Structure and function of the synapsins.

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Effects of the neuronal phosphoprotein synapsin I on actin polymerization. II. Analytical interpretation of kinetic curves.

The general features of the kinetics of actin polymerization are investigated by mathematical models, with the aim of identifying the kinetically relevant parameters in the process and detecting and interpreting the alterations occurring in actin polymerization under various experimental conditions. Polymerization curves, obtained by following the increase in fluorescence of actin derivatized with N-(1-pyrenyl) iodoacetamide, are fitted using analytical equations derived from biochemical models of the actin polymerization process. Particular attention is given to the evaluation of the effects of the neuronal phosphoprotein synapsin I. The models obtained under various ionic conditions reveal that synapsin I interacts with actin in a very complex fashion, sharing some of the properties of classical nucleating proteins but displaying also actions not described previously for other actin-binding proteins. Synapsin I appears to bind G-actin with a very high stoichiometry (1:2-4), and the complex behaves as an F-actin nucleus, producing actin filaments under conditions where spontaneous polymerization is negligible. These actions of synapsin I are markedly affected by site-specific phosphorylation of the protein. An original transformation of the fluorescence data, which estimates the disappearance rate of actin monomer toward the critical concentration, is presented and shown to be of general usefulness for the study of actin-binding proteins

Effects of the neuronal phosphoprotein synapsin I on actin polymerization. I. Evidence for a phosphorylation-dependent nucleating effect.

Synapsin I is a synaptic vesicle-specific phosphoprotein which is able to bind and bundle actin filaments in a phosphorylation-dependent fashion. In the present paper we have analyzed the effects of synapsin I on the kinetics of actin polymerization and their modulation by site-specific phosphorylation of synapsin I. We found that dephosphorylated synapsin I accelerates the initial rate of actin polymerization and decreases the rate of filament elongation. The effect was observed at both low and high ionic strength, was specific for synapsin I, and was still present when polymerization was triggered by F-actin seeds. Dephosphorylated synapsin I was also able to induce actin polymerization and bundle formation in the absence of KCl and MgCl2. The effects of synapsin I were strongly decreased after its phosphorylation by Ca2+/calmodulin-dependent protein kinase II. These observations suggest that synapsin I has a phosphorylation-dependent nucleating effect on actin polymerization. The data are compatible with the view that changes in the phosphorylation state of synapsin I play a functional role in regulating the interactions between the nerve terminal cytoskeleton and synaptic vesicles in various stages of the exoendocytotic cycle

Impaired GABAB-mediated presynaptic inhibition increases excitatory strength and alters short-term plasticity in synapsin knockout mice

Synapsins are a family of synaptic vesicle phosphoproteins regulating synaptic transmission and plasticity. SYN1/2 genes are major epilepsy susceptibility genes in humans. Consistently, synapsin I/II/III triple knockout (TKO) mice are epileptic and exhibit severe impairments in phasic and tonic GABAergic inhibition that precede the appearance of the epileptic phenotype. These changes are associated with an increased strength of excitatory transmission that has never been mechanistically investigated. Here, we observed that an identical effect in excitatory transmission could be induced in wild-type (WT) Schaffer collateral-CA1 pyramidal cell synapses by blockade of GABABreceptors (GABABRs). The same treatment was virtually ineffective in TKO slices, suggesting that the increased strength of the excitatory transmission results from an impairment of GABABpresynaptic inhibition. Exogenous stimulation of GABABRs in excitatory autaptic neurons, where GABA spillover is negligible, demonstrated that GABABRs were effective in inhibiting excitatory transmission in both WT and TKO neurons. These results demonstrate that the decreased GABA release and spillover, previously observed in TKO hippocampal slices, removes the tonic brake of presynaptic GABABRs on glutamate transmission, making the excitation/inhibition imbalance stronger

Transcranial direct current stimulation of the mouse prefrontal cortex modulates serotonergic neural activity of the dorsal raphe nucleus

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Synapsin Is a Novel Rab3 Effector Protein on Small Synaptic Vesicles

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APache Is an AP2-Interacting Protein Involved inSynaptic Vesicle Trafficking and Neuronal Development

ynaptic transmission is critically dependent on synaptic vesicle (SV) recycling. Although the precise mechanisms of SV retrieval are still debated, it is widely accepted that a fundamental role is played by clathrin-mediated endocytosis, a form of endocytosis that capitalizes on the clathrin/adaptor protein complex 2 (AP2) coat and several accessory factors. Here, we show that the previously uncharacterized protein KIAA1107, predicted by bioinformatics analysis to be involved in the SV cycle, is an AP2-interacting clathrin-endocytosis protein (APache). We found that APache is highly enriched in the CNS and is associated with clathrin-coated vesicles via interaction with AP2. APache-silenced neurons exhibit a severe impairment of maturation at early developmental stages, reduced SV density, enlarged endosome-like structures, and defects in synaptic transmission, consistent with an impaired clathrin/AP2-mediated SV recycling. Our data implicate APache as an actor in the complex regulation of SV trafficking, neuronal development, and synaptic plasticity. Piccini et al. uncovered the AP2-interacting protein APache that acts in the clathrin-mediated endocytic machinery and synaptic vesicle trafficking. They found that silencing APache impairs neuronal development and neurotransmitter release during repetitive stimulation by markedly reducing vesicle recycling

Synapsin phosphorylation by SRC tyrosine kinase enhances SRC activity in synaptic vesicles.

