scholarly journals Native α-synuclein induces clustering of synaptic-vesicle mimics via binding to phospholipids and synaptobrevin-2/VAMP2

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Jiajie Diao ◽  
Jacqueline Burré ◽  
Sandro Vivona ◽  
Daniel J Cipriano ◽  
Manu Sharma ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Lipid Binding ◽  
Snare Complex ◽  
Loss Of Function ◽  
Related Point ◽  
Molecular Mechanism Of Action ◽  
Presynaptic Protein ◽  
Single Vesicle

α-Synuclein is a presynaptic protein that is implicated in Parkinson's and other neurodegenerative diseases. Physiologically, native α-synuclein promotes presynaptic SNARE-complex assembly, but its molecular mechanism of action remains unknown. Here, we found that native α-synuclein promotes clustering of synaptic-vesicle mimics, using a single-vesicle optical microscopy system. This vesicle-clustering activity was observed for both recombinant and native α-synuclein purified from mouse brain. Clustering was dependent on specific interactions of native α-synuclein with both synaptobrevin-2/VAMP2 and anionic lipids. Out of the three familial Parkinson's disease-related point mutants of α-synuclein, only the lipid-binding deficient mutation A30P disrupted clustering, hinting at a possible loss of function phenotype for this mutant. α-Synuclein had little effect on Ca2+-triggered fusion in our reconstituted single-vesicle system, consistent with in vivo data. α-Synuclein may therefore lead to accumulation of synaptic vesicles at the active zone, providing a ‘buffer’ of synaptic vesicles, without affecting neurotransmitter release itself.

scholarly journals Dual pools of actin at presynaptic terminals

2012 ◽  
Vol 107 (12) ◽  
pp. 3479-3492 ◽  
Author(s):  
Adam Bleckert ◽  
Huzefa Photowala ◽  
Simon Alford
Keyword(s):  
Synaptic Transmission ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Repetitive Stimulation ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Vesicle Recycling ◽  
Latrunculin A ◽  
Kinetics Of

We investigated actin's function in vesicle recycling and exocytosis at lamprey synapses and show that FM1-43 puncta and phalloidin-labeled filamentous actin (F-actin) structures are colocalized, yet recycling vesicles are not contained within F-actin clusters. Additionally, phalloidin also labels a plasma membrane-associated cortical actin. Injection of fluorescent G-actin revealed activity-independent dynamic actin incorporation into presynaptic synaptic vesicle clusters but not into cortical actin. Latrunculin-A, which sequesters G-actin, dispersed vesicle-associated actin structures and prevented subsequent labeled G-actin and phalloidin accumulation at presynaptic puncta, yet cortical phalloidin labeling persisted. Dispersal of presynaptic F-actin structures by latrunculin-A did not disrupt vesicle clustering or recycling or alter the amplitude or kinetics of excitatory postsynaptic currents (EPSCs). However, it slightly enhanced release during repetitive stimulation. While dispersal of presynaptic actin puncta with latrunculin-A failed to disperse synaptic vesicles or inhibit synaptic transmission, presynaptic phalloidin injection blocked exocytosis and reduced endocytosis measured by action potential-evoked FM1-43 staining. Furthermore, phalloidin stabilization of only cortical actin following pretreatment with latrunculin-A was sufficient to inhibit synaptic transmission. Conversely, treatment of axons with jasplakinolide, which induces F-actin accumulation but disrupts F-actin structures in vivo, resulted in increased synaptic transmission accompanied by a loss of phalloidin labeling of cortical actin but no loss of actin labeling within vesicle clusters. Marked synaptic deficits seen with phalloidin stabilization of cortical F-actin, in contrast to the minimal effects of disruption of a synaptic vesicle-associated F-actin, led us to conclude that two structurally and functionally distinct pools of actin exist at presynaptic sites.

scholarly journals Complexin cooperates with Bruchpilot to tether synaptic vesicles to the active zone cytomatrix

