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Fabio Benfenati - One of the best experts on this subject based on the ideXlab platform.
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Synapsins are expressed at neuronal and non-neuronal locations in Octopus vulgaris
Scientific Reports, 2019Co-Authors: Federica Maiole, Fabio Benfenati, Giulia Tedeschi, Simona Candiani, Luca Maragliano, Letizia ZulloAbstract:Synapsins are a family of phosphoproteins fundamental to the regulation of neurotransmitter release. They are typically neuron-specific, although recent evidence pointed to their expression in non-neuronal cells where they play a role in exocytosis and vesicle trafficking. In this work, we characterized Synapsin transcripts in the invertebrate mollusk Octopus vulgaris and present evidence of their expression not only in the brain but also in male and female reproductive organs. We identified three Synapsin isoforms phylogenetically correlated to that of other invertebrates and with a modular structure characteristic of mammalian Synapsins with a central, highly conserved C domain, important for the protein functions, and less conserved A, B and E domains. Our molecular modeling analysis further provided a solid background for predicting Synapsin functional binding to ATP, actin filaments and secretory vesicles. Interestingly, we found that Synapsin expression in ovary and testis increased during sexual maturation in cells with a known secretory role, potentially matching the occurrence of a secretion process. This might indicate that its secretory role has evolved across animals according to cell activity in spite of cell identity. We believe that this study may yield insights into the convergent evolution of ubiquitously expressed proteins between vertebrates and invertebrates.
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Specific recognition of Synapsin I domains expressed in transfected HEK cells by domain-specific antibodies.
2018Co-Authors: Robert Mertens, Fabio Benfenati, Daniel Gitler, Sarah Melchert, Morten Brix Schou, Sverre Georg Saether, Arne Vaaler, Johannes Piepgras, Elena Kochova, Gudrun Ahnert-hilgerAbstract:(A) Domain structure of the Synapsin isoforms Ia and Ib. The N-terminal domains A-C are highly conserved in all Synapsins. The C-terminal region is variable due to heterogeneous combinations of domains. (B) Diagram of the Synapsin Ia fragments examined to investigate targeting of patient Synapsin autoantibodies. (C-H) HEK293 cells were transfected with rat Synapsin Ia constructs, comprising the eYFP-tagged fragment ABC (C), eGFP-tagged BC (D), eGFP-tagged C (E), eGFP-tagged DE (F), eGFP-tagged D (G), or eGFP-tagged E domain (H). Cells were fixed, permeabilized and incubated with antibodies either directed against the A-domain of Synapsin I and II (rabbit anti-Synapsin A; dilution 1:500), the D-domain of Synapsin I (mouse anti-Synapsin D; dilution 1:500), the C-domain of Synapsin I/II (rabbit anti-Synapsin C; dilution 1:500), or the E-domain of Synapsin Ia (rabbit anti-Synapsin E; dilution 1:200). Antibody binding was visualized using Alexa Fluor 549-coupled secondary antibodies. Transfected cells were correctly recognized by the respective antibodies that colocalized with the signal of the tags, confirming domain identities and detectability of the various domains by anti-Synapsin antibodies.
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intersectin associates with Synapsin and regulates its nanoscale localization and function
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Fabian Gerth, Fabio Benfenati, Franco Onofri, Maria Japel, Arndt Pechstein, Gaga Kochlamazashvili, Martin Lehmann, Dmytro Puchkov, Alexander G NikonenkoAbstract:Neurotransmission is mediated by the exocytic release of neurotransmitters from readily releasable synaptic vesicles (SVs) at the active zone. To sustain neurotransmission during periods of elevated activity, release-ready vesicles need to be replenished from the reserve pool of SVs. The SV-associated Synapsins are crucial for maintaining this reserve pool and regulate the mobilization of reserve pool SVs. How replenishment of release-ready SVs from the reserve pool is regulated and which other factors cooperate with Synapsins in this process is unknown. Here we identify the endocytic multidomain scaffold protein intersectin as an important regulator of SV replenishment at hippocampal synapses. We found that intersectin directly associates with Synapsin I through its Src-homology 3 A domain, and this association is regulated by an intramolecular switch within intersectin 1. Deletion of intersectin 1/2 in mice alters the presynaptic nanoscale distribution of Synapsin I and causes defects in sustained neurotransmission due to defective SV replenishment. These phenotypes were rescued by wild-type intersectin 1 but not by a locked mutant of intersectin 1. Our data reveal intersectin as an autoinhibited scaffold that serves as a molecular linker between the Synapsin-dependent reserve pool and the presynaptic endocytosis machinery.
