Src. Our results suggest that SAPAP3 is an anchoring protein for FAK and PYK2 in post-synaptic densities and may contribute to the synaptic function of these tyrosine kinases.

Animals↗

PubMed2005

Phosphorylation of synapsin I by cAMP-dependent protein kinase controls synaptic vesicle dynamics in developing neurons.

In developing neurons, synaptic vesicles (SVs) undergo cycles of exo-endocytosis along isolated axons. However, it is currently unknown whether SV exocytosis is regulated before synaptogenesis. Here, we show that cAMP-dependent pathways affect SV distribution and recycling in the axonal growth cone and that these effects are mediated by the SV-associated phosphoprotein synapsin I. The presence of synapsin I on SVs is necessary for the correct localization of the vesicles in the central portion of the growth cone. Phosphorylation of synapsin I by cAMP-dependent protein kinase (protein kinase A) causes the dissociation of the protein from the SV membrane, allowing diffusion of the vesicles to the periphery of the growth cone and enhancing their rate of recycling. These results provide new clues as to the bases of the well known activity of synapsin I in synapse maturation and indicate that molecular mechanisms similar to those operating at mature nerve terminals are active in developing neurons to regulate the SV life cycle before synaptogenesis.

Animals↗

PubMed2005

Structural domains involved in the regulation of transmitter release by synapsins.

Synapsins are a family of neuron-specific phosphoproteins that regulate neurotransmitter release by associating with synaptic vesicles. Synapsins consist of a series of conserved and variable structural domains of unknown function. We performed a systematic structure-function analysis of the various domains of synapsin by assessing the actions of synapsin fragments on neurotransmitter release, presynaptic ultrastructure, and the biochemical interactions of synapsin. Injecting a peptide derived from domain A into the squid giant presynaptic terminal inhibited neurotransmitter release in a phosphorylation-dependent manner. This peptide had no effect on vesicle pool size, synaptic depression, or transmitter release kinetics. In contrast, a peptide fragment from domain C reduced the number of synaptic vesicles in the periphery of the active zone and increased the rate and extent of synaptic depression. This peptide also slowed the kinetics of neurotransmitter release without affecting the number of docked vesicles. The domain C peptide, as well as another peptide from domain E that is known to have identical effects on vesicle pool size and release kinetics, both specifically interfered with the binding of synapsins to actin but not with the binding of synapsins to synaptic vesicles. This suggests that both peptides interfere with release by preventing interactions of synapsins with actin. Thus, interactions of domains C and E with the actin cytoskeleton may allow synapsins to perform two roles in regulating release, whereas domain A has an actin-independent function that regulates transmitter release in a phosphorylation-sensitive manner.

Amino Acid Sequence↗

PubMed2005

Synapsin associates with cyclophilin B in an ATP- and cyclosporin A-dependent manner.

Immunophilins are ubiquitous enzymes responsible for proline isomerisation during protein synthesis and for the chaperoning of several membrane proteins. These activities can be blocked by the immunosuppressants cyclosporin A, FK506 and rapamycin. It has been shown that all three immunosuppressants have neurotrophic activity and can modulate neurotransmitter release, but the molecular basis of these effects is currently unknown. Here, we show that synapsin I, a synaptic vesicle-associated protein, can be purified from Torpedo cholinergic synaptosomes through its affinity to cyclophilin B, an immunophilin that is particularly abundant in brain. The interaction is direct and conserved in mammals, and shows a dissociation constant of about 0.5 microM in vitro. The binding between the two proteins can be disrupted by cyclosporin A and inhibited by physiological concentrations of ATP. Furthermore, cyclophilin B co-localizes with synapsin I in rat synaptic vesicle fractions and its levels in synaptic vesicle-containing fractions are decreased in synapsin knockout mice. These results suggest that immunophilins are involved in the complex protein networks operating at the presynaptic level and implicate the interaction between cyclophilin B and synapsins in presynaptic function.

Adenosine Triphosphate↗

PubMed2004

Phosphorylation by cAMP-dependent protein kinase is essential for synapsin-induced enhancement of neurotransmitter release in invertebrate neurons.

