scholarly journals The Zinc Transporter ZnT3 Interacts with AP-3 and It Is Preferentially Targeted to a Distinct Synaptic Vesicle Subpopulation

2004 ◽  
Vol 15 (2) ◽  
pp. 575-587 ◽  
Author(s):  
Gloria Salazar ◽  
Rachal Love ◽  
Erica Werner ◽  
Michele M. Doucette ◽  
Su Cheng ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Zinc Transporter ◽  
Neuroendocrine Cells ◽  
Molecular Composition ◽  
Wild Type ◽  
Overlapping Domains ◽  
Biochemical Analyses

Synaptic vesicles (SV) are generated by two different mechanisms, one AP-2 dependent and one AP-3 dependent. It has been uncertain, however, whether these mechanisms generate SV that differ in molecular composition. We explored this hypothesis by analyzing the targeting of ZnT3 and synaptophysin both to PC12 synaptic-like microvesicles (SLMV) as well as SV isolated from wild-type and AP-3-deficient mocha brains. ZnT3 cytosolic tail interacted selectively with AP-3 in cell-free assays. Accordingly, pharmacological disruption of either AP-2- or AP-3-dependent SLMV biogenesis preferentially reduced synaptophysin or ZnT3 targeting, respectively; suggesting that these antigens were concentrated in different vesicles. As predicted, immuno-isolated SLMV revealed that ZnT3 and synaptophysin were enriched in different vesicle populations. Likewise, morphological and biochemical analyses in hippocampal neurons indicated that these two antigens were also present in distinct but overlapping domains. ZnT3 SV content was reduced in AP-3-deficient neurons, but synaptophysin was not altered in the AP-3 null background. Our evidence indicates that neuroendocrine cells assemble molecularly heterogeneous SV and suggests that this diversity could contribute to the functional variety of synapses.

scholarly journals BDNF mobilizes synaptic vesicles and enhances synapse formation by disrupting cadherin–β-catenin interactions

2006 ◽  
Vol 174 (2) ◽  
pp. 289-299 ◽  
Author(s):  
Shernaz X. Bamji ◽  
Beatriz Rico ◽  
Nikole Kimes ◽  
Louis F. Reichardt
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Synapse Formation ◽  
Time Lapse ◽  
Synapse Density ◽  
Vesicle Mobility ◽  
Small Clusters ◽  
Vertebrate Central Nervous System

Neurons of the vertebrate central nervous system have the capacity to modify synapse number, morphology, and efficacy in response to activity. Some of these functions can be attributed to activity-induced synthesis and secretion of the neurotrophin brain-derived neurotrophic factor (BDNF); however, the molecular mechanisms by which BDNF mediates these events are still not well understood. Using time-lapse confocal analysis, we show that BDNF mobilizes synaptic vesicles at existing synapses, resulting in small clusters of synaptic vesicles “splitting” away from synaptic sites. We demonstrate that BDNF's ability to mobilize synaptic vesicle clusters depends on the dissociation of cadherin–β-catenin adhesion complexes that occurs after tyrosine phosphorylation of β-catenin. Artificially maintaining cadherin–β-catenin complexes in the presence of BDNF abolishes the BDNF-mediated enhancement of synaptic vesicle mobility, as well as the longer-term BDNF-mediated increase in synapse number. Together, this data demonstrates that the disruption of cadherin–β-catenin complexes is an important molecular event through which BDNF increases synapse density in cultured hippocampal neurons.

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 Normal Biogenesis and Cycling of Empty Synaptic Vesicles in Dopamine Neurons of Vesicular Monoamine Transporter 2 Knockout Mice

2005 ◽  
Vol 16 (1) ◽  
pp. 306-315 ◽  
Author(s):  
Benjamin G. Croft ◽  
Gabriel D. Fortin ◽  
Amadou T. Corera ◽  
Robert H. Edwards ◽  
Alain Beaudet ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Knockout Mice ◽  
Synaptic Vesicles ◽  
Dopamine Neurons ◽  
Vesicular Monoamine Transporter ◽  
Wild Type ◽  
Monoamine Transporter ◽  
Vesicle Biogenesis ◽  
And Function ◽  
Activity Dependent

The neuronal isoform of vesicular monoamine transporter, VMAT2, is responsible for packaging dopamine and other monoamines into synaptic vesicles and thereby plays an essential role in dopamine neurotransmission. Dopamine neurons in mice lacking VMAT2 are unable to store or release dopamine from their synaptic vesicles. To determine how VMAT2-mediated filling influences synaptic vesicle morphology and function, we examined dopamine terminals from VMAT2 knockout mice. In contrast to the abnormalities reported in glutamatergic terminals of mice lacking VGLUT1, the corresponding vesicular transporter for glutamate, we found that the ultrastructure of dopamine terminals and synaptic vesicles in VMAT2 knockout mice were indistinguishable from wild type. Using the activity-dependent dyes FM1-43 and FM2-10, we also found that synaptic vesicles in dopamine neurons lacking VMAT2 undergo endocytosis and exocytosis with kinetics identical to those seen in wild-type neurons. Together, these results demonstrate that dopamine synaptic vesicle biogenesis and cycling are independent of vesicle filling with transmitter. By demonstrating that such empty synaptic vesicles can cycle at the nerve terminal, our study suggests that physiological changes in VMAT2 levels or trafficking at the synapse may regulate dopamine release by altering the ratio of fillable-to-empty synaptic vesicles, as both continue to cycle in response to neural activity.

scholarly journals Synaptophysin I Controls the Targeting of VAMP2/Synaptobrevin II to Synaptic Vesicles

2003 ◽  
Vol 14 (12) ◽  
pp. 4909-4919 ◽  
Author(s):  
Maria Pennuto ◽  
Dario Bonanomi ◽  
Fabio Benfenati ◽  
Flavia Valtorta
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Synaptic Vesicle ◽  
Cell Body ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Direct Interaction ◽  
Diffuse Component ◽  
Synaptotagmin I ◽  
Dose Dependent

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.

scholarly journals Munc13 controls the location and efficiency of dense-core vesicle release in neurons

2012 ◽  
Vol 199 (6) ◽  
pp. 883-891 ◽  
Author(s):  
Rhea van de Bospoort ◽  
Margherita Farina ◽  
Sabine K. Schmitz ◽  
Arthur de Jong ◽  
Heidi de Wit ◽  
...  
Keyword(s):  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Dense Core ◽  
Dense Core Vesicle ◽  
Wild Type ◽  
Vesicle Release ◽  
Prolonged Stimulation ◽  
And Function ◽  
Activity Dependent ◽  
Single Vesicle

Neuronal dense-core vesicles (DCVs) contain diverse cargo crucial for brain development and function, but the mechanisms that control their release are largely unknown. We quantified activity-dependent DCV release in hippocampal neurons at single vesicle resolution. DCVs fused preferentially at synaptic terminals. DCVs also fused at extrasynaptic sites but only after prolonged stimulation. In munc13-1/2–null mutant neurons, synaptic DCV release was reduced but not abolished, and synaptic preference was lost. The remaining fusion required prolonged stimulation, similar to extrasynaptic fusion in wild-type neurons. Conversely, Munc13-1 overexpression (M13OE) promoted extrasynaptic DCV release, also without prolonged stimulation. Thus, Munc13-1/2 facilitate DCV fusion but, unlike for synaptic vesicles, are not essential for DCV release, and M13OE is sufficient to produce efficient DCV release extrasynaptically.

scholarly journals Ageing synaptic vesicles are inactivated by contamination with SNAP25

2017 ◽  
Author(s):  
Sven Truckenbrodt ◽  
Abhiyan Viplav ◽  
Sebastian Jähne ◽  
Angela Vogts ◽  
Annette Denker ◽  
...  
Keyword(s):  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Wild Type ◽  
Recycling Endosome ◽  
Vesicle Recycling ◽  
Priming Process ◽  
Recycling Pathway ◽  
Molecular Damage ◽  
Wild Type Form ◽  
Cultured Hippocampal Neurons

AbstractOld organelles can become a hazard to cellular function, by accumulating molecular damage. Mechanisms that identify aged organelles, and prevent them from participating in cellular reactions, are therefore necessary. We describe here one such mechanism for the synaptic vesicle recycling pathway. Using cultured hippocampal neurons, we found that newly synthesized vesicle proteins were incorporated in the active (recycling) pool, and were preferentially employed in neurotransmitter release. They remained in use for up to ~24 hours, during which they recycled up to a few hundred times. We could only detect one change in the molecular composition of the vesicles, an apparent accumulation of SNAP25 in the aged synaptic vesicles. Overexpression of SNAP25, both in wild-type form or in vesicle-bound form, inhibited exocytosis and promoted the co-localization of the vesicle molecules with a recycling endosome marker. This is in line with the hypothesis that the SNAP25 contamination causes the inactivation of the aged vesicles. The SNAP25 overexpression effect could be alleviated by co-expressing the vesicle-associated molecule CSPa, which has been previously shown to be involved in chaperoning SNAP25 in the vesicle priming process. Overall, these results suggest that newly synthesized vesicle molecules are preferred in vesicle recycling, probably through a mechanism that renders their priming more efficient than that of aged vesicles.

scholarly journals Targeting of the Synaptic Vesicle Protein Synaptobrevin in the Axon of Cultured Hippocampal Neurons: Evidence for Two Distinct Sorting Steps

1997 ◽  
Vol 139 (4) ◽  
pp. 917-927 ◽  
Author(s):  
Anne E. West ◽  
Rachael L. Neve ◽  
Kathleen M. Buckley
Keyword(s):  
Amino Acid ◽  
Synaptic Vesicle ◽  
Transferrin Receptor ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Expression System ◽  
Amino Acid Sequences ◽  
Amino Acid Region ◽  
Human Transferrin Receptor ◽  
Cultured Hippocampal Neurons

Synaptic vesicles are concentrated in the distal axon, far from the site of protein synthesis. Integral membrane proteins destined for this organelle must therefore make complex targeting decisions. Short amino acid sequences have been shown to act as targeting signals directing proteins to a variety of intracellular locations. To identify synaptic vesicle targeting sequences and to follow the path that proteins travel en route to the synaptic vesicle, we have used a defective herpes virus amplicon expression system to study the targeting of a synaptobrevin-transferrin receptor (SB-TfR) chimera in cultured hippocampal neurons. Addition of the cytoplasmic domain of synaptobrevin onto human transferrin receptor was sufficient to retarget the transferrin receptor from the dendrites to presynaptic sites in the axon. At the synapse, the SB-TfR chimera did not localize to synaptic vesicles, but was instead found in an organelle with biochemical and functional characteristics of an endosome. The chimera recycled in parallel with synaptic vesicle proteins demonstrating that the nerve terminal efficiently sorts transmembrane proteins into different pathways. The synaptobrevin sequence that controls targeting to the presynaptic endosome was not localized to a single, 10– amino acid region of the molecule, indicating that this targeting signal may be encoded by a more distributed structural conformation. However, the chimera could be shifted to synaptic vesicles by deletion of amino acids 61–70 in synaptobrevin, suggesting that separate signals encode the localization of synaptobrevin to the synapse and to the synaptic vesicle.

scholarly journals Synaptotagmin 7 is targeted to the axonal plasma membrane through γ-secretase processing to promote synaptic vesicle docking in mouse hippocampal neurons

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jason D Vevea ◽  
Grant F Kusick ◽  
Kevin C Courtney ◽  
Erin Chen ◽  
Shigeki Watanabe ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Glutamate Release ◽  
Vesicle Docking ◽  
Presynaptic Function ◽  
Asynchronous Release ◽  
Vesicle Cycle ◽  
Activity Dependent

Synaptotagmin 7 (SYT7) has emerged as a key regulator of presynaptic function, but its localization and precise role in the synaptic vesicle cycle remain the subject of debate. Here, we used iGluSnFR to optically interrogate glutamate release, at the single-bouton level, in SYT7KO-dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired-pulse facilitation, and synaptic vesicle replenishment and found that SYT7 contributes to each of these processes to different degrees. ‘Zap-and-freeze’ electron microscopy revealed that a loss of SYT7 diminishes docking of synaptic vesicles after a stimulus and inhibits the recovery of depleted synaptic vesicles after a stimulus train. SYT7 supports these functions from the axonal plasma membrane, where its localization and stability require both γ-secretase-mediated cleavage and palmitoylation. In summary, SYT7 is a peripheral membrane protein that controls multiple modes of synaptic vesicle (SV) exocytosis and plasticity, in part, through enhancing activity-dependent docking of SVs.

scholarly journals Synaptotagmin 7 is enriched at the plasma membrane through γ-secretase processing to promote vesicle docking and control synaptic plasticity in mouse hippocampal neurons

2021 ◽  
Author(s):  
Jason D. Vevea ◽  
Grant F. Kusick ◽  
Erin Chen ◽  
Kevin C. Courtney ◽  
Shigeki Watanabe ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Synaptic Function ◽  
List Type ◽  
Paired Pulse ◽  
Paired Pulse Facilitation ◽  
Asynchronous Release ◽  
Vesicle Cycle

Abstract Synaptotagmin (SYT) 7 has emerged as key regulator of presynaptic function, but its localization and precise function in the synaptic vesicle cycle remain unclear. Here, we used iGluSnFR to optically and directly interrogate glutamate release, at the single bouton level, in SYT7 KO dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired pulse facilitation, and synaptic vesicle replenishment, and found that SYT7 contributes to each of these processes to different degrees. ‘Zap-and-freeze’ electron microscopy revealed that loss of SYT7 impairs the docking of synaptic vesicles after a stimulus and the recovery of depleted synaptic vesicles after a stimulus train. To execute these functions, SYT7 must be targeted to the plasma membrane via γ-secretase-mediated cleavage of the amino terminus, followed by palmitoylation. The complex sorting itinerary of SYT7 endows this Ca2+-sensor with the ability to control crucial forms of synaptic function and plasticity. SYT7 mediated asynchronous release, paired pulse facilitation, and synaptic vesicle replenishment was observed optically at individual hippocampal synapses Localization, trafficking, and stability of SYT7 is dependent on processing by γ-secretase Short term plasticity defects arise in SYT7KOs due to decreased docking of synaptic vesicles after stimulation SYT7 promotes paired-pulse facilitation and asynchronous release via distinct mechanisms

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