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Synapsins and Synaptic Vesicle Storage
Presynaptic Terminals, 2014Co-Authors: Fabrizia C. Guarnieri, Fabio Benfenati, Flavia ValtortaAbstract:The Synapsins constitute a family of evolutionarily conserved neuronal phosphoproteins associated with the cytosolic surface of synaptic vesicles. In mammals, the family comprises three members encoded by distinct genes that give rise to various splicing isoforms. In the central nervous system, the vast majority of neurons expresses at least one Synapsin isoform. However, the functions of these proteins are not fully understood to date. Given their ability to bind both the vesicular membrane and actin filaments in a phosphorylation-dependent manner, the classical role attributed to Synapsins is the reversible anchorage of synaptic vesicles to the cytoskeletal matrix present in the presynaptic terminal. However, recent evidences suggest the implication of Synapsins in other aspects of the synaptic vesicle life cycle, such as docking, fusion and recycling. Genetic manipulation of Synapsins in various in vitro and in vivo models has proved that they are dispensable for the proper development of functional neuronal networks but are essential modulators of synaptic neurotransmission and play differential roles at excitatory and inhibitory synapses. Indeed, mice lacking Synapsins are viable and do not display gross brain abnormalities but exhibit generalised epileptic seizures as well as autism-related behavioural abnormalities. Consistently, several mutations have been identified in SYN1 and SYN2 genes in patients affected by epilepsy and/or autism spectrum disorders.
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Synapsins: from synapse to network hyperexcitability and epilepsy.
Seminars in cell & developmental biology, 2011Co-Authors: Anna Fassio, Fabio Benfenati, Andrea Raimondi, Gabriele Lignani, Pietro BaldelliAbstract:The Synapsin family in mammals consists of at least 10 isoforms encoded by three distinct genes and composed by a mosaic of conserved and variable domains. Synapsins, although not essential for the basic development and functioning of neuronal networks, are extremely important for the fine-tuning of SV cycling and neuronal plasticity. Single, double and triple Synapsin knockout mice, with the notable exception of the Synapsin III knockout mice, show a severe epileptic phenotype without gross alterations in brain morphology and connectivity. However, the molecular and physiological mechanisms underlying the pathogenesis of the epileptic phenotype observed in Synapsin deficient mice are still far from being elucidated. In this review, we summarize the current knowledge about the role of Synapsins in the regulation of network excitability and about the molecular mechanism leading to epileptic phenotype in mouse lines lacking one or more Synapsin isoforms. The current evidences indicate that Synapsins exert distinct roles in excitatory versus inhibitory synapses by differentially affecting crucial steps of presynaptic physiology and by this mean participate in the determination of network hyperexcitability.
Paul Greengard - One of the best experts on this subject based on the ideXlab platform.
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Synapsin iia controls the reserve pool of glutamatergic synaptic vesicles
The Journal of Neuroscience, 2008Co-Authors: Daniel Gitler, Paul Greengard, Qing Cheng, George J AugustineAbstract:Synapsins regulate synaptic transmission by controlling the reserve pool of synaptic vesicles. Each of the three mammalian Synapsin genes is subject to alternative splicing, yielding several isoforms whose roles are unknown. To investigate the function of these isoforms, we examined the synaptic effects of introducing each isoform into glutamatergic cultured hippocampal neurons from Synapsin triple knock-out mice. Remarkably, we found that Synapsin IIa was the only isoform that could rescue the synaptic depression phenotype of the triple knock-out mice; other isoforms examined, including the well-studied Synapsin Ia isoform, had no significant effect on the kinetics of synaptic depression. The slowing of synaptic depression by Synapsin IIa was quantitatively paralleled by an increase in the density of reserve pool synaptic vesicles, as measured either by fluorescent tagging of the vesicle protein synaptobrevin-2 or by staining with the styryl dye FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-diethylamino)phenyl)-hexatrienyl)pyridinium dibromide]. Our results provide further support for the hypothesis that Synapsins define the kinetics of synaptic depression at glutamatergic synapses by controlling the size of the vesicular reserve pool and identify Synapsin IIa as the isoform primarily responsible for this task.