2019 ◽  
Vol 218 (3) ◽  
pp. 1011-1026 ◽  
Author(s):  
Nicole Scholz ◽  
Nadine Ehmann ◽  
Divya Sachidanandan ◽  
Cordelia Imig ◽  
Benjamin H. Cooper ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Active Zone ◽  
Synaptic Vesicles ◽  
Snare Complex ◽  
C Terminus ◽  
Unknown Protein ◽  
Transmission Part ◽  
Molecular Components ◽  
In Vivo Screen

Information processing by the nervous system depends on neurotransmitter release from synaptic vesicles (SVs) at the presynaptic active zone. Molecular components of the cytomatrix at the active zone (CAZ) regulate the final stages of the SV cycle preceding exocytosis and thereby shape the efficacy and plasticity of synaptic transmission. Part of this regulation is reflected by a physical association of SVs with filamentous CAZ structures via largely unknown protein interactions. The very C-terminal region of Bruchpilot (Brp), a key component of the Drosophila melanogaster CAZ, participates in SV tethering. Here, we identify the conserved SNARE regulator Complexin (Cpx) in an in vivo screen for molecules that link the Brp C terminus to SVs. Brp and Cpx interact genetically and functionally. Both proteins promote SV recruitment to the Drosophila CAZ and counteract short-term synaptic depression. Analyzing SV tethering to active zone ribbons of cpx3 knockout mice supports an evolutionarily conserved role of Cpx upstream of SNARE complex assembly.

scholarly journals A new life for an old pump: V-ATPase and neurotransmitter release

2014 ◽  
Vol 205 (1) ◽  
pp. 7-9 ◽  
Author(s):  
Stefano Vavassori ◽  
Andreas Mayer
Keyword(s):  
Plasma Membrane ◽  
Complex Formation ◽  
Membrane Fusion ◽  
Synaptic Vesicle ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Snare Complex ◽  
Spontaneous Release ◽  
Synaptic Vesicle Fusion ◽  
Snare Complex Formation

Neurons fire by releasing neurotransmitters via fusion of synaptic vesicles with the plasma membrane. Fusion can be evoked by an incoming signal from a preceding neuron or can occur spontaneously. Synaptic vesicle fusion requires the formation of trans complexes between SNAREs as well as Ca2+ ions. Wang et al. (2014. J. Cell Biol. http://dx.doi.org/jcb.201312109) now find that the Ca2+-binding protein Calmodulin promotes spontaneous release and SNARE complex formation via its interaction with the V0 sector of the V-ATPase.

scholarly journals Diverse modes of synaptic signaling, regulation, and plasticity distinguish two classes of C. elegans glutamatergic neurons

2017 ◽  
Author(s):  
Donovan Ventimiglia ◽  
Cornelia I. Bargmann
Keyword(s):  
Synaptic Vesicle ◽  
Neuronal Cell ◽  
Cell Types ◽  
Snare Complex ◽  
C Elegans ◽  
Synaptic Vesicle Exocytosis ◽  
High Calcium ◽  
Vesicle Exocytosis ◽  
Synaptic Dynamics

AbstractSynaptic vesicle release properties vary between neuronal cell types, but in most cases the molecular basis of this heterogeneity is unknown. Here, we compare in vivo synaptic properties of two neuronal classes in the C. elegans central nervous system, using VGLUT-pHluorin to monitor synaptic vesicle exocytosis and retrieval in intact animals. We show that the glutamatergic sensory neurons AWCON and ASH have distinct synaptic dynamics associated with tonic and phasic synaptic properties, respectively. Exocytosis in ASH and AWCON is differentially affected by SNARE-complex regulators that are present in both neurons: phasic ASH release is strongly dependent on UNC-13, whereas tonic AWCON release relies upon UNC-18 and on the protein kinase C homolog PKC-1. Exocytosis and retrieval each have two timescales in AWCON but one major timescale in ASH. Strong stimuli that elicit high calcium levels also increase exocytosis and retrieval rates in AWCON, generating distinct tonic and evoked synaptic modes. These results highlight the differential deployment of shared presynaptic proteins in neuronal cell type-specific functions.

