Loss of synaptotagmin IV results in a reduction in synaptic vesicles and a distortion of the Golgi structure in 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.

Download Full-text

Ageing synaptic vesicles are inactivated by contamination with SNAP25

10.1101/172239 ◽

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.

Download Full-text

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

The Journal of Cell Biology ◽

10.1083/jcb.139.4.917 ◽

1997 ◽

Vol 139 (4) ◽

pp. 917-927 ◽

Cited By ~ 39

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.

Download Full-text

Spatial changes in calcium signaling during the establishment of neuronal polarity and synaptogenesis.

The Journal of Cell Biology ◽

10.1083/jcb.126.6.1527 ◽

1994 ◽

Vol 126 (6) ◽

pp. 1527-1536 ◽

Cited By ~ 34

Author(s):

C Verderio ◽

S Coco ◽

G Fumagalli ◽

M Matteoli

Keyword(s):

Glutamate Receptors ◽

Synaptic Vesicles ◽

Hippocampal Neurons ◽

Imaging Techniques ◽

Indirect Evidence ◽

Receptor Activation ◽

Neuronal Polarity ◽

Spatial Changes ◽

Glutamate Receptor Antagonists ◽

Cultured Hippocampal Neurons

Calcium imaging techniques were used to obtain a clear although indirect evidence about the distribution of functional glutamate receptors of NMDA and non-NMDA type in cultured hippocampal neurons during establishment of polarity and synaptogenesis. Glutamate receptors were expressed and were already functional as early as one day after plating. At this stage NMDA and non-NMDA receptors were distributed in all plasmalemmal areas. During the establishment of neuronal polarity, responses to either types of glutamate receptors became restricted to the soma and dendrites. Compartmentalization of glutamate receptors occurred at stages of development when synaptic vesicles were already fully segregated to the axon. Formation of synapses was accompanied by a further redistribution of receptors, which segregated to synapse-enriched portions of dendrites. Receptor compartmentalization and dendritic redistribution as well as accumulation of synaptic vesicles at synaptic sites occurred also in neurons cultured in the presence of either the sodium channel blocker tetrodotoxin or glutamate receptor antagonists. These results indicate that signals generated by neuronal electrical activity or receptor activation are not involved in the establishment of neuronal polarity and synaptogenesis.

Download Full-text

A Conserved Mechanism of Synaptogyrin Localization

Molecular Biology of the Cell ◽

10.1091/mbc.12.8.2275 ◽

2001 ◽

Vol 12 (8) ◽

pp. 2275-2289 ◽

Cited By ~ 14

Author(s):

Hongjuan Zhao ◽

Michael L. Nonet

Keyword(s):

Synaptic Vesicles ◽

Hippocampal Neurons ◽

Fluorescent Protein ◽

Transmembrane Domain ◽

Mutational Analysis ◽

C Elegans ◽

C Terminus ◽

Green Fluorescent ◽

Cultured Hippocampal Neurons

We have studied the localization of synaptogyrin family members in vivo. Both native and green fluorescent protein (GFP)-taggedCaenorhabditis elegans synaptogyrin (SNG-1) are expressed in neurons and synaptically localized. Deletion and mutational analysis with the use of GFP-tagged SNG-1 has defined a 38 amino acid sequence within the C terminus of SNG-1 and a single arginine in the cytoplasmic loop between transmembrane domain 2 and 3 that are required for SNG-1 localization. These domains may represent components of signals that target synaptogyrin for endocytosis from the plasma membrane and direct synaptogyrin to synaptic vesicles, respectively. In chimeric studies, these regions were sufficient to relocalize cellugyrin, a nonneuronal form of synaptogyrin, from nonsynaptic regions such as the sensory dendrites and the cell body to synaptic vesicles. Furthermore, GFP-tagged rat synaptogyrin is synaptically localized in neurons of C. elegans and in cultured hippocampal neurons. Similarly, the C-terminal domain of rat synaptogyrin is necessary for localization in hippocampal neurons. Our study suggests that the mechanisms for synaptogyrin localization are likely to be conserved from C. elegans to vertebrates.

Download Full-text