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structural domains involved in the regulation of transmitter release by Synapsins
The Journal of Neuroscience, 2005Co-Authors: Sabine Hilfiker, Fabio Benfenati, George J Augustine, Andrew J Czernik, Frederic Doussau, Angus C Nairn, Paul GreengardAbstract: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.
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Different presynaptic roles of Synapsins at excitatory and inhibitory synapses.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2004Co-Authors: Daniel Gitler, Paul Greengard, William C. Wetsel, Jian Feng, Yoshiko Takagishi, Yong Ren, Ramona M. Rodriguiz, George J AugustineAbstract:The functions of Synapsins were examined by characterizing the phenotype of mice in which all three Synapsin genes were knocked out. Although these triple knock-out mice were viable and had normal brain anatomy, they exhibited a number of behavioral defects. Synaptic transmission was altered in cultured neurons from the hippocampus of knock-out mice. At excitatory synapses, loss of Synapsins did not affect basal transmission evoked by single stimuli but caused a threefold increase in the rate of synaptic depression during trains of stimuli. This suggests that Synapsins regulate the reserve pool of synaptic vesicles. This possibility was examined further by measuring synaptic vesicle density in living neurons transfected with green fluorescent protein-tagged synaptobrevin 2, a marker of synaptic vesicles. The relative amount of fluorescent synaptobrevin was substantially lower at synapses of knock-out neurons than of wild-type neurons. Electron microscopy also revealed a parallel reduction in the number of vesicles in the reserve pool of vesicles >150 nm away from the active zone at excitatory synapses. Thus, Synapsins are required for maintaining vesicles in the reserve pool at excitatory synapses. In contrast, basal transmission at inhibitory synapses was reduced by loss of Synapsins, but the kinetics of synaptic depression were unaffected. In these terminals, there was a mild reduction in the total number of synaptic vesicles, but this was not restricted to the reserve pool of vesicles. Thus, Synapsins maintain the reserve pool of glutamatergic vesicles but regulate the size of the readily releasable pool of GABAergic vesicles.
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Synapsin is a novel rab3 effector protein on small synaptic vesicles ii functional effects of the rab3a Synapsin i interaction
Journal of Biological Chemistry, 2004Co-Authors: Silvia Giovedi, Paul Greengard, Flavia Valtorta, François Darchen, Fabio BenfenatiAbstract: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.
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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
The Journal of biological chemistry, 2004Co-Authors: Silvia Giovedi, Paul Greengard, Flavia Valtorta, Paola Vaccaro, Gianni Cesareni, François Darchen, Fabio BenfenatiAbstract: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.
George J Augustine - One of the best experts on this subject based on the ideXlab platform.
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Synapsins and the Synaptic Vesicle Reserve Pool: Floats or Anchors?
Cells, 2021Co-Authors: Minchuan Zhang, George J AugustineAbstract:In presynaptic terminals, synaptic vesicles (SVs) are found in a discrete cluster that includes a reserve pool that is mobilized during synaptic activity. Synapsins serve as a key protein for maintaining SVs within this reserve pool, but the mechanism that allows Synapsins to do this is unclear. This mechanism is likely to involve Synapsins either cross-linking SVs, thereby anchoring SVs to each other, or creating a liquid phase that allows SVs to float within a Synapsin droplet. Here, we summarize what is known about the role of Synapsins in clustering of SVs and evaluate experimental evidence supporting these two models.
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molecular mechanisms of short term plasticity role of Synapsin phosphorylation in augmentation and potentiation of spontaneous glutamate release
Frontiers in Synaptic Neuroscience, 2018Co-Authors: Qing Cheng, George J Augustine, Sang-ho SongAbstract:We used genetic and pharmacological approaches to identify the signaling pathways involved in augmentation and potentiation, two forms of activity dependent, short-term synaptic plasticity that enhance neurotransmitter release. Trains of presynaptic action potentials produced a robust increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs). Following the end of the stimulus, mEPSC frequency followed a bi-exponential decay back to basal levels. The time constants of decay identified these two exponential components as the decay of augmentation and potentiation, respectively. Augmentation increased mEPSC frequency by 9.3-fold, while potentiation increased mEPSC frequency by 2.4-fold. In Synapsin triple-knockout (TKO) neurons, augmentation was reduced by 83% and potentiation was reduced by 74%, suggesting that Synapsins are key signaling elements in both forms of plasticity. To examine the Synapsin isoforms involved, we expressed individual Synapsin isoforms in TKO neurons. Synapsin IIIa was most effective in rescuing both augmentation and potentiation, while Synapsin IIa was slightly less effective and Synapsins Ia, Ib and IIb did not rescue. To determine the involvement of protein kinases in these two forms of short-term plasticity, we examined the effects of inhibitors of protein kinases A and C. While inhibition of PKC had little effect, PKA inhibition reduced augmentation by 76% and potentiation by 60%. Further, elevation of intracellular cAMP concentration, by either forskolin or IBMX, greatly increased mEPSC frequency and occluded the amount of augmentation and potentiation evoked by electrical stimulation. Finally, mutating a PKA phosphorylation site to non-phosphorylatable alanine abolished the ability of either Synapsin IIa or IIIa to rescue both augmentation and potentiation. Together, these results indicate that PKA activation is required for both augmentation and potentiation of spontaneous neurotransmitter release. We conclude that PKA-mediated phosphorylation of Synapsins – specifically either the Synapsin IIa or IIIa isoforms - underlies both forms of presynaptic short-term plasticity.
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Synapsins regulate brain-derived neurotrophic factor-mediated synaptic potentiation and axon elongation by acting on membrane rafts.
European Journal of Neuroscience, 2017Co-Authors: Hung-teh Kao, George J Augustine, Kanghyun Ryoo, Albert Lin, Stephen R. Janoschka, Barbara PortonAbstract:In neurons, intracellular membrane rafts are essential for specific actions of brain-derived neurotrophic factor (BDNF), which include the regulation of axon outgrowth, growth cone turning and synaptic transmission. Virtually, all the actions of BDNF are mediated by binding to its receptor, TrkB. The association of TrkB with the tyrosine kinase, Fyn, is critical for its localization to intracellular membrane rafts. Here, we show that Synapsins, a family of highly amphipathic neuronal phosphoproteins, regulate membrane raft lipid composition and consequently, the ability of BDNF to regulate axon/neurite development and potentiate synaptic transmission. In the brains of mice lacking all Synapsins, the expression of both BDNF and TrkB were increased, suggesting that BDNF/TrkB-mediated signaling is impaired. Consistent with this finding, Synapsin-depleted neurons exhibit altered raft lipid composition, deficient targeting of Fyn to rafts, attenuated TrkB activation, and abrogation of BDNF-stimulated axon outgrowth and synaptic potentiation. Conversely, overexpression of Synapsins in neuroblastoma cells results in corresponding reciprocal changes in raft lipid composition, increased localization of Fyn to rafts and promotion of BDNF-stimulated neurite formation. In the presence of Synapsins, the ratio of cholesterol to estimated total phospholipids converged to 1, suggesting that Synapsins act by regulating the ratio of lipids in intracellular membranes, thereby promoting lipid raft formation. These studies reveal a mechanistic link between BDNF and Synapsins, impacting early development and synaptic transmission.
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Synapsin Isoforms Regulating GABA Release from Hippocampal Interneurons.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2016Co-Authors: Sang-ho Song, George J AugustineAbstract:Although Synapsins regulate GABA release, it is unclear which Synapsin isoforms are involved. We identified the Synapsin isoforms that regulate GABA release via rescue experiments in cultured hippocampal neurons from Synapsin I, II, and III triple knock-out (TKO) mice. In situ hybridization indicated that five different Synapsin isoforms are expressed in hippocampal interneurons. Evoked IPSC amplitude was reduced in TKO neurons compared with triple wild-type neurons and was rescued by introducing any of the five Synapsin isoforms. This contrasts with hippocampal glutamatergic terminals, where only Synapsin IIa rescues the TKO phenotype. Deconvolution analysis indicated that the duration of GABA release was prolonged in TKO neurons and this defect in release kinetics was rescued by each Synapsin isoform, aside from Synapsin IIIa. Because release kinetics remained slow, whereas peak release rate was rescued, there was a 2-fold increase in GABA release in TKO neurons expressing Synapsin IIIa. TKO neurons expressing individual Synapsin isoforms showed normal depression kinetics aside from more rapid depression in neurons expressing Synapsin IIIa. Measurements of the cumulative amount of GABA released during repetitive stimulation revealed that the rate of mobilization of vesicles from the reserve pool to the readily releasable pool and the size of the readily releasable pool of GABAergic vesicles were unaffected by Synapsins. Instead, Synapsins regulate release of GABA from the readily releasable pool, with all isoforms aside from Synapsin IIIa controlling release synchrony. These results indicate that Synapsins play fundamentally distinct roles at different types of presynaptic terminals. SIGNIFICANCE STATEMENT Synapsins are a family of proteins that regulate synaptic vesicle (SV) trafficking within nerve terminals. Here, we demonstrate that release of the inhibitory neurotransmitter GABA is supported by many different Synapsin types. This contrasts with the release of other neurotransmitters, which typically is supported by only one type of Synapsin. We also found that Synapsins serve to synchronize the release of GABA in response to presynaptic action potentials, which is different from the Synapsin-dependent trafficking of SVs in other nerve terminals. Our results establish that different Synapsins play fundamentally different roles at nerve terminals releasing different types of neurotransmitters. This is an important clue to understanding how neurons release their neurotransmitters, a process essential for normal brain function.
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Synapsin Isoforms and Synaptic Vesicle Trafficking.
Molecules and cells, 2015Co-Authors: Sang-ho Song, George J AugustineAbstract:Synapsins were the first presynaptic proteins identified and have served as the flagship of the presynaptic protein field. Here we review recent studies demonstrating that different members of the Synapsin family play different roles at presynaptic terminals employing different types of synaptic vesicles. The structural underpinnings for these functions are just beginning to be understood and should provide a focus for future efforts.
Flavia Valtorta - One of the best experts on this subject based on the ideXlab platform.
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Synapsins and Synaptic Vesicle Storage
Presynaptic Terminals, 2014Co-Authors: Fabrizia C. Guarnieri, Fabio Benfenati, Flavia ValtortaAbstract:The Synapsins constitute a family of evolutionarily conserved neuronal phosphoproteins associated with the cytosolic surface of synaptic vesicles. In mammals, the family comprises three members encoded by distinct genes that give rise to various splicing isoforms. In the central nervous system, the vast majority of neurons expresses at least one Synapsin isoform. However, the functions of these proteins are not fully understood to date. Given their ability to bind both the vesicular membrane and actin filaments in a phosphorylation-dependent manner, the classical role attributed to Synapsins is the reversible anchorage of synaptic vesicles to the cytoskeletal matrix present in the presynaptic terminal. However, recent evidences suggest the implication of Synapsins in other aspects of the synaptic vesicle life cycle, such as docking, fusion and recycling. Genetic manipulation of Synapsins in various in vitro and in vivo models has proved that they are dispensable for the proper development of functional neuronal networks but are essential modulators of synaptic neurotransmission and play differential roles at excitatory and inhibitory synapses. Indeed, mice lacking Synapsins are viable and do not display gross brain abnormalities but exhibit generalised epileptic seizures as well as autism-related behavioural abnormalities. Consistently, several mutations have been identified in SYN1 and SYN2 genes in patients affected by epilepsy and/or autism spectrum disorders.
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the Synapsins key actors of synapse function and plasticity
Progress in Neurobiology, 2010Co-Authors: Fabrizia Cesca, Flavia Valtorta, Pietro Baldelli, Fabio BenfenatiAbstract:The Synapsins are a family of neuronal phosphoproteins evolutionarily conserved in invertebrate and vertebrate organisms. Their best-characterised function is to modulate neurotransmitter release at the pre-synaptic terminal, by reversibly tethering synaptic vesicles (SVs) to the actin cytoskeleton. However, many recent data have suggested novel functions for Synapsins in other aspects of the pre-synaptic physiology, such as SV docking, fusion and recycling. Synapsin activity is tightly regulated by several protein kinases and phosphatases, which modulate the association of Synapsins to SVs as well as their interaction with actin filaments and other synaptic proteins. In this context, Synapsins act as a link between extracellular stimuli and the intracellular signalling events activated upon neuronal stimulation. Genetic manipulation of Synapsins in various in vivo models has revealed that, although not essential for the basic development and functioning of neuronal networks, these proteins are extremely important in the fine-tuning of neuronal plasticity, as shown by the epileptic phenotype and behavioural abnormalities characterising mouse lines lacking one or more Synapsin isoforms. In this review, we summarise the current knowledge about how the various members of the Synapsin family are involved in the modulation of the pre-synaptic physiology. We give a comprehensive description of the molecular basis of Synapsin function, as well as an overview of the more recent evidence linking mutations in the Synapsin proteins to the onset of severe central nervous system diseases such as epilepsy and schizophrenia.
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Tyrosine phosphorylation of Synapsin I by Src regulates synaptic-vesicle trafficking.
Journal of Cell Science, 2010Co-Authors: Mirko Messa, Franco Onofri, Flavia Valtorta, Anna Fassio, Sonia Congia, Enrico Defranchi, Fabio BenfenatiAbstract:Synapsins are synaptic vesicle (SV)-associated phosphoproteins involved in the regulation of neurotransmitter release. Synapsins reversibly tether SVs to the cytoskeleton and their phosphorylation by serine/threonine kinases increases SV availability for exocytosis by impairing their association with SVs and/or actin. We recently showed that Synapsin I, through SH3- or SH2-mediated interactions, activates Src and is phosphorylated by the same kinase at Tyr301. Here, we demonstrate that, in contrast to serine phosphorylation, Src-mediated tyrosine phosphorylation of Synapsin I increases its binding to SVs and actin, and increases the formation of Synapsin dimers, which are both potentially involved in SV clustering. Synapsin I phosphorylation by Src affected SV dynamics and was physiologically regulated in brain slices in response to depolarization. Expression of the non-phosphorylatable (Y301F) Synapsin I mutant in Synapsin-I-knockout neurons increased the sizes of the readily releasable and recycling pools of SVs with respect to the wild-type form, which is consistent with an increased availability of recycled SVs for exocytosis. The data provide a mechanism for the effects of Src on SV trafficking and indicate that tyrosine phosphorylation of Synapsins, unlike serine phosphorylation, stimulates the reclustering of recycled SVs and their recruitment to the reserve pool.
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The role of Synapsins in neuronal development
Cellular and Molecular Life Sciences, 2010Co-Authors: Eugenio F. Fornasiero, Fabio Benfenati, Dario Bonanomi, Flavia ValtortaAbstract:The Synapsins, the first identified synaptic vesicle-specific proteins, are phosphorylated on multiple sites by a number of protein kinases and are involved in neurite outgrowth and synapse formation as well as in synaptic transmission. In mammals, the Synapsin family consists of at least 10 isoforms encoded by 3 distinct genes and composed by a mosaic of conserved and variable domains. The Synapsins are highly conserved evolutionarily, and orthologues have been found in invertebrates and lower vertebrates. Within nerve terminals, Synapsins are implicated in multiple interactions with presynaptic proteins and the actin cytoskeleton. Via these interactions, Synapsins control several mechanisms important for neuronal homeostasis. In this review, we describe the main functional features of the Synapsins, in relation to the complex role played by these phosphoproteins in neuronal development.
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The highly conserved Synapsin domain E mediates Synapsin dimerization and phospholipid vesicle clustering.
The Biochemical journal, 2010Co-Authors: Ilaria Monaldi, Silvia Giovedi, Fabio Benfenati, Flavia Valtorta, Massimo Vassalli, Angela Bachi, Enrico Millo, Roberto Raiteri, Anna FassioAbstract:Synapsins are abundant SV (synaptic vesicle)-associated phosphoproteins that regulate synapse formation and function. The highly conserved C-terminal domain E was shown to contribute to several Synapsin functions, ranging from formation of the SV reserve pool to regulation of the kinetics of exocytosis and SV cycling, although the molecular mechanisms underlying these effects are unknown. In the present study, we used a synthetic 25-mer peptide encompassing the most conserved region of domain E (Pep-E) to analyse the role of domain E in regulating the interactions between Synapsin I and liposomes mimicking the phospholipid composition of SVs (SV-liposomes) and other pre-synaptic protein partners. In affinity-chromatography and cross-linking assays, Pep-E bound to endogenous and purified exogenous Synapsin I and strongly inhibited Synapsin dimerization, indicating a role in Synapsin oligomerization. Consistently, Pep-E (but not its scrambled version) counteracted the ability of holo-Synapsin I to bind and coat phospholipid membranes, as analysed by AFM (atomic force microscopy) topographical scanning, and significantly decreased the clustering of SV-liposomes induced by holo-Synapsin I in FRET (Forster resonance energy transfer) assays, suggesting a causal relationship between Synapsin oligomerization and vesicle clustering. Either Pep-E or a peptide derived from domain C was necessary and sufficient to inhibit both dimerization and vesicle clustering, indicating the participation of both domains in these activities of Synapsin I. The results provide a molecular explanation for the effects of domain E in nerve terminal physiology and suggest that its effects on the size and integrity of SV pools are contributed by the regulation of Synapsin dimerization and SV clustering.
Hung-teh Kao - One of the best experts on this subject based on the ideXlab platform.
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Synapsins regulate brain-derived neurotrophic factor-mediated synaptic potentiation and axon elongation by acting on membrane rafts.
European Journal of Neuroscience, 2017Co-Authors: Hung-teh Kao, George J Augustine, Kanghyun Ryoo, Albert Lin, Stephen R. Janoschka, Barbara PortonAbstract:In neurons, intracellular membrane rafts are essential for specific actions of brain-derived neurotrophic factor (BDNF), which include the regulation of axon outgrowth, growth cone turning and synaptic transmission. Virtually, all the actions of BDNF are mediated by binding to its receptor, TrkB. The association of TrkB with the tyrosine kinase, Fyn, is critical for its localization to intracellular membrane rafts. Here, we show that Synapsins, a family of highly amphipathic neuronal phosphoproteins, regulate membrane raft lipid composition and consequently, the ability of BDNF to regulate axon/neurite development and potentiate synaptic transmission. In the brains of mice lacking all Synapsins, the expression of both BDNF and TrkB were increased, suggesting that BDNF/TrkB-mediated signaling is impaired. Consistent with this finding, Synapsin-depleted neurons exhibit altered raft lipid composition, deficient targeting of Fyn to rafts, attenuated TrkB activation, and abrogation of BDNF-stimulated axon outgrowth and synaptic potentiation. Conversely, overexpression of Synapsins in neuroblastoma cells results in corresponding reciprocal changes in raft lipid composition, increased localization of Fyn to rafts and promotion of BDNF-stimulated neurite formation. In the presence of Synapsins, the ratio of cholesterol to estimated total phospholipids converged to 1, suggesting that Synapsins act by regulating the ratio of lipids in intracellular membranes, thereby promoting lipid raft formation. These studies reveal a mechanistic link between BDNF and Synapsins, impacting early development and synaptic transmission.
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Synapsin III: Role in Neuronal Plasticity and Disease
Seminars in cell & developmental biology, 2011Co-Authors: Barbara Porton, William C. Wetsel, Hung-teh KaoAbstract:Synapsin III was discovered in 1998, more than two decades after the first two Synapsins (Synapsins I and II) were identified. Although the biology of Synapsin III is not as well understood as Synapsins I and II, this gene is emerging as an important factor in the regulation of the early stages of neurodevelopment and dopaminergic neurotransmission, and in certain neuropsychiatric illnesses. Molecular genetic and clinical studies of Synapsin III have determined that its neurodevelopmental effects are exerted at the levels of neurogenesis and axonogenesis. In vitro voltammetry studies have shown that Synapsin III can control dopamine release in the striatum. Since dopaminergic dysfunction is implicated in many neuropsychiatric conditions, one may anticipate that polymorphisms in Synapsin III can exert pervasive effects, especially since it is localized to extrasynaptic sites. Indeed, mutations in this gene have been identified in individuals diagnosed with schizophrenia, bipolar disorder and multiple sclerosis. These and other findings indicate that the roles of Synapsin III differ significantly from those of Synapsins I and II. Here, we focus on the unique roles of the newest Synapsin, and where relevant, compare and contrast these with the actions of Synapsins I and II.
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protein kinase a mediated Synapsin i phosphorylation is a central modulator of ca2 dependent synaptic activity
The Journal of Neuroscience, 2006Co-Authors: Andrea Menegon, Fabio Benfenati, Hung-teh Kao, Pietro Baldelli, Dario Bonanomi, Chiara Albertinazzi, Francesco Lotti, Giuliana Ferrari, Flavia ValtortaAbstract:Protein kinase A (PKA) modulates several steps of synaptic transmission. However, the identification of the mediators of these effects is as yet incomplete. Synapsins are synaptic vesicle (SV)-associated phosphoproteins that represent the major presynaptic targets of PKA. We show that, in hippocampal neurons, cAMP-dependent pathways affect SV exocytosis and that this effect is primarily brought about through Synapsin I phosphorylation. Phosphorylation by PKA, by promoting dissociation of Synapsin I from SVs, enhances the rate of SV exocytosis on stimulation. This effect becomes relevant when neurons are challenged with sustained stimulation, because it appears to counteract synaptic depression and accelerate recovery from depression by fostering the supply of SVs from the reserve pool to the readily releasable pool. In contrast, Synapsin phosphorylation appears to be dispensable for the effects of cAMP on the frequency and amplitude of spontaneous synaptic currents and on the amplitude of evoked synaptic currents. The modulation of depolarization-evoked SV exocytosis by PKA phosphorylation of Synapsin I is primarily caused by calmodulin (CaM)-dependent activation of cAMP pathways rather than by direct activation of CaM kinases. These data define a hierarchical crosstalk between cAMP- and CaM-dependent cascades and point to Synapsin as a major effector of PKA in the modulation of activity-dependent SV exocytosis.
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Synapsin-regulated synaptic transmission from readily releasable synaptic vesicles in excitatory hippocampal synapses in mice.
The Journal of physiology, 2005Co-Authors: Øivind Hvalby, Hung-teh Kao, Vidar Jensen, S Ivar WalaasAbstract:The effects of Synapsin proteins on synaptic transmission from vesicles in the readily releasable vesicle pool have been examined by comparing excitatory synaptic transmission in hippocampal slices from mice devoid of Synapsins I and II and from wild-type control animals. Application of stimulus trains at variable frequencies to the CA3-to-CA1 pyramidal cell synapse suggested that, in both genotypes, synaptic responses obtained within 2 s stimulation originated from readily releasable vesicles. Detailed analysis of the responses during this period indicated that stimulus trains at 2-20 Hz enhanced all early synaptic responses in the CA3-to-CA1 pyramidal cell synapse, but depressed all early responses in the medial perforant path-to-granule cell synapse. The Synapsin-dependent part of these responses, i.e. the difference between the results obtained in the transgene and the wild-type preparations, showed that in the former synapse, the presence of Synapsins I and II minimized the early responses at 2 Hz, but enhanced the early responses at 20 Hz, while in the latter synapse, the presence of Synapsins I and II enhanced all responses at both stimulation frequencies. The results indicate that Synapsins I and II are necessary for full expression of both enhancing and decreasing modulatory effects on synaptic transmission originating from the readily releasable vesicles in these excitatory synapses.
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Molecular Determinants of Synapsin Targeting to Presynaptic Terminals
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2004Co-Authors: Daniel Gitler, Paul Greengard, Hung-teh Kao, Dayu Lin, Sangmi Lim, Jian Feng, George J AugustineAbstract:Although Synapsins are abundant synaptic vesicle proteins that are widely used as markers of presynaptic terminals, the mechanisms that target Synapsins to presynaptic terminals have not been elucidated. We have addressed this question by imaging the targeting of green fluorescent protein-tagged Synapsins in cultured hippocampal neurons. Whereas all Synapsin isoforms targeted robustly to presynaptic terminals in wild-type neurons, Synapsin Ib scarcely targeted in neurons in which all Synapsins were knocked-out. Coexpression of other Synapsin isoforms significantly strengthened the targeting of Synapsin Ib in knock-out neurons, indicating that heterodimerization is required for Synapsin Ib to target. Truncation mutagenesis revealed that Synapsin Ia targets via distributed binding sites that include domains B, C, and E. Although domain A was not necessary for targeting, its presence enhanced targeting. Domain D inhibited targeting, but this inhibition was overcome by domain E. Thus, multiple intermolecular and intramolecular interactions are required for Synapsins to target to presynaptic terminals.