Synapsins are synaptic vesicle-associated phosphoproteins involved in the regulation of neurotransmitter release and synapse formation; they are substrates for multiple protein kinases that phosphorylate them on distinct sites. We have previously found that injection of synapsin into Helix snail neurons cultured under low-release conditions increases the efficiency of neurotransmitter release. In order to investigate the role of phosphorylation in this modulatory action of synapsins, we examined the substrate properties of the snail synapsin orthologue recently cloned in Aplysia (apSyn) for various protein kinases and compared the effects of the intracellular injection of wild-type apSyn with those of its phosphorylation site mutants. ApSyn was found to be an excellent in vitro substrate for cAMP-dependent protein kinase, which phosphorylated it at high stoichiometry on a single site (Ser-9) in the highly conserved domain A, unlike the other kinases reported to phosphorylate mammalian synapsins, which phosphorylated apSyn to a much lesser extent. The functional effect of apSyn phosphorylation by cAMP-dependent protein kinase on neurotransmitter release was studied by injecting wild-type or Ser-9 mutated apSyn into the soma of Helix serotonergic C1 neurons cultured under low-release conditions, i.e. in contact with the non-physiological target neuron C3. In this model of impaired neurotransmitter release, the injection of wild-type apSyn induced a significant enhancement of release. This enhancement was virtually absent after injection of the non-phosphorylatable mutant (Ser-9-->Ala), but it was maintained after injection of the pseudophosphorylated mutant (Ser-9-->Asp). These functional effects of apSyn injection were paralleled by marked ultrastructural changes in the C1 neuron, with the formation of extensive interdigitations of neurite-like processes containing an increased complement of C1 dense core vesicles at the sites of cell-to-cell contact. This structural rearrangement was virtually absent in mock-injected C1 neurons or after injection of the non-phosphorylatable apSyn mutant. These data indicate that phosphorylation of synapsin domain A is essential for the synapsin-induced enhancement of neurotransmitter release and suggest that endogenous kinases phosphorylating this domain play a central role in the regulation of the efficiency of the exocytotic machinery.

Animals↗

PubMed2004

Synapsin is a novel Rab3 effector protein on small synaptic vesicles. I. Identification and characterization of the synapsin I-Rab3 interactions in vitro and in intact nerve terminals.

Synapsins, a family of neuron-specific phosphoproteins, have been demonstrated to regulate the availability of synaptic vesicles for exocytosis by binding to both synaptic vesicles and the actin cytoskeleton in a phosphorylation-dependent manner. Although the above-mentioned observations strongly support a pre-docking role of the synapsins in the assembly and maintenance of a reserve pool of synaptic vesicles, recent results suggest that the synapsins may also be involved in some later step of exocytosis. In order to investigate additional interactions of the synapsins with nerve terminal proteins, we have employed phage display library technology to select peptide sequences binding with high affinity to synapsin I. Antibodies raised against the peptide YQYIETSMQ (syn21) specifically recognized Rab3A, a synaptic vesicle-specific small G protein implicated in multiple steps of exocytosis. The interaction between synapsin I and Rab3A was confirmed by photoaffinity labeling experiments on purified synaptic vesicles and by the formation of a chemically cross-linked complex between synapsin I and Rab3A in intact nerve terminals. Synapsin I could be effectively co-precipitated from synaptosomal extracts by immobilized recombinant Rab3A in a GTP-dependent fashion. In vitro binding assays using purified proteins confirmed the binding preference of synapsin I for Rab3A-GTP and revealed that the COOH-terminal regions of synapsin I and the Rab3A effector domain are required for the interaction with Rab3A to occur. The data indicate that synapsin I is a novel Rab3 interactor on synaptic vesicles and suggest that the synapsin-Rab3 interaction may participate in the regulation of synaptic vesicle trafficking within the nerve terminals.

Amino Acid Sequence↗

PubMed2004

Synapsin is a novel Rab3 effector protein on small synaptic vesicles. II. Functional effects of the Rab3A-synapsin I interaction.

Synapsins, a family of neuron-specific phosphoproteins that play an important role in the regulation of synaptic vesicle trafficking and neurotransmitter release, were recently demonstrated to interact with the synaptic vesicle-associated small G protein Rab3A within nerve terminals (Giovedi, S., Vaccaro, P., Valtorta, F., Darchen, F., Greengard, P., Cesareni, G., and Benfenati, F. (2004) J. Biol. Chem. 279, 43760-43768). We have analyzed the functional consequences of this interaction on the biological activities of both proteins and on their subcellular distribution within nerve terminals. The presence of synapsin I stimulated GTP binding and GTPase activity of both purified and endogenous synaptic vesicle-associated Rab3A. Conversely, Rab3A inhibited synapsin I binding to F-actin, as well as synapsin-induced actin bundling and vesicle clustering. Moreover, the amount of Rab3A associated with synaptic vesicles was decreased in synapsin knockout mice, and the presence of synapsin I prevented RabGDI-induced Rab3A dissociation from synaptic vesicles. The results indicate that an interaction between synapsin I and Rab3A exists on synaptic vesicles that modulates the functional properties of both proteins. Given the well recognized importance of both synapsins and Rab3A in synaptic vesicles exocytosis, this interaction is likely to play a major role in the modulation of neurotransmitter release.