Synapsins are synaptic vesicle-associated phosphoproteins implicated in the regulation of neurotransmitter release. Synapsin I is the major binding protein for the SH3 domain of the kinase c-Src in synaptic vesicles. Its binding leads to stimulation of synaptic vesicle-associated c-Src activity. We investigated the mechanism and role of Src activation by synapsins on synaptic vesicles. We found that synapsin is tyrosine phosphorylated by c-Src in vitro and on intact synaptic vesicles independently of its phosphorylation state on serine. Mass spectrometry revealed a single major phosphorylation site at Tyr(301), which is highly conserved in all synapsin isoforms and orthologues. Synapsin tyrosine phosphorylation triggered its binding to the SH2 domains of Src or Fyn. However, synapsin selectively activated and was phosphorylated by Src, consistent with the specific enrichment of c-Src in synaptic vesicles over Fyn or n-Src. The activity of Src on synaptic vesicles was controlled by the amount of vesicle-associated synapsin, which is in turn dependent on synapsin serine phosphorylation. Synaptic vesicles depleted of synapsin in vitro or derived from synapsin null mice exhibited greatly reduced Src activity and tyrosine phosphorylation of other synaptic vesicle proteins. Disruption of the Src-synapsin interaction by internalization of either the Src SH3 or SH2 domains into synaptosomes decreased synapsin tyrosine phosphorylation and concomitantly increased neurotransmitter release in response to Ca(2+)-ionophores. We conclude that synapsin is an endogenous substrate and activator of synaptic vesicle-associated c-Src and that regulation of Src activity on synaptic vesicles participates in the regulation of neurotransmitter release by synapsin

PhotoMEA: a new optical biosensor for the study of the functional properties of neuronal networks

Technological innovations in the fields of biomedical optics and electronics have lead to an extremely high level of miniaturization. These steps opened important perspectives to interface optoelectronic instruments with cellular systems. In this frame, a patent was registered for a novel optoelectronic technological solution, named PhotoMEA, for the study of the neuronal activity in culture, in order to understand the physiological and pathological functioning of neuronal networks. At present, the neuronal functional properties are investigated either by a large-scale approach (i.e. MicroElectrode Array devices, MEAs) that enables the study of the general activity of a complex neuronal network, or alternatively by a micro-scale approach (i.e. intracellular or patch electrodes) suitable for the detailed analysis of the molecular mechanisms that actively contribute to the generation and modulation of the single neuron activity. Systems based on electrodes have yielded important results in neurophysiology, but now they start to show some severe limits, such as the possibility of inducing cellular damage in the case of intracellular electrodes and the poor spatial resolution in the case of MEAs. The PhotoMEA device combines two optical tools for studying the functional properties of in-vitro neuronal networks. Light stimulation and optical recording of neuronal activity are promising approaches for investigating the molecular mechanisms at the basis of neuronal physiology. In particular, flash photolysis of caged compounds offers the unique advantage of allowing to quickly change the concentration of either intracellular or extracellular bioactive molecules, such as neurotransmitters or second messengers, for the stimulation or modulation of neuronal activity. Moreover, optical recordings of neuronal activity by Voltage-Sensitive Dyes (VSDs) allow to follow changes of neuronal membrane potential with high-spatial resolution. This enables the study of the sub-cellular responses and that of the entire network at the same time. Combining these two optical methods the micro-scale approach (stimulation) meets the large-scale approach (recording). This methodology may be extremely useful for testing the ability of drugs to affect neuronal properties as well as alterations in inter- and intra-neuronal communication

PhotoMEA: An opto-electronic biosensor for monitoring in vitro neuronal networks activity

PhotoMEA is a biosensor useful for the analysis of an in vitro neuronal network, fully based on optical methods. Its function is based on the stimulation of neurons with caged-glutamate and the recording of neuronal activity by fluorescence Voltage-Sensitive Dyes. The main advantage is that it will be possible to stimulate even at sub-single neuron level and to record with high resolution the activity of the entire network in the culture. A large-scale view of neuronal intercommunications offers a unique opportunity for testing the ability of drugs to affect neuronal properties as well as alterations in the behaviour of the entire network. The concept and a prototype for validation is described here in details