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Control of synaptic vesicle release probability via VAMP4 targeting to endolysosomes.

ABSTRACT: Synaptic vesicle (SV) release probability (Pr), determines the steady state and plastic control of neurotransmitter release. However, how diversity in SV composition arises and regulates the Pr of individual SVs is not understood. We found that modulation of the copy number of the noncanonical vesicular SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor), vesicle-associated membrane protein 4 (VAMP4), on SVs is key for regulating Pr. Mechanistically, this is underpinned by its reduced ability to form an efficient SNARE complex with canonical plasma membrane SNAREs. VAMP4 has unusually high synaptic turnover and is selectively sorted to endolysosomes during activity-dependent bulk endocytosis. Disruption of endolysosomal trafficking and function markedly increased the abundance of VAMP4 in the SV pool and inhibited SV fusion. Together, our results unravel a new mechanism for generating SV heterogeneity and control of Pr through coupling of SV recycling to a major clearing system that regulates protein homeostasis.

SUBMITTER: Ivanova D

PROVIDER: S-EPMC8087399 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Control of synaptic vesicle release probability via VAMP4 targeting to endolysosomes

Project description:The probability of synaptic vesicle (SV) fusion in response to an action potential, termed vesicular release probability (Pr), is a stochastic but tightly regulated process. SNARE proteins are the molecular executors of SV fusion and therefore, key modulators of Pr. The non-canonical SNARE VAMP4 is a cargo for activity-dependent bulk endocytosis (ADBE), a major SV retrieval mode operating during intense neuronal activity. Using optical imaging in primary neuronal cultures and slice electrophysiology from wild-type and VAMP4 knockout mice, we reveal that modulation of VAMP4 copy number on SVs is an important mechanism to regulate Pr. The control of VAMP4 copy number is determined by its clearance from axons via the endolysosomal system after its retrieval by ADBE. This activity-dependent regulatory mechanism enables synapses to 1) adapt their Pr dynamically to changing input and 2) couple regulation of synaptic strength to neuronal proteostasis.

2021-03-11 | PXD021437 | Pride

Elevated synaptic vesicle release probability in synaptophysin/gyrin family quadruple knockouts.

Project description:Synaptophysins 1 and 2 and synaptogyrins 1 and 3 constitute a major family of synaptic vesicle membrane proteins. Unlike other widely expressed synaptic vesicle proteins such as vSNAREs and synaptotagmins, the primary function has not been resolved. Here, we report robust elevation in the probability of release of readily releasable vesicles with both high and low release probabilities at a variety of synapse types from knockout mice missing all four family members. Neither the number of readily releasable vesicles, nor the timing of recruitment to the readily releasable pool was affected. The results suggest that family members serve as negative regulators of neurotransmission, acting directly at the level of exocytosis to dampen connection strength selectively when presynaptic action potentials fire at low frequency. The widespread expression suggests that chemical synapses may play a frequency filtering role in biological computation that is more elemental than presently envisioned.Editorial noteThis article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

| S-EPMC6519982 | biostudies-literature

Influence of synaptic vesicle position on release probability and exocytotic fusion mode.

Project description:Neurotransmission depends on movements of transmitter-laden synaptic vesicles, but accurate, nanometer-scale monitoring of vesicle dynamics in presynaptic terminals has remained elusive. Here, we report three-dimensional, real-time tracking of quantum dot-loaded single synaptic vesicles with an accuracy of 20 to 30 nanometers, less than a vesicle diameter. Determination of the time, position, and mode of fusion, aided by trypan blue quenching of Qdot fluorescence, revealed that vesicles starting close to their ultimate fusion sites tended to fuse earlier than those positioned farther away. The mode of fusion depended on the prior motion of vesicles, with long-dwelling vesicles preferring kiss-and-run rather than full-collapse fusion. Kiss-and-run fusion events were concentrated near the center of the synapse, whereas full-collapse fusion events were broadly spread.

| S-EPMC3776413 | biostudies-literature

Position of UNC-13 in the active zone regulates synaptic vesicle release probability and release kinetics.