scholarly journals Alpha-Synuclein and Calpains Disrupt SNARE-Mediated Synaptic Vesicle Fusion During Manganese Exposure in SH-SY5Y Cells

Cells ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 258 ◽  
Author(s):  
Can Wang ◽  
Zhuo Ma ◽  
Dong-Ying Yan ◽  
Chang Liu ◽  
Yu Deng ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Vesicle Fusion ◽  
Alpha Synuclein ◽  
Convincing Evidence ◽  
Snare Complex ◽  
Attachment Protein ◽  
Over Expression ◽  
Synaptic Vesicle Fusion

Synaptic vesicle fusion is mediated by an assembly of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs), composed of syntaxin 1, soluble NSF-attachment protein (SNAP)-25, and synaptobrevin-2/VAMP-2. Previous studies have suggested that over-exposure to manganese (Mn) could disrupt synaptic vesicle fusion by influencing SNARE complex formation, both in vitro and in vivo. However, the mechanisms underlying this effect remain unclear. Here we employed calpeptin, an inhibitor of calpains, along with a lentivirus vector containing alpha-synuclein (α-Syn) shRNA, to examine whether specific SNAP-25 cleavage and the over-expression of α-Syn disturbed the formation of the SNARE complex in SH-SY5Y cells. After cells were treated with Mn for 24 h, fragments of SNAP-25-N-terminal protein began to appear; however, this effect was reduced in the group of cells which were pre-treated with calpeptin. FM1-43-labeled synaptic vesicle fusion decreased with Mn treatment, which was consistent with the formation of SNARE complexes. The interaction of VAMP-2 and α-Syn increased significantly in normal cells in response to 100 μM Mn treatment, but decreased in LV-α-Syn shRNA cells treated with 100 μM Mn; similar results were observed in terms of the formation of SNARE complexes and FM1-43-labeled synaptic vesicle fusion. Our data suggested that Mn treatment could increase [Ca2+]i, leading to abnormally excessive calpains activity, which disrupted the SNARE complex by cleaving SNAP-25. Our data also provided convincing evidence that Mn could induce the over-expression of α-Syn; when combined with VAMP-2, α-Syn prevented VAMP-2 from joining the SNARE complex cycle.

scholarly journals The synaptic vesicle cycle: a single vesicle budding step involving clathrin and dynamin.

1996 ◽  
Vol 133 (6) ◽  
pp. 1237-1250 ◽  
Author(s):  
K Takei ◽  
O Mundigl ◽  
L Daniell ◽  
P De Camilli
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Nerve Terminals ◽  
Vesicle Budding ◽  
Vesicle Cycle ◽  
Single Vesicle

Strong evidence implicates clathrin-coated vesicles and endosome-like vacuoles in the reformation of synaptic vesicles after exocytosis, and it is generally assumed that these vacuoles represent a traffic station downstream from clathrin-coated vesicles. To gain insight into the mechanisms of synaptic vesicle budding from endosome-like intermediates, lysed nerve terminals and nerve terminal membrane subfractions were examined by EM after incubations with GTP gamma S. Numerous clathrin-coated budding intermediates that were positive for AP2 and AP180 immunoreactivity and often collared by a dynamin ring were seen. These were present not only on the plasma membrane (Takei, K., P.S. McPherson, S.L.Schmid, and P. De Camilli. 1995. Nature (Lond.). 374:186-190), but also on internal vacuoles. The lumen of these vacuoles retained extracellular tracers and was therefore functionally segregated from the extracellular medium, although narrow connections between their membranes and the plasmalemma were sometimes visible by serial sectioning. Similar observations were made in intact cultured hippocampal neurons exposed to high K+ stimulation. Coated vesicle buds were generally in the same size range of synaptic vesicles and positive for the synaptic vesicle protein synaptotagmin. Based on these results, we suggest that endosome-like intermediates of nerve terminals originate by bulk uptake of the plasma membrane and that clathrin- and dynamin-mediated budding takes place in parallel from the plasmalemma and from these internal membranes. We propose a synaptic vesicle recycling model that involves a single vesicle budding step mediated by clathrin and dynamin.

