ORCID Profile
0000-0002-1762-160X
Current Organisation
The University of Edinburgh
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Publisher: Wiley
Date: 16-07-2019
DOI: 10.1111/JNC.14797
Publisher: Portland Press Ltd.
Date: 11-1994
DOI: 10.1042/BST0220970
Publisher: Elsevier BV
Date: 08-2003
Publisher: Cold Spring Harbor Laboratory
Date: 16-03-2022
DOI: 10.1101/2022.03.15.484308
Abstract: Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a severe early-onset epileptic encephalopathy resulting mainly from de novo mutations in the X-linked CDKL5 gene. To determine whether loss of presynaptic CDKL5 function contributes to CDD, we examined synaptic vesicle (SV) recycling in primary hippoc al neurons generated from a Cdkl5 knockout rat model. Using a genetically-encoded reporter, we revealed that CDKL5 is selectively required for efficient SV endocytosis. We showed that CDKL5 kinase activity is both necessary and sufficient for optimal SV endocytosis, since kinase-inactive mutations failed to correct endocytosis in Cdkl5 knockout neurons, whereas the isolated CDKL5 kinase domain fully restored SV endocytosis kinetics. Finally, we demonstrated that CDKL5-mediated phosphorylation of hiphysin 1, a putative presynaptic target, is not required for CDKL5-dependent control of SV endocytosis. Overall, our findings reveal a key presynaptic role for CDKL5 kinase activity and enhance our insight into how its dysfunction may culminate in CDD.
Publisher: Society for Neuroscience
Date: 07-01-2022
DOI: 10.1523/JNEUROSCI.0852-21.2021
Abstract: Synaptic vesicle (SV) recycling is essential for the maintenance of neurotransmission, with a number of neurodevelopmental disorders linked to defects in this process. Fragile X syndrome (FXS) results from a loss of fragile X mental retardation protein (FMRP) encoded by the FMR1 gene. Hyperexcitability of neuronal circuits is a key feature of FXS, therefore we investigated whether SV recycling was affected by the absence of FMRP during increased neuronal activity. We revealed that primary neuronal cultures from male Fmr1 knock-out (KO) rats display a specific defect in activity-dependent bulk endocytosis (ADBE). ADBE is dominant during intense neuronal activity, and this defect resulted in an inability of Fmr1 KO neurons to sustain SV recycling during trains of high-frequency stimulation. Using a molecular replacement strategy, we also revealed that a human FMRP mutant that cannot bind BK channels failed to correct ADBE dysfunction in KO neurons, however this dysfunction was corrected by BK channel agonists. Therefore, FMRP performs a key role in sustaining neurotransmitter release via selective control of ADBE, suggesting intervention via this endocytosis mode may correct the hyperexcitability observed in FXS. SIGNIFICANCE STATEMENT Loss of fragile X mental retardation protein (FMRP) results in fragile X syndrome (FXS), however whether its loss has a direct role in neurotransmitter release remains a matter of debate. We demonstrate that neurons lacking FMRP display a specific defect in a mechanism that sustains neurotransmitter release during intense neuronal firing, called activity-dependent bulk endocytosis (ADBE). This discovery provides key insights into mechanisms of brain communication that occur because of loss of FMRP function. Importantly it also reveals ADBE as a potential therapeutic target to correct the circuit hyperexcitability observed in FXS.
Publisher: Springer Science and Business Media LLC
Date: 03-09-2013
DOI: 10.1038/NCOMMS3394
Publisher: Elsevier BV
Date: 2016
Publisher: Wiley
Date: 1999
DOI: 10.1046/J.1460-9568.1999.00412.X
Abstract: The role of protein kinase C (PKC) in the control of neurotransmitter release from cultured rat cerebellar granule cells was investigated. Release of preloaded [3H]-D-aspartate which is incorporated into synaptic vesicles in this preparation was evoked by electrical field stimulation or elevated KCl. PKC activation by phorbol esters resulted in a large facilitation of field-evoked Ca(2+)-dependent [3H]-D-aspartate release and a lesser enhancement of KCl-stimulated release. Inhibition of PKC by Ro 31-8220 or staurosporine virtually abolished field-evoked release but had no effect on KCl-evoked release. Field-evoked, but not KCl-evoked, synaptic vesicle exocytosis monitored by the fluorescent vesicle probe FM2-10 was inhibited by staurosporine. PKC was not directly modulating neurite Ca2+ channels coupled to release, as Ro 31-8220 did not inhibit these channels. Activation or inhibition of PKC modulated field-evoked plasma membrane depolarization, but had no effect on KCl-evoked depolarization, consistent with a regulation of Na+ or K+ channels activated by field stimulation. No modulation of field-evoked neurite Na+ influx was seen using phorbol esters. Phorbol ester-induced facilitation of field-evoked [3H]-D-aspartate release and neurite Ca2+ entry was non-additive with that produced by the specific K+ channel antagonist dendrotoxin-1, suggesting that PKC modulates transmitter release from field-stimulated cerebellar granule cells by inhibiting a dendrotoxin-1-sensitive K+ channel.
Publisher: Society for Neuroscience
Date: 16-10-2019
DOI: 10.1523/JNEUROSCI.1158-19.2019
Abstract: Neurotransmission is sustained by endocytosis and refilling of synaptic vesicles (SVs) locally within the presynapse. Until recently, a consensus formed that after exocytosis, SVs are recovered by either fusion pore closure (kiss-and-run) or clathrin-mediated endocytosis directly from the plasma membrane. However, recent data have revealed that SV formation is more complex than previously envisaged. For ex le, two additional recycling pathways have been discovered, ultrafast endocytosis and activity-dependent bulk endocytosis, in which SVs are regenerated from the internalized membrane and synaptic endosomes. Furthermore, these erse modes of endocytosis appear to influence both the molecular composition and subsequent physiological role of in idual SVs. In addition, previously unknown complexity in SV refilling and reclustering has been revealed. This review presents a modern view of the SV life cycle and discusses how neuronal subtype, physiological temperature, and in idual activity patterns can recruit different endocytic modes to generate new SVs and sculpt subsequent presynaptic performance.
Publisher: Proceedings of the National Academy of Sciences
Date: 16-02-2010
Abstract: Clathrin-mediated synaptic vesicle (SV) recycling involves the spatiotemporally controlled assembly of clathrin coat components at phosphatidylinositiol (4, 5)-bisphosphate [PI(4,5)P 2 ]-enriched membrane sites within the periactive zone. Such spatiotemporal control is needed to coordinate SV cargo sorting with clathrin/AP2 recruitment and to restrain membrane fission and synaptojanin-mediated uncoating until membrane deformation and clathrin coat assembly are completed. The molecular events underlying these control mechanisms are unknown. Here we show that the endocytic SH3 domain-containing accessory protein intersectin 1 scaffolds the endocytic process by directly associating with the clathrin adaptor AP2. Acute perturbation of the intersectin 1-AP2 interaction in l rey synapses in situ inhibits the onset of SV recycling. Structurally, complex formation can be attributed to the direct association of hydrophobic peptides within the intersectin 1 SH3A-B linker region with the “side sites” of the AP2 α- and β-appendage domains. AP2 appendage association of the SH3A-B linker region inhibits binding of the inositol phosphatase synaptojanin 1 to intersectin 1. These data identify the intersectin-AP2 complex as an important regulator of clathrin-mediated SV recycling in synapses.
Publisher: Society for Neuroscience
Date: 21-08-2013
DOI: 10.1523/JNEUROSCI.0636-13.2013
Abstract: Synaptophysin is an integral synaptic vesicle (SV) protein that accounts for ∼10% of total SV protein cargo. Deletion of synaptophysin results in the defective retrieval of synaptobrevin II (sybII) from the plasma membrane during endocytosis, coupled with a slowing in the speed of endocytosis. Synaptophysin has been implicated in X-linked intellectual disability, with a recent study identifying four separate synaptophysin gene mutations in families affected by the disorder. To determine how these mutations may affect synaptophysin function, we expressed them in cultured neurons derived from synaptophysin knock-out mice. Two distinct truncating mutants were mislocalized throughout the axon and phenocopied the arrest of sybII retrieval in synaptophysin knock-out cultures. The remaining two mutants displayed a nerve terminal localization but did not support efficient sybII retrieval. Interestingly, one mutant fully rescued SV endocytosis kinetics, suggesting that sybII retrieval and endocytosis speed are independent from each other. These studies suggest that the efficient retrieval of sybII by synaptophysin may be key to maintaining synaptic health and perturbation of this event may contribute to the pathogenesis underlying neurodevelopmental disorders such as X-linked intellectual disability.
Publisher: Oxford University Press (OUP)
Date: 13-08-2018
DOI: 10.1093/BRAIN/AWY209
Abstract: Baker, Gordon et al. present the first international case series describing the neurodevelopmental disorder associated with Synaptotagmin 1 (SYT1) de novo missense mutations. Key features include movement abnormalities, severe intellectual disability, and hallmark EEG alterations. Expression of patients’ SYT1 mutations in mouse neurons disturbs presynaptic vesicle dynamics in a mutation-specific manner.
Publisher: Elsevier BV
Date: 11-2001
DOI: 10.1016/S0166-2236(00)01930-5
Abstract: When nerve terminals in the brain are stimulated, a group of phosphoproteins called the dephosphins are coordinately dephosphorylated by calcineurin, the Ca(2+)-dependent protein phosphatase. Amazingly, the seven presently known dephosphins are not structurally related, yet each has been independently shown to be essential for synaptic vesicle endocytosis (SVE). Nowhere else in biology is there a similar ex le of the coordinated dephosphorylation of such a large group of proteins each sharing roles in the same biological response. This suggests that dephosphorylation and phosphorylation of the dephosphins is essential for SVE. Recent studies in synaptosomes have confirmed this view, with calcineurin-mediated dephosphorylation of the dephosphins essential for triggering SVE. The phosphorylation cycle of the dephosphins might regulate SVE by targeting the proteins to sites of action and by stimulating the assembly of several large essential endocytic protein complexes.
Publisher: Wiley
Date: 19-11-2007
DOI: 10.1111/J.1742-4658.2007.06145.X
Abstract: Chloride intracellular channels (CLICs) are soluble, signal peptide-less proteins that are distantly related to Omega-type glutathione-S-transferases. Although some CLICs bypass the classical secretory pathway and autoinsert into cell membranes to form ion channels, their cellular roles remain unclear. Many CLICs are strongly associated with cytoskeletal proteins, but the role of these associations is not known. In this study, we incorporated purified, recombinant mammalian CLIC1, CLIC4 and (for the first time) CLIC5 into planar lipid bilayers, and tested the hypothesis that the channels are regulated by actin. CLIC5 formed multiconductance channels that were almost equally permeable to Na(+), K(+) and Cl(-), suggesting that the 'CLIC' nomenclature may need to be revised. CLIC1 and CLIC5, but not CLIC4, were strongly and reversibly inhibited (or inactivated) by 'cytosolic' F-actin in the absence of any other protein. This inhibition effect on channels could be reversed by using cytochalasin to disrupt the F-actin. We suggest that actin-regulated membrane CLICs could modify solute transport at key stages during cellular events such as apoptosis, cell and organelle ision and fusion, cell-volume regulation, and cell movement.
Publisher: Elsevier BV
Date: 08-2007
Publisher: Elsevier BV
Date: 11-1993
DOI: 10.1016/0028-3908(93)90012-R
Abstract: The increase in cytosolic calcium, [Ca2+]c, evoked with 50 mM KCl in cerebellar granule cells consists of four components (1) a rapidly inactivating transient or spike (2) a nifedipine-sensitive non-inactivating plateau (3) an Aga-GI (spider toxin) sensitive non-inactivating plateau (4) a residual non-inactivating plateau insensitive to nifedipine and Aga-GI. None of these components is blocked by synthetic arginine polyamine toxin, spermine, (+)-MK-801 hydrogen maleate, D(-)-2-amino-5-phosphonopentanoic acid or omega-conotoxin-GVIA. The proposed P-type channel antagonist, omega-agatoxin-IVA, has a limited but non-significant effect on the elevated plateau [CA2+]c.L-type Ca2+ channels are located primarily on the soma whereas the component of the plateau which is blocked specifically by Aga-GI is localized primarily on the cell neurites. The latter component is coupled to the exocytosis of endogenous glutamate evoked with 50 mM KCl.
Publisher: Society for Neuroscience
Date: 27-04-2020
DOI: 10.1523/JNEUROSCI.0210-20.2020
Abstract: The epilepsy-linked gene SV2A , has a number of potential roles in the synaptic vesicle (SV) life cycle. However, how loss of SV2A function translates into presynaptic dysfunction and ultimately seizure activity is still undetermined. In this study, we examined whether the first SV2A mutation identified in human disease (R383Q) could provide information regarding which SV2A-dependent events are critical in the translation to epilepsy. We utilized a molecular replacement strategy in which exogenous SV2A was expressed in mouse neuronal cultures of either sex, which had been depleted of endogenous SV2A to mimic the homozygous human condition. We found that the R383Q mutation resulted in a mislocalization of SV2A from SVs to the plasma membrane, but had no effect on its activity-dependent trafficking. This SV2A mutant displayed reduced mobility when stranded on the plasma membrane and reduced binding to its interaction partner synaptotagmin-1 (Syt1). Furthermore, the R383Q mutant failed to rescue reduced expression and dysfunctional activity-dependent trafficking of Syt1 in the absence of endogenous SV2A. This suggests that the inability to control Syt1 expression and trafficking at the presynapse may be key in the transition from loss of SV2A function to seizure activity. SIGNIFICANCE STATEMENT SV2A is a synaptic vesicle (SV) protein, the absence or dysfunction of which is linked to epilepsy. However, the series of molecular events that result in this neurological disorder is still undetermined. We demonstrate here that the first human mutation in SV2A identified in an in idual with epilepsy displays reduced binding to synaptotagmin-1 (Syt1), an SV protein essential for synchronous neurotransmitter release. Furthermore, this mutant cannot correct alterations in both Syt1 expression and trafficking when expressed in the absence of endogenous SV2A (to mimic the homozygous human condition). This suggests that the inability to control Syt1 expression and trafficking may be key in the transition from loss of SV2A function to seizure activity.
Publisher: Wiley
Date: 14-12-2007
Publisher: Springer New York
Date: 2018
DOI: 10.1007/978-1-4939-8719-1_18
Abstract: This protocol utilizes lipophilic FM dyes to monitor membrane recycling in real time. FM dyes are virtually nonfluorescent in solution but when membrane bound are intensely fluorescent, combined with the flexibility of different emission wavelengths make these dyes an excellent choice for investigating clathrin-mediated endocytosis, among other membrane trafficking and recycling pathways.
Publisher: Portland Press Ltd.
Date: 08-1996
DOI: 10.1042/BST024430S
Publisher: Wiley
Date: 09-09-2021
DOI: 10.1111/JNC.15499
Abstract: Synaptobrevin‐2 (Syb2) is a soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) that is essential for neurotransmitter release. It is the most numerous protein on a synaptic vesicle (SV) and drives SV fusion via interactions with its cognate SNARE partners on the presynaptic plasma membrane. Synaptophysin (Syp) is the second most abundant protein on SVs however, in contrast to Syb2, it has no obligatory role in neurotransmission. Syp interacts with Syb2 on SVs, and the molecular nature of its interaction with Syb2 and its physiological role has been debated for decades. However, recent studies have revealed that the sole physiological role of Syp at the presynapse is to ensure the efficient retrieval of Syb2 during SV endocytosis. In this review, current theories surrounding the role of Syp in Syb2 trafficking will be discussed, in addition to the debate regarding the molecular nature of their interaction. A unifying model is presented that describes how Syp controls Syb2 function as part of an integrated mechanism involving key molecular players such as intersectin‐1 and AP180/CALM. Finally, key future questions surrounding the role of Syp‐dependent Syb2 trafficking will be posed, with respect to brain function in health and disease. image
Publisher: Elsevier BV
Date: 08-2001
Publisher: Society for Neuroscience
Date: 25-06-2008
Publisher: Hindawi Limited
Date: 2011
DOI: 10.4061/2011/263673
Abstract: The past ten years of research have identified a number of key roles for glycogen synthase kinase 3 (GSK3) at the synapse. In terms of presynaptic physiology, critical roles for GSK3 have been revealed in the growth and maturation of the nerve terminal and more recently a key role in the control of activity-dependent bulk endocytosis of synaptic vesicles. This paper will summarise the major roles assigned to GSK3 in both immature and mature nerve terminals, the substrates GSK3 phosphorylates to exert its action, and how GSK3 activity is regulated by different presynaptic signalling cascades. The number of essential roles for GSK3, coupled with the numerous signalling cascades all converging to regulate its activity, suggests that GSK3 is a key integrator of multiple inputs to modulate the strength of neurotransmission. Modulation of these pathways may point to potential mechanisms to overcome synaptic failure in neurodegenerative disorders such as Alzheimer's disease.
Publisher: Public Library of Science (PLoS)
Date: 25-01-2016
Publisher: EMBO
Date: 25-05-2023
Abstract: The unique nerve terminal targeting of botulinum neurotoxin type A (BoNT/A) is due to its capacity to bind two receptors on the neuronal plasma membrane: polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Whether and how PSGs and SV2 may coordinate other proteins for BoNT/A recruitment and internalization remains unknown. Here, we demonstrate that the targeted endocytosis of BoNT/A into synaptic vesicles (SVs) requires a tripartite surface nanocluster. Live‐cell super‐resolution imaging and electron microscopy of catalytically inactivated BoNT/A wildtype and receptor‐binding‐deficient mutants in cultured hippoc al neurons demonstrated that BoNT/A must bind coincidentally to a PSG and SV2 to target synaptic vesicles. We reveal that BoNT/A simultaneously interacts with a preassembled PSG‐synaptotagmin‐1 (Syt1) complex and SV2 on the neuronal plasma membrane, facilitating Syt1‐SV2 nanoclustering that controls endocytic sorting of the toxin into synaptic vesicles. Syt1 CRISPRi knockdown suppressed BoNT/A‐ and BoNT/E‐induced neurointoxication as quantified by SNAP‐25 cleavage, suggesting that this tripartite nanocluster may be a unifying entry point for selected botulinum neurotoxins that hijack this for synaptic vesicle targeting.
Publisher: The Company of Biologists
Date: 2014
DOI: 10.1242/DMM.015222
Abstract: Mutations in RAB18 have been shown to cause the heterogeneous autosomal recessive disorder Warburg Micro syndrome (WARBM). Patients with WARBM present with a range of clinical symptoms including ocular and neurological abnormalities. However, the underlying cellular and molecular pathogenesis of the disorder remains unclear, largely due to the lack of any robust animal models phenocopying both ocular and neurological features of the disease. We report here the generation and characterisation of a novel Rab18 mutant mouse model of WARBM. Rab18 mutant mice are viable and fertile. They present with congenital nuclear cataracts and atonic pupils, recapitulating characteristic ocular features associated with WARBM. In addition, Rab18 mutant cells have an increase in lipid droplet size following treatment with oleic acid. Lipid droplet abnormalities are a characteristic feature of WARBM patient cells, as well as cells from patients with other neurodegenerative conditions. Neurological dysfunction is also apparent in Rab18 mutant mice, including progressive weakness of the hind limbs. We show that the neurological defects are most likely not due to gross perturbations in synaptic vesicle recycling in the central or peripheral nervous system. Rather, loss of Rab18 is associated with widespread disruption of the neuronal cytoskeleton, including abnormal accumulations of neurofilament and microtubule proteins in synaptic terminals and gross disorganisation of the cytoskeleton in peripheral nerves. Global proteomic profiling of peripheral nerve in Rab18 mutant mice reveals significant alterations in several core molecular pathways regulating cytoskeletal dynamics in neurons. The clear similarities between WARBM and the phenotype we describe indicate that the Rab18 mutant mouse provides an important platform for investigating the disease pathogenesis and therapeutic interventions.
Publisher: Springer Science and Business Media LLC
Date: 30-04-2006
DOI: 10.1038/NN1695
Publisher: Springer Science and Business Media LLC
Date: 06-06-2010
DOI: 10.1038/NN.2571
Publisher: Springer Science and Business Media LLC
Date: 30-08-2023
DOI: 10.1038/S41467-023-41035-W
Abstract: Dynamin-1 is a large GTPase with an obligatory role in synaptic vesicle endocytosis at mammalian nerve terminals. Heterozygous missense mutations in the dynamin-1 gene ( DNM1 ) cause a novel form of epileptic encephalopathy, with pathogenic mutations clustering within regions required for its essential GTPase activity. We reveal the most prevalent pathogenic DNM1 mutation, R237W, disrupts dynamin-1 enzyme activity and endocytosis when overexpressed in central neurons. To determine how this mutation impacted cell, circuit and behavioural function, we generated a mouse carrying the R237W mutation. Neurons from heterozygous mice display dysfunctional endocytosis, in addition to altered excitatory neurotransmission and seizure-like phenotypes. Importantly, these phenotypes are corrected at the cell, circuit and in vivo level by the drug, BMS-204352, which accelerates endocytosis. Here, we demonstrate a credible link between dysfunctional endocytosis and epileptic encephalopathy, and importantly reveal that synaptic vesicle recycling may be a viable therapeutic target for monogenic intractable epilepsies.
Publisher: Wiley
Date: 16-10-2009
DOI: 10.1111/J.1471-4159.2009.06384.X
Abstract: Central nerve terminals release neurotransmitter in response to a wide variety of stimuli. Because maintenance of neurotransmitter release is dependent on the continual supply of synaptic vesicles (SVs), nerve terminals possess an array of endocytosis modes to retrieve and recycle SV membrane and proteins. During mild stimulation conditions, single SV retrieval modes such as clathrin‐mediated endocytosis predominate. However, during increased neuronal activity, additional SV retrieval capacity is required, which is provided by activity‐dependent bulk endocytosis (ADBE). ADBE is the dominant SV retrieval mechanism during elevated neuronal activity. It is a high capacity SV retrieval mode that is immediately triggered during such stimulation conditions. This review will summarize the current knowledge regarding the molecular mechanism of ADBE, including molecules required for its triggering and subsequent steps, including SV budding from bulk endosomes. The molecular relationship between ADBE and the SV reserve pool will also be discussed. It is becoming clear that an understanding of the molecular physiology of ADBE will be of critical importance in attempts to modulate both normal and abnormal synaptic function during intense neuronal activity.
Publisher: Wiley
Date: 16-12-2013
DOI: 10.1111/TRA.12140
Publisher: Elsevier BV
Date: 12-2009
Publisher: Elsevier BV
Date: 08-1995
DOI: 10.1016/0306-4522(95)00061-M
Abstract: When cerebellar granule cells in the presence of 1.3 mM calcium chloride (Ca2+) are depolarized by high potassium chloride (KCl), the release of endogenous glutamate is coupled to a high threshold Ca2+ channel blocked by the spider toxin omega Agatoxin-glutamate-release-inhibitor (Aga-GI) and insensitive to the L-type voltage-dependent Ca2+ channel-inhibitor nifedipine. A prolonged KCl depolarization in the absence of Ca2+ followed by addition of 5 mM Ca2+ results in an enhanced nifedipine-sensitive Ca2+ entry glutamate exocytosis retains sensitivity to tetanus toxin and bafilomycin A1, is now totally inhibited by nifedipine and shows greatly reduced sensitivity to AGA-GI. Single cell Ca2+ imaging indicates that the L-type channel modulating release is preferentially located at somatic regions rather than neurites. A different pattern of vesicle endocytosis monitored with the fluorescent indicator FM1-43 is seen in response to the two depolarization protocols. Furthermore, vesicles loaded during depolarization with high KCl in the presence of 5 mM Ca2+ extensively exocytose dye in a nifedipine-insensitive manner in response to a second similar stimulation but release little dye in response to stimulus with high KCl in the absence of Ca2+ followed by the addition of 5 mM Ca2+. In contrast, vesicles loaded by stimulating with KCl in the absence of Ca2+ followed by the addition of 5 mM Ca2+ can be released by a second similar stimulus and this release is sensitive to nifedipine. Nifedipine sensitivity is not induced in cerebellar synaptosomes subjected to stimulation with high KCl in the absence of Ca2+ followed by the re-addition of 5 mM Ca2+. The results indicate that different populations of channels and vesicles may be functional during two depolarization protocols.
Publisher: Elsevier BV
Date: 09-2022
Publisher: Elsevier BV
Date: 02-2013
Publisher: Springer Science and Business Media LLC
Date: 13-07-2003
DOI: 10.1038/NCB1020
Publisher: Frontiers Media SA
Date: 09-02-2016
Publisher: Springer Science and Business Media LLC
Date: 2000
Publisher: Wiley
Date: 02-06-2005
DOI: 10.1111/J.1471-4159.2005.03213.X
Abstract: Synaptic vesicle endocytosis is stimulated by calcium influx in mature central nerve terminals via activation of the calcium‐dependent protein phosphatase, calcineurin. However, in different neuronal preparations calcineurin activity is either inhibitory, stimulatory or irrelevant to the process. We addressed this inconsistency by investigating the requirement for calcineurin activity in synaptic vesicle endocytosis during development, using vesicle recycling assays in isolated nerve terminals. We show that endocytosis occurs independently of calcineurin activity in immature nerve terminals, and that a calcineurin requirement develops 2–4 weeks after birth. Calcineurin‐independent endocytosis is not due to the absence of calcineurin activity, since calcineurin is present in immature nerve terminals and its substrate, dynamin I, is dephosphorylated on depolarization. Calcineurin‐independent endocytosis is calcium‐dependent, since substitution of the alent cation, barium, inhibits the process. Finally, we demonstrated that in primary neuronal cultures derived from neonatal rats, endocytosis that was initially calcineurin‐independent developed a calcineurin requirement on maturation in culture. Our data account for the apparent inconsistencies regarding the role of calcineurin in synaptic vesicle endocytosis, and we propose that an unidentified calcium sensor exists to couple calcium influx to endocytosis in immature nerve terminals.
Publisher: Wiley
Date: 15-02-2017
DOI: 10.1113/JP273596
Publisher: Portland Press Ltd.
Date: 08-1995
DOI: 10.1042/BST0230648
Publisher: Wiley
Date: 11-12-2021
DOI: 10.1111/JNC.15551
Abstract: Mutations in the ESCRT‐III subunit CHMP2B cause frontotemporal dementia (FTD) and lead to impaired endolysosomal trafficking and lysosomal storage pathology in neurons. We investigated the effect of mutant CHMP2B on synaptic pathology, as ESCRT function was recently implicated in the degradation of synaptic vesicle (SV) proteins. We report here that expression of C‐terminally truncated mutant CHMP2B results in a novel synaptopathy. This unique synaptic pathology is characterised by selective retention of presynaptic SV trafficking proteins in aged mutant CHMP2B transgenic mice, despite significant loss of postsynaptic proteins. Furthermore, ultrastructural analysis of primary cortical cultures from transgenic CHMP2B mice revealed a significant increase in the number of presynaptic endosomes, while neurons expressing mutant CHMP2B display defective SV recycling and alterations to functional SV pools. Therefore, we reveal how mutations in CHMP2B affect specific presynaptic proteins and SV recycling, identifying CHMP2B FTD as a novel synaptopathy. This novel synaptopathic mechanism of impaired SV physiology may be a key early event in multiple forms of FTD, since proteins that mediate the most common genetic forms of FTD all localise at the presynapse. image
Publisher: Cold Spring Harbor Laboratory
Date: 18-01-2023
DOI: 10.1101/2023.01.15.524101
Abstract: The multidomain adaptor protein hiphysin-1 (Amph1) is an important coordinator of clathrin-mediated endocytosis in non-neuronal cells and synaptic vesicle (SV) endocytosis at central nerve terminals. Amph1 contains a lipid-binding N-BAR (Bin/Amphiphysin/Rvs) domain, central proline-rich (PRD) and clathrin/AP2 (CLAP) domains, and a C-terminal SH3 domain. All domains interact with either lipids or SV endocytosis proteins, with all of these interactions required for SV endocytosis, apart from the Amph1 PRD. In this study, we determined this role and confirmed requirements for established Amph1 interactions in SV endocytosis at typical small central synapses. Domain-specific interactions of Amph1 were validated using in vitro GST pull-down assays, with the role of these interactions in SV endocytosis determined in molecular replacement experiments in primary neuronal culture. Using this approach, we confirmed important roles for CLAP and SH3 domain interactions in the control of SV endocytosis. Furthermore, we identified an interaction site for the endocytosis protein endophilin A1 in the Amph1 PRD and revealed a key role for this interaction in SV endocytosis. Finally, we discovered that the phosphorylation status of Amph1-S293 within the PRD dictates the formation of the Amph1-endophilin A1 complex and is essential for efficient SV regeneration. This work therefore identifies an activity-dependent dephosphorylation-dependent interaction that is key for efficient SV endocytosis.
Publisher: Wiley
Date: 02-07-2020
DOI: 10.1111/JNC.15035
Abstract: The activity‐dependent fusion, retrieval and recycling of synaptic vesicles is essential for the maintenance of neurotransmission. Until relatively recently it was believed that most mutations in genes that were essential for this process would be incompatible with life, because of this fundamental role. However, an ever‐expanding number of mutations in this very cohort of genes are being identified in in iduals with neurodevelopmental disorders, including autism, intellectual disability and epilepsy. This article will summarize the current state of knowledge linking mutations in presynaptic genes to neurodevelopmental disorders by sequentially covering the various stages of the synaptic vesicle life cycle. It will also discuss how perturbations of specific stages within this recycling process could translate into human disease. Finally, it will also provide perspectives on the potential for future therapy that are targeted to presynaptic function. image
Publisher: Wiley
Date: 14-05-2015
DOI: 10.1111/JNC.13132
Publisher: Rockefeller University Press
Date: 19-11-2021
Abstract: The regulation of activity-dependent bulk endocytosis, the dominant mode of membrane retrieval in response to intense neuronal activity, is poorly understood. In this JCB issue, Peng et al. (2021. J. Cell. Biol.0.1083/jcb.202011028) propose a novel molecular mechanism for the coordination of activity-dependent bulk endocytosis that builds on Minibrain kinase and its presynaptic substrate synaptojanin-1.
Publisher: Wiley
Date: 17-10-2019
DOI: 10.1111/JNC.14862
Publisher: Wiley
Date: 04-2011
Publisher: Elsevier BV
Date: 06-2023
Publisher: Wiley
Date: 12-1995
DOI: 10.1111/J.1460-9568.1995.TB01035.X
Abstract: The free calcium concentration, [Ca2+]c, in fura-2-loaded rat cerebellar granule cells was investigated by digital imaging during trains of uniform field stimuli in order to compare the ability of calcium channels in somata and neurites to respond to brief, physiologically relevant depolarizations. Very few somata responded to 20 Hz trains of 1 ms pulses, while virtually all neurites showed an extensive increase which was rapidly reversed when stimulation was terminated. In contrast, both somata and neurites responded when cells were depolarized with 50 mM KCI. The field stimuli evoked a tetrodotoxin-sensitive increase in Na+ concentration in both somata and neurites. When 4-aminopyridine, which inhibits delayed K+ currents in these cells, was present during the field stimulus both somata and neurites increased their [Ca2+]c, suggesting that prolongation of the duration of depolarization is required for somatic Ca2+ channel activation. The neurite response did not depend on the orientation of the neurite relative to the applied field. The neurite response was insensitive to nifedipine (1 microM) and omega-agatoxin-IVA (30 nM) but was uniformly inhibited by omega-conotoxin-GVIA (30% inhibition at 1 microM) and omega-conotoxin-MVIIC (44% inhibition at 5 microM). The two inhibitors were not additive. The neurite [Ca2+]c response was insensitive to the combination of ionotropic glutamate receptor antagonists. Field stimulation caused the exocytosis of the fluorescent probe FM1-43 previously loaded during KCI depolarization, suggesting that presynaptic Ca2+ channels contribute to the field-evoked neurite response.
Publisher: Elsevier BV
Date: 08-1998
DOI: 10.1016/S0304-3940(98)00610-7
Abstract: To investigate whether any specific requirement for extracellular Ca2+ exists in the control synaptic vesicle retrieval, we examined the ability of the alent cation Ba2+ to substitute for Ca2+ in both vesicle exocytosis and endocytosis. Ba2+ stimulated glutamate release from rat cerebrocortical synaptosomes. Ba2+-evoked release was inhibited by bafilomycin A1, indicating release was via exocytosis of synaptic vesicles. However, Ba2+ did not stimulate vesicle retrieval, monitored by a FM2-10-based retrieval assay. Therefore synaptic vesicle retrieval in central nerve terminals has a specific requirement for extracellular Ca2+ and the Ca2+ receptor for retrieval has a different cation specificity to the Ca2+ receptor for exocytosis.
Publisher: Wiley
Date: 28-06-2004
Publisher: Wiley
Date: 17-03-2021
DOI: 10.1111/JNC.15319
Abstract: The synapse is formed between a presynapse (which releases neurotransmitter) and the postsynapse (which transduces this chemical signal). Over the past decade, presynaptic dysfunction has emerged as a key mediator of a series of neurodevelopmental and neurodegenerative disorders. This special issue will highlight some of the important presynaptic molecules and mechanisms that are disrupted in these conditions and reveal potential routes for therapy.
Publisher: Hindawi Limited
Date: 2011
DOI: 10.4061/2011/279234
Publisher: Society for Neuroscience
Date: 09-02-2023
DOI: 10.1523/JNEUROSCI.1537-22.2023
Abstract: Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a severe early-onset epileptic encephalopathy resulting mainly from de novo mutations in the X-linked CDKL5 gene. To determine whether loss of presynaptic CDKL5 function contributes to CDD, we examined synaptic vesicle (SV) recycling in primary hippoc al neurons generated from Cdkl5 knockout rat males. Using a genetically encoded reporter, we revealed that CDKL5 is selectively required for efficient SV endocytosis. We showed that CDKL5 kinase activity is both necessary and sufficient for optimal SV endocytosis, since kinase-inactive mutations failed to correct endocytosis in Cdkl5 knockout neurons, whereas the isolated CDKL5 kinase domain fully restored SV endocytosis kinetics. Finally, we demonstrated that CDKL5-mediated phosphorylation of hiphysin 1, a putative presynaptic target, is not required for CDKL5-dependent control of SV endocytosis. Overall, our findings reveal a key presynaptic role for CDKL5 kinase activity and enhance our insight into how its dysfunction may culminate in CDD. SIGNIFICANCE STATEMENT Loss of cyclin-dependent kinase like 5 (CDKL5) function is a leading cause of monogenic childhood epileptic encephalopathy. However, information regarding its biological role is scarce. In this study, we reveal a selective presynaptic role for CDKL5 in synaptic vesicle endocytosis and that its protein kinase activity is both necessary and sufficient for this role. The isolated protein kinase domain is sufficient to correct this loss of function, which may facilitate future gene therapy strategies if presynaptic dysfunction is proven to be central to the disorder. It also reveals that a CDKL5-specific substrate is located at the presynapse, the phosphorylation of which is required for optimal SV endocytosis.
Publisher: Elsevier BV
Date: 03-1993
DOI: 10.1016/0006-8993(93)90989-Z
Abstract: Flunarizine, an established Ca2+ channel antagonist, blocks both exocytotic glutamate release from mammalian cultured cerebellar granule cells and isolated presynaptic nerve endings (synaptosomes) prepared from two distinct areas of the mammalian brain. This blockade of release displays the same flunarizine concentration dependency in synaptosomes in the presence or absence of Ca2+, with total inhibition at a concentration of 10 microM. In cultured neurones, a selective effect on the L-channel-coupled component of the KCl-evoked rise in intracellular Ca2+, [Ca2+]c, can be demonstrated between flunarizine concentrations of 100 nM and 10 microM, while at concentrations above 10 microM, the remaining residual and transient components are affected. In synaptosomes, flunarizine blocks the KCl-evoked elevation in [Ca2+]c in a concentration-dependent manner. Additionally, 10 microM flunarizine directly antagonises ouabain-induced tetrodotoxin (TTX)-sensitive Na+ influx, glutamate, aspartate and GABA release from synaptosomes, whilst inhibiting veratridine-induced Ca(2+)-independent TTX-sensitive Na+ influx and glutamate release at 15 microM and 10 microM in cells and synaptosomes, respectively. In both cultured neurones and synaptosomes, the ability of flunarizine to block both neurotransmitter and cytoplasmic glutamate release is due to a direct antagonism of both voltage dependent Ca2+ channels and tetrodotoxin-sensitive Na+ channels.
Publisher: Portland Press Ltd.
Date: 12-2005
DOI: 10.1042/BST20051350
Publisher: Society for Neuroscience
Date: 02-2000
DOI: 10.1523/JNEUROSCI.20-03-00949.2000
Abstract: Ca 2+ entry into nerve terminals through clusters of voltage-dependent Ca 2+ channels (VDCCs) at active zones creates a microdomain of elevated intracellular free Ca 2+ concentration ([Ca 2+ ] i ) that stimulates exocytosis. We show that this VDCC-mediated [Ca 2+ ] i elevation has no specific role in stimulating endocytosis but can inhibit endocytosis evoked by three different methods in isolated mammalian nerve terminals. The inhibition can be relieved by using either VDCC antagonists or fast, but not slow, binding intracellular Ca 2+ chelators. The Ca 2+ -dependent inhibition of endocytosis is mimicked in vitro by a low-affinity inhibition of dynamin I vesiculation of phospholipids. Increased [Ca 2+ ] i also inhibits dynamin II GTPase activity and receptor-mediated endocytosis in non-neuronal cells. VDCC-meditated Ca 2+ entry inhibits dynamin-mediated endocytosis at the active zone and provides neurons with a mechanism to clear recycling vesicles to nonactive zone regions during periods of high activity.
Publisher: Elsevier BV
Date: 08-2001
Publisher: American Association for the Advancement of Science (AAAS)
Date: 30-04-2021
Abstract: VAMP4 is a molecular rheostat that adjusts neurotransmitter release to both the activity and degradative status of neurons.
Publisher: Society for Neuroscience
Date: 20-02-2013
DOI: 10.1523/JNEUROSCI.4697-12.2013
Abstract: Activity-dependent bulk endocytosis (ADBE) is the dominant mode of synaptic vesicle (SV) endocytosis during high-frequency stimulation in central nerve terminals. ADBE generates endosomes direct from the plasma membrane, meaning that high concentrations of calcium will be present in their interior due to fluid phase uptake from the extracellular space. Morphological and fluorescent assays were used to track the generation of SVs from bulk endosomes in primary neuronal culture. This process was functionally uncoupled from both SV exocytosis and plasma membrane retrieval events by intervening only after SV fusion and endocytosis were completed. Either intracellular (BAPTA-AM) or intra-endosomal (Rhod-dextran) calcium chelation inhibited SV generation from bulk endosomes, indicating that calcium efflux from this compartment is critical for this process. The V-type ATPase antagonist bafilomycin A1 also arrested SV generation from bulk endosomes, indicating endosomal acidification may be required for calcium efflux. Finally, pharmacological inhibition of the calcium-dependent protein phosphatase calcineurin blocked endosomal SV generation, identifying it as a key downstream effector in this process. These results reveal a novel and key role for the fluid phase uptake of extracellular calcium and its subsequent efflux in the SV lifecycle.
Publisher: Wiley
Date: 27-05-2023
DOI: 10.1111/JNC.15848
Abstract: The multidomain adaptor protein hiphysin‐1 (Amph1) is an important coordinator of clathrin‐mediated endocytosis in non‐neuronal cells and synaptic vesicle (SV) endocytosis at central nerve terminals. Amph1 contains a lipid‐binding N‐BAR (Bin/Amphiphysin/Rvs) domain, central proline‐rich (PRD) and clathrin/AP2 (CLAP) domains, and a C‐terminal SH3 domain. Amph1 interacts with both lipids and proteins, with all of these interactions required for SV endocytosis, with the exception of the Amph1 PRD. The Amph1 PRD associates with the endocytosis protein endophilin A1, however, the role of this interaction in SV endocytosis has not been investigated. In this study, we set out to determine whether the Amph1 PRD and its interaction with endophilin A1 was essential for efficient SV endocytosis at typical small central synapses. To achieve this, domain‐specific interactions of Amph1 were validated using in vitro GST pull‐down assays, with the role of these interactions in SV endocytosis determined in molecular replacement experiments in primary neuronal culture. Using this approach, we confirmed important roles for CLAP and SH3 domain interactions of Amph1 in the control of SV endocytosis. Importantly, we identified the interaction site for endophilin A1 within the Amph1 PRD and exploited specific binding mutants to reveal a key role for this interaction in SV endocytosis. Finally, we determined that the formation of the Amph1‐endophilin A1 complex is dependent on the phosphorylation status of Amph1‐S293 within the PRD and that the phosphorylation status of this residue is essential for efficient SV regeneration. This work, therefore, reveals a key role for the dephosphorylation‐dependent Amph1‐endophilin A1 interaction in efficient SV endocytosis. image
Publisher: Society for Neuroscience
Date: 16-06-2010
Publisher: Wiley
Date: 2001
DOI: 10.1046/J.1471-4159.2001.00049.X
Abstract: Dynamin I and at least five other nerve terminal proteins, hiphysins I and II, synaptojanin, epsin and eps15 (collectively called dephosphins), are coordinately dephosphorylated by calcineurin during endocytosis of synaptic vesicles. Here we have identified a new dephosphin, the essential endocytic protein AP180. Blocking dephosphorylation of the dephosphins is known to inhibit endocytosis, but the role of phosphorylation has not been determined. We show that the protein kinase C (PKC) antagonists Ro 31-8220 and Go 7874 block the rephosphorylation of dynamin I and synaptojanin that occurs during recovery from an initial depolarizing stimulus (S1). The rephosphorylation of AP180 and hiphysins 1 and 2, however, were unaffected by Ro 31-8220. Although these dephosphins share a single phosphatase, different protein kinases phosphorylated them after nerve terminal stimulation. The inhibitors were used to selectively examine the role of dynamin I and/or synaptojanin phosphorylation in endocytosis. Ro 31-8220 and Go 7874 did not block the initial S1 cycle of endocytosis, but strongly inhibited endocytosis following a second stimulus (S2). Therefore, phosphorylation of a subset of dephosphins, which includes dynamin I and synaptojanin, is required for the next round of stimulated synaptic vesicle retrieval.
Publisher: Wiley
Date: 10-2000
DOI: 10.1046/J.1471-4159.2000.0751645.X
Abstract: KCl and 4-aminopyridine (4-AP) evoke glutamate release from rat brain cortical nerve terminals by voltage cl ing or by Na(+) channel-generated repetitive action potentials, respectively. Stimulation by 4-AP but not KCl is largely mediated by protein kinase C (PKC). To determine whether KCl and 4-AP utilise the same mechanism to release glutamate, we correlated glutamate release with release of the hydrophobic synaptic vesicle (SV) marker FM2-10. A strong correlation was observed for increasing concentrations of KCl and after application of phorbol 12-myristate 13-acetate (PMA) or staurosporine. The parallel increase in exocytosis measured by two approaches suggested it occurred by a PKC-independent mechanism involving complete fusion of SVs with the plasma membrane. At low concentrations of 4-AP, alone or with staurosporine, glutamate and FM2-10 release also correlated. However, higher concentrations of 4-AP or of 4-AP plus PMA greatly increased glutamate release but did not further increase FM2-10 release. This ergence suggests that 4-AP recruits an additional mechanism of release during strong stimulation that is PKC dependent and is superimposed upon the first mechanism. This second mechanism is characteristic of kiss-and-run, which is not detectable by styryl dyes. Our data suggest that glutamate release in nerve terminals occurs via two mechanisms: (1) complete SV fusion, which is PKC independent and (2) a kiss-and-run-like mechanism, which is PKC dependent. Recruitment of a second release mechanism may be a widespread means to facilitate neurotransmitter release in central neurons.
Publisher: Society for Neuroscience
Date: 11-02-2015
DOI: 10.1523/JNEUROSCI.4248-14.2015
Abstract: Synaptic vesicle protein 2A (SV2A) is a ubiquitous component of synaptic vesicles (SVs). It has roles in both SV trafficking and neurotransmitter release. We demonstrate that Casein kinase 1 family members, including isoforms of Tau–tubulin protein kinases (TTBK1 and TTBK2), phosphorylate human SV2A at two constellations of residues, namely Cluster-1 (Ser42, Ser45, and Ser47) and Cluster-2 (Ser80, Ser81, and Thr84). These residues are also phosphorylated in vivo , and the phosphorylation of Thr84 within Cluster-2 is essential for triggering binding to the C2B domain of human synaptotagmin-1. We show by crystallographic and other analyses that the phosphorylated Thr84 residue binds to a pocket formed by three conserved Lys residues (Lys314, Lys326, and Lys328) on the surface of the synaptotagmin-1 C2B domain. Finally, we observed dysfunctional synaptotagmin-1 retrieval during SV endocytosis by ablating its phospho-dependent interaction with SV2A, knockdown of SV2A, or rescue with a phosphorylation-null Thr84 SV2A mutant in primary cultures of mouse neurons. This study reveals fundamental details of how phosphorylation of Thr84 on SV2A controls its interaction with synaptotagmin-1 and implicates SV2A as a phospho-dependent chaperone required for the specific retrieval of synaptotagmin-1 during SV endocytosis.
Publisher: Proceedings of the National Academy of Sciences
Date: 21-02-2012
Abstract: Syndapin I (PACSIN 1) is a synaptically enriched membrane tubulating protein that plays important roles in activity-dependent bulk endocytosis and neuronal morphogenesis. While syndapin I is an in vitro phosphoprotein, it is not known to be phosphorylated in neurons. Here, we report the identification of two phosphorylation sites, S76 and T181, of syndapin I from nerve terminals. Both residues are located at the N-terminal helix-capping motifs (N-Cap) of different α-helices in the F-BAR domain, important for F-BAR homodimer curvature and dimer-dimer filament assembly, respectively. Phospho-mimetic mutations of these residues regulate lipid-binding and tubulation both in vitro and in cells. Neither phosphosite regulated syndapin I function in activity-dependent bulk endocytosis. Rather, T181 phosphorylation was developmentally regulated and inhibited syndapin I function in neuronal morphogenesis. This suggests a novel mechanism for phosphorylation control of an F-BAR function through the regulation of α-helix interactions and stability within the folded F-BAR domain.
Publisher: Elsevier BV
Date: 06-2012
DOI: 10.1016/J.NEURON.2012.05.013
Abstract: In this issue of Neuron reveal that weak base antipsychotic drugs inhibit presynaptic function in an activity-dependent manner via their release from synaptic vesicles.
Publisher: Elsevier BV
Date: 12-2015
Publisher: MyJove Corporation
Date: 11-11-2011
DOI: 10.3791/3143
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 16-08-2007
Abstract: Dynamin is a GTPase enzyme involved in membrane constriction and fission during endocytosis. Phospholipid binding via its pleckstrin homology domain maximally stimulates dynamin activity. We developed a series of surface-active small-molecule inhibitors, such as myristyl trimethyl ammonium bromide (MiTMAB) and octadecyltrimethyl ammonium bromide (OcTMAB), and we now show MiTMAB targets the dynamin-phospholipid interaction. MiTMAB inhibited dynamin GTPase activity, with a Ki of 940 +/- 25 nM. It potently inhibited receptor-mediated endocytosis (RME) of transferrin or epidermal growth factor (EGF) in a range of cells without blocking EGF binding, receptor number, or autophosphorylation. RME inhibition was rapidly reversed after washout. The rank order of potency for a variety of MiTMAB analogs on RME matched the rank order for dynamin inhibition, suggesting dynamin recruitment to the membrane is a primary cellular target. MiTMAB also inhibited synaptic vesicle endocytosis in rat brain nerve terminals (synaptosomes) without inducing depolarization or morphological defects. Therefore, the drug rapidly and reversibly blocks multiple forms of endocytosis with no acute cellular damage. The unique mechanism of action of MiTMAB provides an important tool to better understand dynamin-mediated membrane trafficking events in a variety of cells.
Publisher: Society for Neuroscience
Date: 25-04-2012
DOI: 10.1523/JNEUROSCI.6305-11.2012
Abstract: Activity-dependent bulk endocytosis is the dominant synaptic vesicle retrieval mode during high intensity stimulation in central nerve terminals. A key event in this endocytosis mode is the generation of new vesicles from bulk endosomes, which replenish the reserve vesicle pool. We have identified an essential requirement for both adaptor protein complexes 1 and 3 in this process by employing morphological and optical tracking of bulk endosome-derived synaptic vesicles in rat primary neuronal cultures. We show that brefeldin A inhibits synaptic vesicle generation from bulk endosomes and that both brefeldin A knockdown and shRNA knockdown of either adaptor protein 1 or 3 subunits inhibit reserve pool replenishment from bulk endosomes. Conversely, no plasma membrane function was found for adaptor protein 1 or 3 in either bulk endosome formation or clathrin-mediated endocytosis. Simultaneous knockdown of both adaptor proteins 1 and 3 indicated that they generated the same population of synaptic vesicles. Thus, adaptor protein complexes 1 and 3 play an essential dual role in generation of synaptic vesicles during activity-dependent bulk endocytosis.
Publisher: American Chemical Society (ACS)
Date: 30-05-2013
DOI: 10.1021/CB400137P
Publisher: Society for Neuroscience
Date: 28-09-2011
DOI: 10.1523/JNEUROSCI.3162-11.2011
Abstract: The integral synaptic vesicle (SV) protein synaptophysin forms ∼10% of total SV protein content, but has no known function in SV physiology. Synaptobrevin (sybII) is another abundant integral SV protein with an essential role in SV exocytosis. Synaptophysin and sybII form a complex in nerve terminals, suggesting this interaction may have a key role in presynaptic function. To determine how synaptophysin controls sybII traffic in nerve terminals, we used a combination of optical imaging techniques in cultures derived from synaptophysin knock-out mice. We show that synaptophysin is specifically required for the retrieval of the pH-sensitive fluorescent reporter sybII-pHluorin from the plasma membrane during endocytosis. The retrieval of other SV protein cargo reporters still occurred however, their recapture proceeded with slower kinetics. This slowing of SV retrieval kinetics in the absence of synaptophysin did not impact on global SV turnover. These results identify a specific and selective requirement for synaptophysin in the retrieval of sybII during SV endocytosis and suggest that their interaction may act as an adjustable regulator of SV retrieval efficiency.
Publisher: Wiley
Date: 27-11-2014
DOI: 10.1111/TRA.12235
Abstract: Central nerve terminals contain a small number of synaptic vesicles (SVs) that must sustain the fidelity of neurotransmission across a wide range of stimulation intensities. For this to be achieved, nerve terminals integrate a number of complementary endocytosis modes whose activation spans the breadth of these neuronal stimulation patterns. Two such modes are ultrafast endocytosis and activity-dependent bulk endocytosis, which are triggered by stimuli at either end of the physiological range. Both endocytosis modes generate endosomes directly from the nerve terminal plasma membrane, before the subsequent production of SVs from these structures. This review will discuss the current knowledge relating to the molecular mechanisms involved in the generation of SVs from nerve terminal endosomes, how this relates to other mechanisms of SV production and the functional role of such SVs.
Publisher: Society for Neuroscience
Date: 10-01-2007
DOI: 10.1523/JNEUROSCI.3809-06.2007
Abstract: The stimulated dephosphorylation of the dephosphin group of endocytic proteins by calcineurin and their subsequent rephosphorylation by cyclin-dependent kinase 5 (cdk5) is required for synaptic vesicle (SV) retrieval in central nerve terminals. However, the specific endocytic pathway(s) controlled by these enzymes is unknown. To address this issue, we combined functional and morphological assays of endocytosis in primary neuronal cultures with pharmacological and molecular ablation of calcineurin and cdk5 activity. During strong stimulation, inhibition of calcineurin or cdk5 blocked uptake of the activity-dependent membrane marker FM1–43, but not the more hydrophilic FM2–10. However, FM2–10 uptake-measured poststimulation was sensitive to cdk5 and calcineurin inhibition, indicating that a slow form of endocytosis persists after termination of stimulation. In parallel EM studies, inhibition of cdk5 during strong stimulation greatly reduced horseradish peroxidase labeling of plasma membrane-derived nerve terminal endosomes, but not SVs. Furthermore, during mild stimulation, FM1–43 uptake was unaffected by cdk5 inhibition and the SV membrane was exclusively retrieved via a single SV route, suggesting that recruitment of the endosomal route of membrane retrieval is activity dependent. Thus, we propose that the calcineurin/cdk5-dependent phosphorylation cycle of the dephosphins specifically controls a slow endocytic pathway that proceeds via endosomal intermediates and is activated by strong physiological stimulation in central nerve terminals.
Publisher: SAGE Publications
Date: 15-07-2014
Abstract: Synaptic vesicle (SV) retrieval from the presynaptic plasma membrane occurs via a variety of different and complementary modes. The dominant retrieval mode during high-intensity stimulation is activity-dependent bulk endocytosis (ADBE). ADBE involves the generation of endosomes direct from the plasma membrane which then donate membrane and cargo to form SVs that replenish the reserve SV pool. Recent evidence has suggested that ADBE may involve an additional endosomal processing step to produce a mature, functional SV. This suggests that ADBE may utilize key molecules or indeed whole pathways from classical endocytic recycling routes that are ubiquitous across all cell types. This review will assess the current evidence for a contribution of endocytic recycling to the SV life cycle, with a particular focus on ADBE. In doing so it highlights points where both routes may either converge or exploit existing mechanisms to ensure efficient generation of SVs during high-intensity stimulation.
Publisher: Society for Neuroscience
Date: 17-06-2009
Publisher: Public Library of Science (PLoS)
Date: 19-05-2016
Publisher: Proceedings of the National Academy of Sciences
Date: 09-10-2018
Abstract: The maintenance of neurotransmission by synaptic vesicle (SV) recycling is critical to brain function. The dominant SV recycling mode during intense activity is activity-dependent bulk endocytosis (ADBE), suggesting it will perform a pivotal role in neurotransmission. However, the role of ADBE is still undetermined, due to the absence of identified molecules specific for this process. The determination of the bulk endosome proteome (a key ADBE organelle) revealed that it has a unique molecular signature and identified a role for Rab11 in presynaptic function. This work provides the molecular inventory of ADBE, a resource that will be of significant value to researchers wishing to modulate neurotransmission during intense neuronal activity in both health and disease.
Publisher: Wiley
Date: 04-2008
DOI: 10.1002/0471142301.NS0206S43
Abstract: The fluorescent dye FM1‐43 and its derivatives can be used to monitor the physiology of synaptic vesicle turnover in central nerve terminals. They do so by their ability to reversibly partition into membranes, a process that results in a huge increase in fluorescence in comparison to their quantum yield in solution. This unit provides protocols for quantifying total synaptic vesicle turnover, the kinetics and extent of synaptic vesicle exocytosis, and the kinetics and mode of synaptic vesicle endocytosis. Descriptions of other ways these protocols have been used to derive information about the life cycle of the synaptic vesicle are also provided. Curr. Protoc. Neurosci . 43:2.6.1‐2.6.12. © 2008 by John Wiley & Sons, Inc.
Publisher: Wiley
Date: 12-1999
DOI: 10.1046/J.1471-4159.1999.0732227.X
Abstract: The recycling of synaptic vesicles in nerve terminals involves multiple steps, underlies all aspects of synaptic transmission, and is a key to understanding the basis of synaptic plasticity. The development of styryl dyes as fluorescent molecules that label recycling synaptic vesicles has revolutionized the way in which synaptic vesicle recycling can be investigated, by allowing an examination of processes in neurons that have long been inaccessible. In this review, we evaluate the major aspects of synaptic vesicle recycling that have been revealed and advanced by studies with styryl dyes, focussing upon synaptic vesicle fusion, retrieval, and trafficking. The greatest impact of styryl dyes has been to allow the routine visualization of endocytosis in central nerve terminals for the first time. This has revealed the kinetics of endocytosis, its underlying sequential steps, and its regulation by Ca2+. In studies of exocytosis, styryl dyes have helped distinguish between different modes of vesicle fusion, provided direct support for the quantal nature of exocytosis and endocytosis, and revealed how the probability of exocytosis varies enormously from one nerve terminal to another. Synaptic vesicle labelling with styryl dyes has helped our understanding of vesicle trafficking by allowing better understanding of different synaptic vesicle pools within the nerve terminal, vesicle intermixing, and vesicle clustering at release sites. Finally, the dyes are now being used in innovative ways to reveal further insights into synaptic plasticity.
Publisher: Springer Science and Business Media LLC
Date: 22-07-2015
Publisher: Elsevier BV
Date: 03-2007
Publisher: Public Library of Science (PLoS)
Date: 04-06-2012
Publisher: Frontiers Media SA
Date: 03-08-2017
Publisher: Elsevier BV
Date: 09-2008
DOI: 10.1016/J.NEUINT.2008.06.002
Abstract: Bulk endocytosis is triggered in central nerve terminals during intense physiological stimulation. This endocytosis pathway can be labelled by the dye FM1-43 but not its more hydrophilic counterpart FM2-10. This selective labelling was proposed to be due to the retention of FM1-43, but not FM2-10, in slowly retrieving structures after washout of the dye. However, this explanation assumed that bulk endocytosis was a slow process that persisted after stimulation. We have recently shown that the great majority of bulk endocytosis occurs during stimulation, therefore another explanation for the specific labelling of this pathway by FM1-43 must be found. In this paper we show that the ability of FM dyes to label bulk endocytosis is dependent on the concentration of dye used and not their washout properties. When the loading concentration of FM1-43 was reduced 10-fold, its ability to label bulk endocytosis was lost. Conversely when the loading concentration of FM2-10 was increased 10-fold, it now labelled the pathway. This suggests that a difference in affinity of bulk endosome membranes for FM1-43 and FM2-10 underlies the disparity in labelling.
Publisher: Elsevier BV
Date: 2021
Publisher: Cold Spring Harbor Laboratory
Date: 10-09-2020
DOI: 10.1101/2020.09.10.291062
Abstract: Synaptic vesicle (SV) recycling defects are linked to neurodevelopmental disorders, including fragile X syndrome (FXS), which results from loss of fragile X mental retardation protein (FMRP) encoded by the FMR1 gene. Hyperexcitability of neuronal circuits is a key feature of FXS, therefore we investigated whether SV recycling was affected by the absence of FMRP during increased neuronal activity. We revealed that primary neuronal cultures from a Fmr1 knockout rat model display a specific defect in activity-dependent bulk endocytosis (ADBE). This defect resulted in an inability of Fmr1 knockout neurons to sustain SV recycling during high frequency stimulation. Using a molecular replacement strategy, we also revealed that a human FMRP mutant that cannot bind BK channels failed to correct ADBE dysfunction in knockout neurons, however this dysfunction was corrected by BK channel agonists. Therefore, FMRP performs a key role in sustaining neurotransmitter release via selective control of ADBE, suggesting intervention via this endocytosis mode may correct hyperexcitabiltiy observed in FXS. Fragile X syndrome (FXS) is caused by loss of fragile X mental retardation protein (FMRP). Bonnycastle et al show that FMRP is specifically required for activity-dependent bulk endocytosis (ADBE), revealing 1) FMRP sustains neurotransmitter release and 2) intervention via ADBE may correct circuit hyperexcitabilty in FXS.
Publisher: Elsevier BV
Date: 08-1997
DOI: 10.1016/S0306-4522(97)00047-X
Abstract: Regulated exocytosis from cultured rat cerebellar granule cells can be localized by the vesicle specific marker FM2-10 to specific sites, the highest density of which are at visible varicosities coinciding with neurite-neurite contacts. Exocytosis can be evoked by uniform electrical field pulses, which initiate tetrodotoxin-sensitive action potentials, or by elevated KCl. [3H]D-Aspartate is an authentic false transmitter in this preparation, judged by sensitivity of release to bafilomycin A1 and tetanus toxin. The coupling of presynaptic voltage-activated Ca2+ channels to [3H]D-aspartate exocytosis was determined during field stimulation. The peak cytoplasmic free Ca2+ concentration achieved in the varicosities was proportional to Ca2+ entry during a 10 strain of pulses. L-type Ca2+ channels did not contribute to either Ca2+ entry or [3H]D-aspartate exocytosis. The P-type Ca2+ channel antagonist omega-agatoxin-IVA (30 nM) only inhibited at 75% of the varicosities, although a mean 15% inhibition of Ca2+ entry caused a 39% inhibition of exocytosis. In contrast the N-type Ca2+ channel inhibitor omega-conotoxin-GVIA (1 microM), which inhibited at virtually all varicosities, caused mean inhibitions of Ca2+ entry and exocytosis of 26% and 24% respectively. The toxin omega-conotoxin-MVIIC (5 microM), which inhibits N-, P- and Q-type Ca2+ channels, was effective at all varicosities. The Q-type component of Ca2+ entry was calculated to be only 5-10% however, the additional inhibition of exocytosis was 30%. Thus P-type and particularly Q-type channels appear to be more closely coupled to exocytosis than N-type Ca2+ channels. The residual Ca2+ entry following 5 microM omega-conotoxin-MVIIC is scarcely coupled to release. The omega-agatoxin-IVA and omega-conotoxin-GVIA inhibitions of both Ca2+ entry and exocytosis were additive and varied stochastically between in idual varicosities. These results demonstrate that both Q- and P-type Ca2+ channels are highly efficient in their coupling to amino acid exocytosis, with N-type less efficient, and L-type channels not at all. The Ca2+ channel types coupled to exocytosis are also able to support exocytosis when evoked by either brief field-evoked action potentials or prolonged depolarization with KCl, indicating that these presynaptic channels, in contrast to those on the somata of the cells, can respond to widely different patterns of activation.
Publisher: Cold Spring Harbor Laboratory
Date: 14-04-2023
DOI: 10.1101/2023.04.14.536870
Abstract: Pathogenic heterozygous missense mutations in the DNM1 gene result in a novel form of epileptic encephalopathy. DNM1 encodes for the large GTPase dynamin-1, an enzyme with an obligatory role in the endocytosis of synaptic vesicles (SVs) at mammalian nerve terminals. Pathogenic DNM1 mutations cluster within regions required for its essential GTPase activity, implicating disruption of this enzyme activity as being central to epileptic encephalopathy. We reveal that the most prevalent pathogenic mutation of DNM1 , R237W, disrupts dynamin-1 enzyme activity and SV endocytosis when overexpressed in central neurons. To determine how this dominant-negative heterozygous mutant impacted cell, circuit and behaviour when expressed from its endogenous locus, we generated a mouse carrying the R237W mutation. Neurons isolated from heterozygous mice displayed dysfunctional SV endocytosis, which translated into altered excitatory neurotransmission and seizure-like phenotypes. Importantly, these phenotypes were corrected at the cell, circuit and in vivo level by the drug, BMS-204352, which accelerates SV endocytosis. This study therefore provides the first direct link between dysfunctional SV endocytosis and epilepsy, and importantly reveals that SV endocytosis is a viable therapeutic route for monogenic intractable epilepsies.
Publisher: Frontiers Media SA
Date: 21-07-2017
Publisher: Wiley
Date: 29-04-2012
Publisher: Springer Science and Business Media LLC
Date: 17-12-2014
DOI: 10.1038/NCOMMS6774
Abstract: Neuronal synapses are among the most scrutinized of cellular systems, serving as a model for all membrane trafficking studies. Despite this, synaptic biology has proven difficult to interrogate directly in situ due to the small size and dynamic nature of central synapses and the molecules within them. Here we determine the spatial and temporal interaction status of presynaptic proteins, imaging large cohorts of single molecules inside active synapses. Measuring rapid interaction dynamics during synaptic depolarization identified the small number of syntaxin1a and munc18-1 protein molecules required to support synaptic vesicle exocytosis. After vesicle fusion and subsequent SNARE complex disassembly, a prompt switch in syntaxin1a and munc18-1-binding mode, regulated by charge alteration on the syntaxin1a N-terminal, sequesters monomeric syntaxin1a from other disassembled fusion complex components, preventing ectopic SNARE complex formation, readying the synapse for subsequent rounds of neurotransmission.
Publisher: Elsevier BV
Date: 12-2017
Publisher: Humana Press
Date: 2008
DOI: 10.1007/978-1-59745-261-8_25
Abstract: Neurons transmit information by exocytosis of synaptic vesicles (SV), which contain neurotransmitter. Exocytosis is followed by efficient retrieval of the plasma membrane by endocytosis to generate a new SV. SV retrieval supports multiple cycles of synaptic transmission. Over the years, styryl dyes have been widely used to probe the mechanism of SV recycling in the processes of cultured neurons. The styryl dye method is complementary to electrophysiological measurements or genetic reporter approaches. Owing to their ease to culture, cerebellar granule neurons provide a robust neuronal model system for the assay. These cells are readily transfected with various DNA constructs to study the function of exocytic or endocytic proteins in SV recycling.
Publisher: Public Library of Science (PLoS)
Date: 12-02-2016
Publisher: Elsevier BV
Date: 06-2016
Publisher: Wiley
Date: 13-08-2021
DOI: 10.1111/JNC.15484
Abstract: Fragile X mental retardation protein (FMRP) is a neuronal protein mediating multiple functions, with its absence resulting in one of the most common monogenic causes of autism, Fragile X syndrome (FXS). Analyses of FXS pathophysiology have identified a range of aberrations in synaptic signaling pathways and plasticity associated with group I metabotropic glutamate (mGlu) receptors. These studies, however, have mostly focused on the post‐synaptic functions of FMRP and mGlu receptor activation, and relatively little is known about their presynaptic effects. Neurotransmitter release is mediated via multiple forms of synaptic vesicle (SV) fusion, each of which contributes to specific neuronal functions. The impacts of mGlu receptor activation and loss of FMRP on these SV fusion events remain unexplored. Here we combined electrophysiological and fluorescence imaging analyses on primary hippoc al cultures prepared from an Fmr1 knockout (KO) rat model. Compared to wild‐type (WT) hippoc al neurons, KO neurons displayed an increase in the frequency of spontaneous excitatory post‐synaptic currents (sEPSCs), as well as spontaneous SV fusion events. Pharmacological activation of mGlu receptors in WT neurons caused a similar increase in spontaneous SV fusion and sEPSC frequency. Notably, this increase in SV fusion was not observed when spontaneous activity was blocked using the sodium channel antagonist tetrodotoxin. Importantly, the effect of mGlu receptor activation on spontaneous SV fusion was occluded in Fmr1 KO neurons. Together, our results reveal that FMRP represses spontaneous presynaptic SV fusion, whereas mGlu receptor activation increases this event. This reciprocal control appears to be mediated via their regulation of intrinsic neuronal excitability. image
Publisher: American Society for Clinical Investigation
Date: 23-02-2015
DOI: 10.1172/JCI79765
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
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Funder: Wellcome Trust
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