Actins↗

PubMed2004

Inhibition of neurotransmitter release by a nonphysiological target requires protein synthesis and involves cAMP-dependent and mitogen-activated protein kinases.

During the development of neuronal circuits, axonal growth cones can contact many inappropriate targets before they reach an appropriate postsynaptic partner. Although it is well known that the contact with synaptic partners upregulates the secretory machinery of the presynaptic neuron, little is known about the signaling mechanisms involved in preventing the formation of connections with inappropriate target cells. Here, we show that the contact with a nonphysiological postsynaptic target inhibits neurotransmitter release from axonal terminals of the Helix serotonergic neuron C1 by means of an active mechanism requiring ongoing protein synthesis and leading to the inhibition of cAMP-dependent protein kinase (PKA) and mitogen-activated protein kinase (MAPK)-extracellular signal-related kinase (Erk) pathways. The reversal of the inhibitory effect of the nonphysiological target by blockade of protein synthesis was prevented by cAMP-PKA or MAPK-Erk inhibitors, whereas disinhibition of neurotransmitter release promoted by cAMP-PKA activation was not affected by MAPK-Erk inhibitors. The data indicate that the inhibitory effect of the nonphysiological target on neurotransmitter release is an active process that requires protein synthesis and involves the downregulation of the MAPK-Erk and cAMP-PKA pathways, the same protein kinases that are activated after contact with a physiological target neuron. These mechanisms could play a relevant role in the prevention of synapse formation between inappropriate partners by modulating the neurotransmitter release capability of growing nerve terminals according to the nature of the targets contacted during their development.

Animals↗

PubMed2004

Synaptophysin: leading actor or walk-on role in synaptic vesicle exocytosis?

Synaptophysin (Syp) was the first synaptic vesicle (SV) protein to be cloned. Since its discovery in 1985, it has been used by us and by many laboratories around the world as an invaluable marker to study the distribution of synapses in the brain and to uncover the basic features of the life cycle of SVs. Although single gene ablation of Syp does not lead to an overt phenotype, a large body of experimental data both in vitro and in vivo indicate that Syp (alone or in association with homologous proteins) is involved in multiple, important aspects of SV exo-endocytosis, including regulation of SNARE assembly into the fusion core complex, formation of the fusion pore initiating neurotransmitter release, activation of SV endocytosis and SV biogenesis. In this article, we summarise the main results of the studies on Syp carried out by our and other laboratories, and explain why we believe that Syp plays a major role in SV trafficking.

Animals↗

PubMed2004

S100A1 codistributes with synapsin I in discrete brain areas and inhibits the F-actin-bundling activity of synapsin I.

The Ca2+-sensor protein S100A1 was recently shown to bind in vitro to synapsins, a family of synaptic vesicle phosphoproteins involved in the regulation of neurotransmitter release. In this paper, we analyzed the distribution of S100A1 and synapsin I in the CNS and investigated the effects of the S100A1/synapsin binding on the synapsin functional properties. Subcellular fractionation of rat brain homogenate revealed that S100A1 is present in the soluble fraction of isolated nerve endings. Confocal laser scanning microscopy and immunogold immunocytochemistry demonstrated that S100A1 and synapsin codistribute in a subpopulation (5-20%) of nerve terminals in the mouse cerebral and cerebellar cortices. By forming heterocomplexes with either dephosphorylated or phosphorylated synapsin I, S100A1 caused a dose- and Ca2+-dependent inhibition of synapsin-induced F-actin bundling and abolished synapsin dimerization, without affecting the binding of synapsin to F-actin, G-actin or synaptic vesicles. These data indicate that: (i) synapsins and S100A1 can interact in the nerve terminals where they are coexpresssed; (ii) S100A1 is unable to bind to SV-associated synapsin I and may function as a cytoplasmic store of monomeric synapsin I; and (iii) synapsin dimerization and interaction with S100A1 are mutually exclusive, suggesting an involvement of S100A1 in the Ca2+-dependent regulation of synaptic vesicle trafficking.

Actins↗

PubMed2004

Using the atomic force microscope to study the interaction between two solid supported lipid bilayers and the influence of synapsin I.

To measure the interaction between two lipid bilayers with an atomic force microscope one solid supported bilayer was formed on a planar surface by spontaneous vesicle fusion. To spontaneously adsorb lipid bilayers also on the atomic force microscope tip, the tips were first coated with gold and a monolayer of mercapto undecanol. Calculations indicate that long-chain hydroxyl terminated alkyl thiols tend to enhance spontaneous vesicle fusion because of an increased van der Waals attraction as compared to short-chain thiols. Interactions measured between dioleoylphosphatidylcholine, dioleoylphosphatidylserine, and dioleoyloxypropyl trimethylammonium chloride showed the electrostatic double-layer force plus a shorter-range repulsion which decayed exponentially with a decay length of 0.7 nm for dioleoylphosphatidylcholine, 1.2 nm for dioleoylphosphatidylserine, and 0.8 nm for dioleoyloxypropyl trimethylammonium chloride. The salt concentration drastically changed the interaction between dioleoyloxypropyl trimethylammonium chloride bilayers. As an example for the influence of proteins on bilayer-bilayer interaction, the influence of the synaptic vesicle-associated, phospholipid binding protein synapsin I was studied. Synapsin I increased membrane stability so that the bilayers could not be penetrated with the tip.

Adsorption↗

PubMed2003

Synaptophysin I controls the targeting of VAMP2/synaptobrevin II to synaptic vesicles.

Synaptic vesicle (SV) proteins are synthesized at the level of the cell body and transported down the axon in membrane precursors of SVs. To investigate the mechanisms underlying sorting of proteins to SVs, fluorescent chimeras of vesicle-associated membrane protein (VAMP) 2, its highly homologous isoform VAMP1 and synaptotagmin I (SytI) were expressed in hippocampal neurons in culture. Interestingly, the proteins displayed a diffuse component of distribution along the axon. In addition, VAMP2 was found to travel in vesicles that constitutively fuse with the plasma membrane. Coexpression of VAMP2 with synaptophysin I (SypI), a major resident of SVs, restored the correct sorting of VAMP2 to SVs. The effect of SypI on VAMP2 sorting was dose dependent, being reversed by increasing VAMP2 expression levels, and highly specific, because the sorting of the SV proteins VAMP1 and SytI was not affected by SypI. The cytoplasmic domain of VAMP2 was found to be necessary for both the formation of VAMP2-SypI hetero-dimers and for VAMP2 sorting to SVs. These data support a role for SypI in directing the correct sorting of VAMP2 in neurons and demonstrate that a direct interaction between the two proteins is required for SypI in order to exert its effect.

Animals↗

PubMed2003

Phosphatidylinositol 4-kinase type IIalpha is responsible for the phosphatidylinositol 4-kinase activity associated with synaptic vesicles.

Phosphorylation of inositol phospholipids plays a key role in cellular regulation via the generation of intracellular second messengers. In addition, it represents a mechanism to regulate interactions of the lipid bilayer with proteins and protein scaffolds involved in vesicle budding, cytoskeletal organization, and signaling. Generation of phosphatidylinositol 4-phosphate [PI(4)P] from phosphatidylinositol (PI) is an important step in this metabolic pathway because PI(4)P is a precursor of other important phosphoinositides and has protein binding properties of its own. We report here that a PI 4-kinase (PI4K) activity previously reported on synaptic vesicles is accounted for by the alpha isoform of the recently characterized type II PI4K (PI4KII) family. PI4KIIalpha, which also accounts for the bulk of PI4K activity in brain extracts, is concentrated at synapses and in the region of the Golgi complex in neuronal perikarya. Our results provide new evidence for the occurrence of a cycle of phosphoinositide synthesis and hydrolysis nested within the exo-endocytic cycle of synaptic vesicles and point to PI4KIIalpha as a critical player in this cycle.

1-Phosphatidylinositol 4-Kinase↗

PubMed2003

Regulated delivery of AMPA receptor subunits to the presynaptic membrane.

In recent years, a role for AMPA receptors as modulators of presynaptic functions has emerged. We have investigated the presence of AMPA receptor subunits and the possible dynamic control of their surface exposure at the presynaptic membrane. We demonstrate that the AMPA receptor subunits GluR1 and GluR2 are expressed and organized in functional receptors in axonal growth cones of hippocampal neurons. AMPA receptors are actively internalized upon activation and recruited to the surface upon depolarization. Pretreatment of cultures with botulinum toxin E or tetanus toxin prevents the receptor insertion into the plasma membrane, whereas treatment with alpha-latrotoxin enhances the surface exposure of GluR2, both in growth cones of cultured neurons and in brain synaptosomes. Purification of small synaptic vesicles through controlled-pore glass chromatography, revealed that both GluR2 and GluR1, but not the GluR2 interacting protein GRIP, copurify with synaptic vesicles. These data indicate that, at steady state, a major pool of AMPA receptor subunits reside in synaptic vesicle membranes and can be recruited to the presynaptic membrane as functional receptors in response to depolarization.

Animals↗

PubMed2003

Specific interactions of neuronal focal adhesion kinase isoforms with Src kinases and amphiphysin.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that activates Src family kinases via SH2- and SH3-mediated interactions. Specific FAK isoforms (FAK+), responsive to depolarization and neurotransmitters, are enriched in neurons. We analyzed the interactions of endogenous FAK+ and recombinant FAK+ isoforms containing amino acid insertions (boxes 6,7,28) with an array of SH3 domains and the c-Src SH2/SH3 domain tandem. Endogenous FAK+ bound specifically to the SH3 domains of c-Src (but not n-Src), Fyn, Yes, phosphtidylinositol-3 kinase, amphiphysin II, amphiphysin I, phospholipase Cgamma and NH2-terminal Grb2. The inclusion of boxes 6,7 was associated with a significant decrease in the binding of FAK+ to the c-Src and Fyn SH3 domains, and a significant increase in the binding to the Src SH2 domain, as a consequence of the higher phosphorylation of Tyr-397. The novel interaction with the amphiphysin SH3 domain, involving the COOH-terminal proline-rich region of FAK, was confirmed by coimmunoprecipitation of the two proteins and a closely similar response to stimuli affecting the actin cytoskeleton. Moreover, an impairment of endocytosis was observed in synaptosomes after internalization of a proline-rich peptide corresponding to the site of interaction. The data account for the different subcellular distribution of FAK and Src kinases and the specific regulation of the transduction pathways linked to FAK activation in the brain and implicate FAK in the regulation of membrane trafficking in nerve terminals.

Animals↗

PubMed2002

Phosphorylation of spinophilin modulates its interaction with actin filaments.

Spinophilin is a protein phosphatase 1 (PP1)- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We report that spinophilin is phosphorylated in vitro by protein kinase A (PKA). Phosphorylation of spinophilin was stimulated by treatment of neostriatal neurons with a dopamine D1 receptor agonist or with forskolin, consistent with spinophilin being a substrate for PKA in intact cells. Using tryptic phosphopeptide mapping, site-directed mutagenesis, and microsequencing analysis, we identified two major sites of phosphorylation, Ser-94 and Ser-177, that are located within the actin-binding domain of spinophilin. Phosphorylation of spinophilin by PKA modulated the association between spinophilin and the actin cytoskeleton. Following subcellular fractionation, unphosphorylated spinophilin was enriched in the postsynaptic density, whereas a pool of phosphorylated spinophilin was found in the cytosol. F-actin co-sedimentation and overlay analysis revealed that phosphorylation of spinophilin reduced the stoichiometry of the spinophilin-actin interaction. In contrast, the ability of spinophilin to bind to PP1 remained unchanged. Taken together, our studies suggest that phosphorylation of spinophilin by PKA modulates the anchoring of the spinophilin-PP1 complex within dendritic spines, thereby likely contributing to the efficacy and plasticity of synaptic transmission.

Actins↗

PubMed2002

Spatial and temporal regulation of Ca2+/calmodulin-dependent protein kinase II activity in developing neurons.

We have studied Ca2+/calmodulin-dependent protein kinase II (CaMKII) isoform distribution and activity in embryonic hippocampal neurons developing in culture. We have found a strong correlation between the expression of the alpha subunit of the enzyme and the ability to undergo depolarization-dependent phosphorylation, which in young neurons is limited to the somatodendritic pool of the kinase. The lack of responsiveness of the axons of young alphaCaMKII-positive neurons is not caused by a lower Ca2+ influx but rather by a differential balance between kinase and phosphatase activities in this compartment. After the establishment of synaptic contacts, the presynaptic pool of the kinase displays an increasing level of activity and acquires the parallel ability to phosphorylate synapsin I, which represents one of the major CaMKII presynaptic targets in mature nerve terminals. In contrast, the activity of the postsynaptic pool of the kinase remains constant throughout synaptogenesis. In the presence of a nearly homogeneous subcellular distribution, this highly regionalized regulation of activity may reflect the multifunctional roles of CaMKII in both developing and mature neurons.

Animals↗