scholarly journals Clathrin coat controls synaptic vesicle acidification by blocking vacuolar ATPase activity

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Zohreh Farsi ◽  
Sindhuja Gowrisankaran ◽  
Matija Krunic ◽  
Burkhard Rammner ◽  
Andrew Woehler ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Synaptic Vesicle ◽  
Mouse Brain ◽  
Synaptic Vesicles ◽  
Vacuolar Atpase ◽  
Coated Vesicles ◽  
Electrochemical Gradient ◽  
Proper Timing ◽  
Adenosine Triphosphatases ◽  
Single Vesicle

Newly-formed synaptic vesicles (SVs) are rapidly acidified by vacuolar adenosine triphosphatases (vATPases), generating a proton electrochemical gradient that drives neurotransmitter loading. Clathrin-mediated endocytosis is needed for the formation of new SVs, yet it is unclear when endocytosed vesicles acidify and refill at the synapse. Here, we isolated clathrin-coated vesicles (CCVs) from mouse brain to measure their acidification directly at the single vesicle level. We observed that the ATP-induced acidification of CCVs was strikingly reduced in comparison to SVs. Remarkably, when the coat was removed from CCVs, uncoated vesicles regained ATP-dependent acidification, demonstrating that CCVs contain the functional vATPase, yet its function is inhibited by the clathrin coat. Considering the known structures of the vATPase and clathrin coat, we propose a model in which the formation of the coat surrounds the vATPase and blocks its activity. Such inhibition is likely fundamental for the proper timing of SV refilling.

scholarly journals BETA- AND GAMMA-SYNUCLEINS MODULATE SYNAPTIC VESICLE-BINDING OF ALPHA-SYNUCLEIN

2020 ◽  
Author(s):  
Kathryn E. Carnazza ◽  
Lauren Komer ◽  
André Pineda ◽  
Yoonmi Na ◽  
Trudy Ramlall ◽  
...  
Keyword(s):  
Nervous System ◽  
Synaptic Vesicle ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Direct Interaction ◽  
Alpha Synuclein ◽  
Snare Complex ◽  
Complex Assembly ◽  
Mutual Compensation ◽  
Conflicting Evidence

SUMMARYα-Synuclein (αSyn), β-synuclein (βSyn), and γ-synuclein (γSyn) are abundantly expressed in the vertebrate nervous system. αSyn functions in neurotransmitter release via binding to and clustering synaptic vesicles and chaperoning of SNARE-complex assembly. The functions of βSyn and γSyn are unknown. Functional redundancy of the three synucleins and mutual compensation when one synuclein is deleted have been proposed, but with conflicting evidence. Here, we demonstrate that βSyn and γSyn have a reduced affinity towards membranes compared to αSyn, and that direct interaction of βSyn or γSyn with αSyn results in reduced membrane binding of αSyn. Our data suggest that all three synucleins affect synapse function, but only αSyn mediates the downstream function of vesicle clustering and SNARE-complex assembly, while βSyn and γSyn modulate the activity of αSyn through regulating its binding to synaptic vesicles.

VAMP (synaptobrevin) is present in the plasma membrane of nerve terminals

1999 ◽  
Vol 112 (20) ◽  
pp. 3559-3567
Author(s):  
P. Taubenblatt ◽  
J.C. Dedieu ◽  
T. Gulik-Krzywicki ◽  
N. Morel
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Specific Interaction ◽  
Snare Complex ◽  
Nerve Endings ◽  
Morphological Data ◽  
Synaptosomal Plasma Membrane ◽  
Presynaptic Plasma Membrane ◽  
Vesicle Proteins

Synaptic vesicle docking and exocytosis require the specific interaction of synaptic vesicle proteins (such as VAMP/synaptobrevin) with presynaptic plasma membrane proteins (such as syntaxin and SNAP 25). These proteins form a stable, SDS-resistant, multimolecular complex, the SNARE complex. The subcellular distribution of VAMP and syntaxin within Torpedo electric organ nerve endings was studied by immunogoldlabeling of SDS-digested freeze-fracture replicas (Fujimoto, 1995). This technique allowed us to visualize large surface areas of the presynaptic plasma membrane and numerous synaptic vesicles from rapidly frozen nerve endings and synaptosomes. VAMP was found associated with synaptic vesicles, as also shown by conventional electron microscopy immunolabeling, and to the presynaptic plasma membrane (P leaflet). Syntaxin was also detected in the nerve ending plasma membrane, without gold labeling of synaptic vesicles. Comparison of gold particle densities suggests that the presynaptic plasma membrane contains 3 VAMP molecules per molecule of syntaxin. After biotinylation of intact synaptosomes, the synaptosomal plasma membrane was isolated on Streptavidin coated magnetic beads. Its antigenic content was compared to that of purified synaptic vesicles. VAMP was present in both membranes whereas syntaxin and SNAP 25 were highly enriched in the synaptosomal plasma membrane. This membrane has a low content of classical synaptic vesicle proteins (synaptophysin, SV2 and the vesicular acetylcholine transporter). The VAMP to syntaxin stoichiometry in the isolated synaptosomal membrane was estimated by comparison with purified antigens and close to 2, in accordance with morphological data. SDS-resistant SNARE complexes were detected in the isolated presynaptic membrane but absent in purified synaptic vesicles. Taken together, these results show that the presence of VAMP in the plasma membrane of nerve endings cannot result from exocytosis of synaptic vesicles, a process which could, as far as SNAREs are concerned, very much resemble homotypic fusion.

Role of C2 domain proteins during synaptic vesicle exocytosis

2010 ◽  
Vol 38 (1) ◽  
pp. 213-216 ◽  
Author(s):  
Sascha Martens
Keyword(s):  
Membrane Fusion ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Calcium Concentration ◽  
Membrane Curvature ◽  
Snare Complex ◽  
C2 Domain ◽  
C2 Domains ◽  
Synaptic Vesicle Exocytosis ◽  
Vesicle Exocytosis

Neurotransmitter release is mediated by the fusion of synaptic vesicles with the presynaptic plasma membrane. Fusion is triggered by a rise in the intracellular calcium concentration and is dependent on the neuronal SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) complex. A plethora of molecules such as members of the MUNC13, MUNC18, complexin and synaptotagmin families act along with the SNARE complex to enable calcium-regulated synaptic vesicle exocytosis. The synaptotagmins are localized to synaptic vesicles by an N-terminal transmembrane domain and contain two cytoplasmic C2 domains. Members of the synaptotagmin family are thought to translate the rise in intracellular calcium concentration into synaptic vesicle fusion. The C2 domains of synaptotagmin-1 bind membranes in a calcium-dependent manner and in response induce a high degree of membrane curvature, which is required for its ability to trigger membrane fusion in vitro and in vivo. Furthermore, members of the soluble DOC2 (double-C2 domain) protein family have similar properties. Taken together, these results suggest that C2 domain proteins such as the synaptotagmins and DOC2s promote membrane fusion by the induction of membrane curvature in the vicinity of the SNARE complex. Given the widespread expression of C2 domain proteins in secretory cells, it is proposed that promotion of SNARE-dependent membrane fusion by the induction of membrane curvature is a widespread phenomenon.

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