Project description:The presynaptic active zone proteins UNC-13/Munc13s are essential for synaptic vesicle (SV) exocytosis by directly interacting with SV fusion apparatus. An open question is how their association with active zones, hence their position to Ca(2+) entry sites, regulates SV release. The N-termini of major UNC-13/Munc13 isoforms contain a non-calcium binding C2A domain that mediates protein homo- or hetero-meric interactions. Here, we show that the C2A domain of Caenorhabditis elegans UNC-13 regulates release probability of evoked release and its precise active zone localization. Kinetics analysis of SV release supports that the proximity of UNC-13 to Ca(2+) entry sites, mediated by the C2A-domain containing N-terminus, is critical for accelerating neurotransmitter release. Additionally, the C2A domain is specifically required for spontaneous release. These data reveal multiple roles of UNC-13 C2A domain, and suggest that spontaneous release and the fast phase of evoked release may involve a common pool of SVs at the active zone. DOI: http://dx.doi.org/10.7554/eLife.01180.001.

| S-EPMC3821175 | biostudies-literature

Bassoon controls synaptic vesicle release via regulation of presynaptic phosphorylation and cAMP.

Project description:Neuronal presynaptic terminals contain hundreds of neurotransmitter-filled synaptic vesicles (SVs). The morphologically uniform SVs differ in their release competence segregating into functional pools that differentially contribute to neurotransmission. The presynaptic scaffold bassoon is required for neurotransmission, but the underlying molecular mechanisms are unknown. We report that glutamatergic synapses lacking bassoon feature decreased SV release competence and increased resting pool of SVs as assessed by imaging of SV release in cultured neurons. CDK5/calcineurin and cAMP/PKA presynaptic signalling are dysregulated, resulting in an aberrant phosphorylation of their downstream effectors synapsin1 and SNAP25, well-known regulators of SV release competence. An acute pharmacological restoration of physiological CDK5 and cAMP/PKA activity fully normalises the SV pools in neurons lacking bassoon. Finally, we demonstrate that CDK5-dependent regulation of PDE4 activity interacts with cAMP/PKA signalling and thereby controls SV release competence. These data reveal that bassoon organises SV pools in glutamatergic synapses via regulation of presynaptic phosphorylation and cAMP homeostasis and indicate a role of CDK5/PDE4/cAMP axis in the control of neurotransmitter release.

| S-EPMC9346490 | biostudies-literature

Heterodimerization of UNC-13/RIM regulates synaptic vesicle release probability but not priming in <i>C. elegans</i>.

Project description:UNC-13 proteins play an essential role in synaptic transmission by recruiting synaptic vesicles (SVs) to become available for release, which is termed SV priming. Here we show that the C2A domain of UNC-13L, like the corresponding domain in mammalian Munc13-1, displays two conserved binding modes: forming C2A/C2A homodimers, or forming a heterodimer with the zinc finger domain of UNC-10/RIM (C2A/RIM). Functional analysis revealed that UNC-13L's C2A promotes synaptic transmission by regulating a post-priming process. Stimulus-evoked release but not SV priming, was impaired in unc-10 mutants deficient for C2A/RIM heterodimerization, leading to decreased release probability. Disrupting C2A/C2A homodimerization in UNC-13L-rescued animals had no effect on synaptic transmission, but fully restored the evoked release and the release probability of unc-10/RIM mutants deficient for C2A/RIM heterodimerization. Thus, our results support the model that RIM binding C2A releases UNC-13L from an autoinhibitory homodimeric complex to become fusion-competent by functioning as a switch only.

| S-EPMC6389284 | biostudies-literature

Release activity-dependent control of vesicle endocytosis by the synaptic adhesion molecule N-cadherin.

Project description:At synapses in the mammalian brain, continuous information transfer requires the long-term maintenance of homeostatic coupling between exo- and endocytosis of synaptic vesicles. Because classical endocytosis is orders of magnitude slower than the millisecond-range exocytosis of vesicles, high frequency vesicle fusion could potentially compromise structural stability of synapses. However, the molecular mechanisms mediating the tight coupling of exo- and endocytosis are largely unknown. Here, we investigated the role of the transsynaptic adhesion molecules N-cadherin and Neuroligin1 in regulating vesicle exo- and endocytosis by using activity-induced FM4-64 staining and by using synaptophysin-pHluorin fluorescence imaging. The synaptic adhesion molecules N-cadherin and Neuroligin1 had distinct impacts on exo- and endocytosis at mature cortical synapses. Expression of Neuroligin1 enhanced vesicle release in a N-cadherin-dependent way. Most intriguingly, expression of N-cadherin enhanced both vesicle exo- and endocytosis. Further detailed analysis of N-cadherin knockout neurons revealed that the boosting of endocytosis by N-cadherin was largely dependent on preceding high levels of vesicle release activity. In summary, regulation of vesicle endocytosis was mediated at the molecular level by N-cadherin in a release activity-dependent manner. Because of its endocytosis enhancing function, N-cadherin might play an important role in the coupling of vesicle exo- and endocytosis.

| S-EPMC5247765 | biostudies-literature

A Hyperactive Form of unc-13 Enhances Ca<sup>2+</sup> Sensitivity and Synaptic Vesicle Release Probability in C. elegans.

Project description:Munc13 proteins play several roles in regulating short-term synaptic plasticity. However, the underlying molecular mechanisms remain largely unclear. Here we report that C. elegans UNC-13L, a Munc13-1 ortholog, has three domains that inhibit synaptic vesicle (SV) exocytosis. These include the X (sequence between C2A and C1), C1, and C2B domains. Deleting all three inhibitory domains produces a hyperactive UNC-13 (sUNC-13) that exhibits dramatically increased neurotransmitter release, Ca2+ sensitivity of release, and release probability. The vesicular pool in unc-13 mutants rescued by sUNC-13 exhibits a faster synaptic recovery and replenishment rate, demonstrating an important role of sUNC-13 in regulating synaptic plasticity. Analysis of double mutants suggests that sUNC-13 enhances tonic release by increasing the open probability of UNC-64/syntaxin-1A, whereas its effects on evoked release appear to be mediated by additional functions, presumably by further regulating the activity of the assembled soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) complex.

| S-EPMC6779330 | biostudies-literature

Ammonium chloride alters neuronal excitability and synaptic vesicle release.

Project description:Genetically encoded pH-sensors are widely used in studying cell membrane trafficking and membrane protein turnover because they render exo-/endocytosis-associated pH changes to fluorescent signals. For imaging and analysis purposes, high concentration ammonium chloride is routinely used to alkalize intracellular membrane compartments under the assumption that it does not cause long-term effects on cellular processes being studied like neurotransmission. However, pathological studies about hyperammonemia have shown that ammonium is toxic to brain cells especially astrocytes and neurons. Here, we focus on ammonium's physiological impacts on neurons including membrane potential, cytosolic Ca2+ and synaptic vesicles. We have found that extracellularly applied ammonium chloride as low as 5 mM causes intracellular Ca2+-increase and a reduction of vesicle release even after washout. The often-used 50 mM ammonium chloride causes more extensive and persistent changes, including membrane depolarization, prolonged elevation of intracellular Ca2+ and diminution of releasable synaptic vesicles. Our findings not only help to bridge the discrepancies in previous studies about synaptic vesicle release using those pH-sensors or other vesicle specific reporters, but also suggest an intriguing relationship between intracellular pH and neurotransmission.

| S-EPMC5505971 | biostudies-literature

Presynaptic GCaMP expression decreases vesicle release probability at the calyx of Held.

Project description:Synaptic vesicle (SV) exocytosis is intimately dependent on free local Ca2+ near active zones. Genetically encoded calcium indicators (GECIs) have become an indispensable tool to monitor calcium dynamics during physiological responses, and they are widely used as a proxy to monitor activity in neuronal ensembles and at synaptic terminals. However, GECIs' ability to bind Ca2+ at physiologically relevant concentration makes them strong candidates to affect calcium homeostasis and alter synaptic transmission by exogenously increasing Ca2+ buffering. In the present study, we show that genetically expressed GCaMP6m modulates SV release probability at the mouse calyx of Held synapse. GCaMP6m expression for approximately three weeks decreased initial SV release for both low-frequency stimulation and high-frequency stimulation trains, and slowed presynaptic short-term depression. However, GCaMP6m does not affect quantal events during spontaneous activity at this synapse. This study emphasizes the careful use of GECIs as monitors of neuronal activity and inspects the role of these transgenic indicators which may alter calcium-dependent physiological responses.

| S-EPMC6186185 | biostudies-literature

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