Spontaneous and evoked synaptic vesicle release arises from a single releasable pool.

The quantal content of an evoked postsynaptic response is typically determined by dividing it by the average spontaneous miniature response. However, this approach is challenged by the notion that different synaptic vesicle pools might drive spontaneous and evoked release. Here, we "silence" synaptic vesicles through pharmacological alkalinization and subsequently rescue them by optogenetic acidification. We find that such silenced synaptic vesicles, retrieved during evoked or spontaneous activity, cross-deplete the complementary release mode in a fully reversible manner. A fluorescently tagged version of the endosomal SNARE protein Vti1a, which has been suggested to identify a separate pool of spontaneously recycling synaptic vesicles, is trafficked to synaptic vesicles significantly only upon overexpression but not when endogenously tagged by CRISPR-Cas9. Thus, both release modes draw synaptic vesicles from the same readily releasable pool.

Delayed recruitment of activity-dependent bulk endocytosis in Fmr1 knockout neurons.

The presynapse performs an essential role in brain communication via the activity-dependent release of neurotransmitters. However, the sequence of events through which a presynapse acquires functionality is relatively poorly understood, which is surprising, since mutations in genes essential for its operation are heavily implicated in neurodevelopmental disorders. We addressed this gap in knowledge by determining the developmental trajectory of synaptic vesicle (SV) recycling pathways in primary cultures of rat hippocampal neurons. Exploiting a series of optical and morphological assays, we revealed that the majority of nerve terminals displayed activity-dependent calcium influx from 3 days in vitro (DIV), immediately followed by functional evoked exocytosis and endocytosis, although the number of responsive nerve terminals continued to increase until the second week in vitro. However, the most intriguing discovery was that activity-dependent bulk endocytosis (ADBE) was only observed from DIV 14 onwards. Importantly, optimal ADBE recruitment was delayed until DIV 21 in Fmr1 knockout neurons, which model Fragile X Syndrome (FXS). This implicates the delayed recruitment of ADBE as a potential contributing factor in the development of circuit dysfunction in FXS, and potentially other neurodevelopmental disorders.

Synaptic Vesicle-Related Proteins and Ubiquilin 2 in Cortical Synaptosomes Mediate Cognitive Impairment in Vascular Dementia Rats.

Synaptic dysfunction is considered the best neuropathological correlate of cognitive decline in vascular dementia (VaD). However, the alterations of synaptic proteins at the synaptosomal level in VaD remain unclear. In this study, a VaD model was established in male rats using bilateral common carotid artery occlusion (2VO). We performed a novel object recognition task to evaluate cognitive impairment. Immunohistochemistry was used to assess the expression of neuron-specific nuclear binding protein (NeuN). Brain synaptosomes were isolated and subjected to label-free proteomic analysis to quantify and identify the synaptic features of differentially expressed proteins (DEPs). Synaptic and hub protein expression was detected in synaptosomes using western blotting. We found that male rats with VaD presented impaired memory and decreased NeuN protein expression in the cortex. Synaptosome proteomic analysis revealed 604 DEPs, with 493 and 111 markedly downregulated and upregulated proteins, respectively. KEGG analysis and SynGO annotation revealed that the synaptic vesicle (SV) cycle may be a key signaling pathway in VaD. Hub protein analysis of the main nodes in the protein network identified UBQLN2 and SV-related proteins, including CLTC, SNAP91, AP2S1, CLTA, VAMP2, EPN1, UBQLN2, AP2B1, AP2A2, and AP2M1. Western blotting showed that the levels of SV2A, CLTC, AP2S1, and VAMP2 decreased in the synaptosomes of 2VO rats, while UBQLN2 expression significantly increased. Our results suggest that the disruption in the presynaptic SV cycle is a key event in male rats with VaD, which could be characterized by the aberrant SV2A expression. SV-related proteins and UBQLN2 may be essential in synaptopathy. Thus, targeting the specific molecular markers in synaptosomes may be critical for the development of mechanism-directed therapies against VaD.

STED Imaging of Vesicular Endocytosis in the Synapse.

Endocytosis is a fundamental biological process that couples exocytosis to maintain the homeostasis of the plasma membrane and sustained neurotransmission. Super-resolution microscopy enables optical imaging of exocytosis and endocytosis in live cells and makes an essential contribution to understanding molecular mechanisms of endocytosis in neuronal somata and other types of cells. However, visualization of exo-endocytic events at the single vesicular level in a synapse with optical imaging remains a great challenge to reveal mechanisms governing the synaptic exo-endocytotic coupling. In this protocol, we describe the technical details of stimulated emission depletion (STED) imaging of synaptic endocytosis at the single-vesicle level, from sample preparation and microscopy calibration to data acquisition and analysis.

A Reduction in the Readily Releasable Vesicle Pool Impairs GABAergic Inhibition in the Hippocampus after Blood-Brain Barrier Dysfunction.

Major burdens for patients suffering from stroke are cognitive co-morbidities and epileptogenesis. Neural network disinhibition and deficient inhibitive pulses for fast network activities may result from impaired presynaptic release of the inhibitory neurotransmitter GABA. To test this hypothesis, a cortical photothrombotic stroke was induced in Sprague Dawley rats, and inhibitory currents were recorded seven days later in the peri-infarct blood-brain barrier disrupted (BBBd) hippocampus via patch-clamp electrophysiology in CA1 pyramidal cells (PC). Miniature inhibitory postsynaptic current (mIPSC) frequency was reduced to about half, and mIPSCs decayed faster in the BBBd hippocampus. Furthermore, the paired-pulse ratio of evoked GABA release was increased at 100 Hz, and train stimulations with 100 Hz revealed that the readily releasable pool (RRP), usually assumed to correspond to the number of tightly docked presynaptic vesicles, is reduced by about half in the BBBd hippocampus. These pathophysiologic changes are likely to contribute significantly to disturbed fast oscillatory activity, like cognition-associated gamma oscillations or sharp wave ripples and epileptogenesis in the BBBd hippocampus.

A lever hypothesis for Synaptotagmin-1 action in neurotransmitter release.

Neurotransmitter release is triggered in microseconds by Ca2+-binding to the Synaptotagmin-1 C2 domains and by SNARE complexes that form four-helix bundles between synaptic vesicles and plasma membranes, but the coupling mechanism between Ca2+-sensing and membrane fusion is unknown. Release requires extension of SNARE helices into juxtamembrane linkers that precede transmembrane regions (linker zippering) and binding of the Synaptotagmin-1 C2B domain to SNARE complexes through a 'primary interface' comprising two regions (I and II). The Synaptotagmin-1 Ca2+-binding loops were believed to accelerate membrane fusion by inducing membrane curvature, perturbing lipid bilayers or helping bridge the membranes, but SNARE complex binding orients the Ca2+-binding loops away from the fusion site, hindering these putative activities. Molecular dynamics simulations now suggest that Synaptotagmin-1 C2 domains near the site of fusion hinder SNARE action, providing an explanation for this paradox and arguing against previous models of Sytnaptotagmin-1 action. NMR experiments reveal that binding of C2B domain arginines to SNARE acidic residues at region II remains after disruption of region I. These results and fluorescence resonance energy transfer assays, together with previous data, suggest that Ca2+ causes reorientation of the C2B domain on the membrane and dissociation from the SNAREs at region I but not region II. Based on these results and molecular modeling, we propose that Synaptotagmin-1 acts as a lever that pulls the SNARE complex when Ca2+ causes reorientation of the C2B domain, facilitating linker zippering and fast membrane fusion. This hypothesis is supported by the electrophysiological data described in the accompanying paper.

Neurotransmitter release is triggered by a calcium-induced rearrangement in the Synaptotagmin-1/SNARE complex primary interface.

The Ca2+ sensor synaptotagmin-1 triggers neurotransmitter release together with the neuronal SNARE complex formed by syntaxin-1, SNAP25 and synaptobrevin. Moreover, synaptotagmin-1 increases synaptic vesicle priming and impairs spontaneous vesicle release. The synaptotagmin-1 C2B domain binds to the SNARE complex through a primary interface via two regions (I and II), but how exactly this interface mediates distinct functions of synaptotagmin-1, and the mechanism underlying Ca2+-triggering of release is unknown. Using mutagenesis and electrophysiological experiments, we show that region II is functionally and spatially subdivided: binding of C2B domain arginines to SNAP-25 acidic residues at one face of region II is crucial for Ca2+-evoked release but not for vesicle priming or clamping of spontaneous release, whereas other SNAP-25 and syntaxin-1 acidic residues at the other face mediate priming and clamping of spontaneous release but not evoked release. Mutations that disrupt region I impair the priming and clamping functions of synaptotagmin-1 while, strikingly, mutations that enhance binding through this region increase vesicle priming and clamping of spontaneous release, but strongly inhibit evoked release and vesicle fusogenicity. These results support previous findings that the primary interface mediates the functions of synaptotagmin-1 in vesicle priming and clamping of spontaneous release, and, importantly, show that Ca2+-triggering of release requires a rearrangement of the primary interface involving dissociation of region I, while region II remains bound. Together with modeling and biophysical studies presented in the accompanying paper, our data suggest a model whereby this rearrangement pulls the SNARE complex to facilitate fast synaptic vesicle fusion.

Role of adaptor protein complexes in generating functionally distinct synaptic vesicle pools.

The synaptic vesicle (SV) cycle ensures the release of neurotransmitters and the replenishment of SVs to sustain neuronal activity. Multiple endocytosis and sorting pathways contribute to the recapture of the SV membrane and proteins after fusion. Adaptor protein (AP) complexes are among the critical components of the SV retrieval machinery. The canonical clathrin adaptor AP2 ensures the replenishment of most SVs across many neuronal populations. An alternative AP1/AP3-dependent process mediates the formation of a subset of SVs that differ from AP2 vesicles in molecular composition and respond preferentially during higher frequency firing. Furthermore, recent studies show that vesicular transporters for different neurotransmitters depend to a different extent on the AP3 pathway and this affects the release properties of the respective neurotransmitters. This review focuses on the current understanding of the AP-dependent molecular and functional diversity among SVs. We also discuss the contribution of these pathways to the regulation of neurotransmitter release across neuronal populations.

Overlapping role of synaptophysin and synaptogyrin family proteins in determining the small size of synaptic vesicles.

Members of the synaptophysin and synaptogyrin family are vesicle proteins with four transmembrane domains. In spite of their abundance in synaptic vesicle (SV) membranes, their role remains elusive and only mild defects at the cellular and organismal level are observed in mice lacking one or more family members. Here, we show that coexpression with synapsin in fibroblasts of each of the four brain-enriched members of this family-synaptophysin, synaptoporin, synaptogyrin 1, and synaptogyrin 3-is sufficient to generate clusters of small vesicles in the same size range of SVs. Moreover, mice lacking all these four proteins have larger SVs. We conclude that synaptophysin and synaptogyrin family proteins play an overlapping function in the biogenesis of SVs and in determining their small size.

The intracellular C-terminus confers compartment-specific targeting of voltage-gated calcium channels.

To achieve the functional polarization that underlies brain computation, neurons sort protein material into distinct compartments. Ion channel composition, for example, differs between axons and dendrites, but the molecular determinants for their polarized trafficking remain obscure. Here, we identify mechanisms that target voltage-gated Ca2+ channels (CaVs) to distinct subcellular compartments. In hippocampal neurons, CaV2s trigger neurotransmitter release at the presynaptic active zone, and CaV1s localize somatodendritically. After knockout of all three CaV2s, expression of CaV2.1, but not CaV1.3, restores neurotransmitter release. We find that chimeric CaV1.3s with CaV2.1 intracellular C-termini localize to the active zone, mediate synaptic vesicle exocytosis, and render release sensitive to CaV1 blockers. This dominant targeting function of the CaV2.1 C-terminus requires the first EF hand in its proximal segment, and replacement of the CaV2.1 C-terminus with that of CaV1.3 abolishes CaV2.1 active zone localization and function. We conclude that CaV intracellular C-termini mediate compartment-specific targeting.

A role for synapsin tetramerization in synaptic vesicle clustering.

Although synapsins have long been proposed to be key regulators of synaptic vesicle (SV) clustering, their mechanism of action has remained mysterious and somewhat controversial. Here, we review synapsins and their associations with each other and with SVs. We highlight the recent hypothesis that synapsin tetramerization is a mechanism for SV clustering. This hypothesis, which aligns with numerous experimental results, suggests that the larger size of synapsin tetramers, in comparison to dimers, allows tetramers to form optimal bridges between SVs that overcome the repulsive force associated with the negatively charged membrane of SVs and allow synapsins to form a reserve pool of SVs within presynaptic terminals.

Interplay between Copper, Phosphatidylserine, and α-Synuclein Suggests a Link between Copper Homeostasis and Synaptic Vesicle Cycling.

Copper homeostasis is critical to the functioning of the brain, and its breakdown is linked with many brain diseases. Copper is also known to interact with the negatively charged lipid, phosphatidylserine (PS), as well as α-synuclein, an aggregation-prone protein enriched in the synapse, which plays a role in synaptic vesicle docking and fusion. However, the interplay between copper, PS lipid, and α-synuclein is not known. Herein, we report a detailed and predominantly kinetic study of the interactions among these three components pertinent to copper homeostasis and neurotransmission. We found that synaptic vesicle-mimicking small unilamellar vesicles (SUVs) can sequester any excess free Cu2+ within milliseconds, and bound Cu2+ on SUVs can be reduced to Cu+ by GSH at a nearly constant rate under physiological conditions. Moreover, we revealed that SUV-bound Cu2+ does not affect the binding between wild-type α-synuclein and SUVs but affect that between N-terminal acetylated α-synuclein and SUVs. In contrast, Cu2+ can effectively displace both types of α-synuclein from the vesicles. Our results suggest that synaptic vesicles may mediate copper transfer in the brain, while copper could participate in synaptic vesicle docking to the plasma membrane via its regulation of the interaction between α-synuclein and synaptic vesicle.

A Transdermal Prion-Bionics Supermolecule as a RAB3A Antagonist for Enhancing Facial Youthfulness.

The mechanism research of skin wrinkles, conducted on volunteers underwent high-intensity desk work and mice subjected to partial sleep deprivation, revealed a significant reduction in dermal thickness associated with the presence of wrinkles. This can be attributed to the activation of facial nerves in a state of hysteria due to an abnormally elevated interaction between SNAP25 and RAB3A proteins involved in the synaptic vesicle cycle (SVC). Facilitated by AI-assisted structural design, a refined peptide called RSIpep is developed to modulate this interaction and normalize SVC. Drawing inspiration from prions, which possess the ability to protect themselves against proteolysis and invade neighboring nerve cells through macropinocytosis, RSIpep is engineered to demonstrate a GSH-responsive reversible self-assembly into a prion-like supermolecule (RSIprion). RSIprion showcases protease resistance, micropinocytosis-dependent cellular internalization, and low adhesion with constituent molecules in the cuticle, thereby endowing it with the transdermic absorption and subsequent biofunction in redressing the frenzied SVC. As a facial mud mask, it effectively reduces periorbital and perinasal wrinkles in the human face. Collectively, RSIprion not only presents a clinical potential as an anti-wrinkle prion-like supermolecule, but also exemplifies a reproducible instance of bionic strategy-guided drug development that bestows transdermal ability upon the pharmaceutical molecule.

A Dual Strategy-In Vitro and In Silico-To Evaluate Human Antitetanus mAbs Addressing Their Potential Protective Action on TeNT Endocytosis in Primary Rat Neuronal Cells.

Tetanus disease, caused by C. tetani, starts with wounds or mucous layer contact. Prevented by vaccination, the lack of booster shots throughout life requires prophylactic treatment in case of accidents. The incidence of tetanus is high in underdeveloped countries, requiring the administration of antitetanus antibodies, usually derived from immunized horses or humans. Heterologous sera represent risks such as serum sickness. Human sera can carry unknown viruses. In the search for human monoclonal antibodies (mAbs) against TeNT (Tetanus Neurotoxin), we previously identified a panel of mAbs derived from B-cell sorting, selecting two nonrelated ones that binded to the C-terminal domain of TeNT (HCR/T), inhibiting its interaction with the cellular receptor ganglioside GT1b. Here, we present the results of cellular assays and molecular docking tools. TeNT internalization in neurons is prevented by more than 50% in neonatal rat spinal cord cells, determined by quantitative analysis of immunofluorescence punctate staining of Alexa Fluor 647 conjugated to TeNT. We also confirmed the mediator role of the Synaptic Vesicle Glycoprotein II (SV2) in TeNT endocytosis. The molecular docking assays to predict potential TeNT epitopes showed the binding of both antibodies to the HCR/T domain. A higher incidence was found between N1153 and W1297 when evaluating candidate residues for conformational epitope.

Rab6-Mediated Polarized Transport of Synaptic Vesicle Precursors Is Essential for the Establishment of Neuronal Polarity and Brain Formation.

Neurons are highly polarized cells that are composed of a single axon and multiple dendrites. Axon-dendrite polarity is essential for proper tissue formation and brain functions. Intracellular protein transport plays an important role in the establishment of neuronal polarity. However, the regulatory mechanism of polarized transport remains unclear. Here, we show that Rab6, a small GTPase that acts on the regulation of intracellular vesicular trafficking, plays key roles in neuronal polarization and brain development. Central nervous system-specific Rab6a/b double knock-out (Rab6 DKO) mice of both sexes exhibit severe dysplasia of the neocortex and the cerebellum. In the Rab6 DKO neocortex, impaired axonal extension of neurons results in hypoplasia of the intermediate zone. In vitro, deletion of Rab6a and Rab6b in cultured neurons from both sexes causes the abnormal accumulation of synaptic vesicle precursors (SVPs) adjacent to the Golgi apparatus, which leads to defects in axonal extension and the loss of axon-dendrite polarity. Moreover, Rab6 DKO causes significant expansion of lysosomes in the soma in neurons. Overall, our results reveal that Rab6-mediated polarized transport of SVPs is crucial for neuronal polarization and subsequent brain formation.

Evoked resonant neural activity long-term dynamics can be reproduced by a computational model with vesicle depletion.

Subthalamic deep brain stimulation (DBS) robustly generates high-frequency oscillations known as evoked resonant neural activity (ERNA). Recently the importance of ERNA has been demonstrated through its ability to predict the optimal DBS contact in the subthalamic nucleus in patients with Parkinson's disease. However, the underlying mechanisms of ERNA are not well understood, and previous modelling efforts have not managed to reproduce the wealth of published data describing the dynamics of ERNA. Here, we aim to present a minimal model capable of reproducing the characteristics of the slow ERNA dynamics published to date. We make biophysically-motivated modifications to the Kuramoto model and fit its parameters to the slow dynamics of ERNA obtained from data. Our results demonstrate that it is possible to reproduce the slow dynamics of ERNA (over hundreds of seconds) with a single neuronal population, and, crucially, with vesicle depletion as one of the key mechanisms behind the ERNA frequency decay in our model. We further validate the proposed model against experimental data from Parkinson's disease patients, where it captures the variations in ERNA frequency and amplitude in response to variable stimulation frequency, amplitude, and to stimulation pulse bursting. We provide a series of predictions from the model that could be the subject of future studies for further validation.

Physiological roles of endocytosis and presynaptic scaffold in vesicle replenishment at fast and slow central synapses.

After exocytosis, release sites are cleared of vesicular residues to replenish with transmitter-filled vesicles. Endocytic and scaffold proteins are thought to underlie this site-clearance mechanism. However, the physiological significance of this mechanism at diverse mammalian central synapses remains unknown. Here, we tested this in a physiologically optimized condition using action potential evoked EPSCs at fast calyx synapse and relatively slow hippocampal CA1 synapse, in post-hearing mice brain slices at 37°C and in 1.3 mM [Ca2+]. Pharmacological block of endocytosis enhanced synaptic depression at the calyx synapse, whereas it attenuated synaptic facilitation at the hippocampal synapse. Block of scaffold protein activity likewise enhanced synaptic depression at the calyx but had no effect at the hippocampal synapse. At the fast calyx synapse, block of endocytosis or scaffold protein activity significantly enhanced synaptic depression as early as 10 ms after the stimulation onset. Unlike previous reports, neither endocytic blockers nor scaffold protein inhibitors prolonged the recovery from short-term depression. We conclude that the release-site clearance by endocytosis can be a universal phenomenon supporting vesicle replenishment at both fast and slow synapses, whereas the presynaptic scaffold mechanism likely plays a specialized role in vesicle replenishment predominantly at fast synapses.

Synaptic vesicle release regulates pre-myelinating oligodendrocyte-axon interactions in a neuron subtype-specific manner.

Oligodendrocyte-lineage cells are central nervous system (CNS) glia that perform multiple functions including the selective myelination of some but not all axons. During myelination, synaptic vesicle release from axons promotes sheath stabilization and growth on a subset of neuron subtypes. In comparison, it is unknown if pre-myelinating oligodendrocyte process extensions selectively interact with specific neural circuits or axon subtypes, and whether the formation and stabilization of these neuron-glia interactions involves synaptic vesicle release. In this study, we used fluorescent reporters in the larval zebrafish model to track pre-myelinating oligodendrocyte process extensions interacting with spinal axons utilizing in vivo imaging. Monitoring motile oligodendrocyte processes and their interactions with individually labeled axons revealed that synaptic vesicle release regulates the behavior of subsets of process extensions. Specifically, blocking synaptic vesicle release decreased the longevity of oligodendrocyte process extensions interacting with reticulospinal axons. