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.

CCK-8S increased the filopodia and spines density in cultured hippocampal neurons of APP/PS1 and wild-type mice

2013 ◽  
Vol 542 ◽  
pp. 47-52 ◽  
Author(s):  
Lu-lu Zhang ◽  
Xiao-fei Wei ◽  
Yang-hui Zhang ◽  
Shu-jun Xu ◽  
Xiao-wei Chen ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Wild Type ◽  
Cultured Hippocampal Neurons

scholarly journals Delayed Retraction of Filopodia in Gelsolin Null Mice

1997 ◽  
Vol 138 (6) ◽  
pp. 1279-1287 ◽  
Author(s):  
Mei Lu ◽  
Walter Witke ◽  
David J. Kwiatkowski ◽  
Kenneth S. Kosik
Keyword(s):  
Growth Cone ◽  
Hippocampal Neurons ◽  
Neurite Growth ◽  
Time Lapse ◽  
Growth Cones ◽  
Wild Type ◽  
Dynamic Studies ◽  
Shape Changes ◽  
Time Lapse Video ◽  
Cultured Hippocampal Neurons

Growth cones extend dynamic protrusions called filopodia and lamellipodia as exploratory probes that signal the direction of neurite growth. Gelsolin, as an actin filament-severing protein, may serve an important role in the rapid shape changes associated with growth cone structures. In wild-type (wt) hippocampal neurons, antibodies against gelsolin labeled the neurite shaft and growth cone. The behavior of filopodia in cultured hippocampal neurons from embryonic day 17 wt and gelsolin null (Gsn−) mice (Witke, W., A.H. Sharpe, J.H. Hartwig, T. Azuma, T.P. Stossel, and D.J. Kwiatkowski. 1995. Cell. 81:41–51.) was recorded with time-lapse video microscopy. The number of filopodia along the neurites was significantly greater in Gsn− mice and gave the neurites a studded appearance. Dynamic studies suggested that most of these filopodia were formed from the region of the growth cone and remained as protrusions from the newly consolidated shaft after the growth cone advanced. Histories of individual filopodia in Gsn− mice revealed elongation rates that did not differ from controls but an impaired retraction phase that probably accounted for the increased number of filopodia long the neutrite shaft. Gelsolin appears to function in the initiation of filopodial retraction and in its smooth progression.

Pregabalin Reduces the Release of Synaptic Vesicles from Cultured Hippocampal Neurons

2006 ◽  
Vol 70 (2) ◽  
pp. 467-476 ◽  
Author(s):  
Kristina D. Micheva ◽  
Charles P. Taylor ◽  
Stephen J Smith
Keyword(s):  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Cultured Hippocampal Neurons

scholarly journals Regulation of presynaptic phosphatidylinositol 4,5-biphosphate by neuronal activity

2001 ◽  
Vol 154 (2) ◽  
pp. 355-368 ◽  
Author(s):  
Kristina D. Micheva ◽  
Ronald W. Holz ◽  
Stephen J. Smith
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Confocal Imaging ◽  
Resting Cells ◽  
Ph Domain ◽  
Cellular Processes ◽  
Vesicle Recycling ◽  
Green Fluorescent

Phosphatidylinositol 4,5-biphosphate (PIP2) has been implicated in a variety of cellular processes, including synaptic vesicle recycling. However, little is known about the spatial distribution of this phospholipid in neurons and its dynamics. In this study, we have focused on these questions by transiently expressing the phospholipase C (PLC)-δ1 pleckstrin homology (PH) domain fused to green fluorescent protein (GFP) in cultured hippocampal neurons. This PH domain binds specifically and with high affinity to PIP2. Live confocal imaging revealed that in resting cells, PH-GFP is localized predominantly on the plasma membrane. Interestingly, no association of PH-GFP with synaptic vesicles in quiescent neurons was observed, indicating the absence of detectable PIP2 on mature synaptic vesicles. Electrical stimulation of hippocampal neurons resulted in a decrease of the PH-GFP signal at the plasma membrane, most probably due to a PLC-mediated hydrolysis of PIP2. This was accompanied in the majority of presynaptic terminals by a marked increase in the cytoplasmic PH-GFP signal, localized most probably on freshly endocytosed membranes. Further investigation revealed that the increase in PH-GFP signal was dependent on the activation of N-methyl-D-aspartate receptors and the consequent production of nitric oxide (NO). Thus, PIP2 in the presynaptic terminal appears to be regulated by postsynaptic activity via a retrograde action of NO.

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 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 KCC2 activity is critical in limiting the onset and severity of status epilepticus

2015 ◽  
Vol 112 (11) ◽  
pp. 3523-3528 ◽  
Author(s):  
Liliya Silayeva ◽  
Tarek Z. Deeb ◽  
Rochelle M. Hines ◽  
Matt R. Kelley ◽  
Michaelanne B. Munoz ◽  
...  
Keyword(s):  
Status Epilepticus ◽  
Neuronal Activity ◽  
Hippocampal Neurons ◽  
Critical Role ◽  
Aminobutyric Acid ◽  
Synaptic Inhibition ◽  
Wild Type ◽  
Expression And Activity ◽  
Cultured Hippocampal Neurons

The K+/Cl– cotransporter (KCC2) allows adult neurons to maintain low intracellular Cl– levels, which are a prerequisite for efficient synaptic inhibition upon activation of γ-aminobutyric acid receptors. Deficits in KCC2 activity are implicated in epileptogenesis, but how increased neuronal activity leads to transporter inactivation is ill defined. In vitro, the activity of KCC2 is potentiated via phosphorylation of serine 940 (S940). Here we have examined the role this putative regulatory process plays in determining KCC2 activity during status epilepticus (SE) using knockin mice in which S940 is mutated to an alanine (S940A). In wild-type mice, SE induced by kainate resulted in dephosphorylation of S940 and KCC2 internalization. S940A homozygotes were viable and exhibited comparable basal levels of KCC2 expression and activity relative to WT mice. However, exposure of S940A mice to kainate induced lethality within 30 min of kainate injection and subsequent entrance into SE. We assessed the effect of the S940A mutation in cultured hippocampal neurons to explore the mechanisms underlying this phenotype. Under basal conditions, the mutation had no effect on neuronal Cl– extrusion. However, a selective deficit in KCC2 activity was seen in S940A neurons upon transient exposure to glutamate. Significantly, whereas the effects of glutamate on KCC2 function could be ameliorated in WT neurons with agents that enhance S940 phosphorylation, this positive modulation was lost in S940A neurons. Collectively our results suggest that phosphorylation of S940 plays a critical role in potentiating KCC2 activity to limit the development of SE.

scholarly journals Q-SNARE Syntaxin 7 confers actin-dependent rapidly replenishing synaptic vesicles upon high activity

2020 ◽  
Author(s):  
Yasunori Mori ◽  
Koichiro Takenaka ◽  
Yugo Fukazawa ◽  
Shigeo Takamori
Keyword(s):  
High Frequency ◽  
Transmitter Release ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Actin Polymerization ◽  
Repetitive Stimulation ◽  
Snare Proteins ◽  
Endosomal Membrane ◽  
Kinetics Of ◽  
Cultured Hippocampal Neurons

AbstractReplenishment of readily releasable synaptic vesicles (SVs) with vesicles in the recycling pool is important for sustained transmitter release during repetitive stimulation. Kinetics of replenishment and available pool size define synaptic performance. However, whether all SVs in the recycling pool are recruited for release with equal probability is unknown. Here, using comprehensive optical imaging for various presynaptic endosomal SNARE proteins in cultured hippocampal neurons, we demonstrate that part of the recycling pool bearing the endosomal Q–SNARE Syntaxin 7 (Stx7) is preferentially mobilized for release during high–frequency repetitive stimulation. Recruitment of the SV pool marked with the Stx7–reporter requires high intra–terminal Ca2+ concentrations and actin polymerization. Furthermore, disruption of Stx7 function by overexpressing the N–terminal domain selectively abolished this pool. Thus, our data indicate that endosomal membrane fusion involving Stx7 is essential for adaptation of synapses to respond high-frequency repetitive stimulation.

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.

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