Showing papers by "Fabio Benfenati published in 2004"

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

Abstract: SummarySynaptophysin (Syp) was the first synaptic vesicle (SV)protein to be cloned. Since its discovery in 1985, it hasbeen used by us and by many laboratories around theworldasaninvaluablemarkertostudythedistributionofsynapses in the brain and to uncover the basic featuresof the life cycle of SVs. Although single gene ablation ofSyp does not lead to an overt phenotype, a large body ofexperimental data both in vitro and in vivo indicate thatSyp (alone or in association with homologous proteins)is involved in multiple, important aspects of SV exo-endocytosis, including regulation of SNARE assemblyintothefusioncorecomplex,formationofthefusionporeinitiatingneurotransmitterrelease,activationofSVendo-cytosis and SV biogenesis. In this article, we summarisethe main results of the studies on Syp carried out by ourand other laboratories, and explain why we believe thatSyp plays a major role in SV trafficking. BioEssays26:445–453, 2004. 2004 Wiley Periodicals, Inc.IntroductionNeurotransmitter release is the main mechanism of informa-tion transfer among neurons. Neurotransmitter molecules arestored in specialised organelles, the synaptic vesicles (SVs),from which they are released at the arrival of the nerveimpulse. SVs are small translucent organelles of about 40–60 nm in diameter and are concentrated in clusters in definedsubplasmalemmal areas of the nerve terminal. These areas,named ‘‘active zones’’, are the sites where neurotransmitterrelease takes place.The release of neurotransmitter by SVs involves an exo-endocytotic cycle which comprising several steps: (1) thetargettingofSVstothereleasesites,(2)docking,i.e.anchoringofSVstotheplasmamembraneattheactivezone,(3)priming,i.e. preparation to fuse with high speed at the arrival of theimpulse,(4)Fusion/exocytosispermittingthedischargeoftheSV content, and (5) finally retrieval of the SV membrane byendocytosis.

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

Abstract: 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.

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

Abstract: 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.

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

Abstract: 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.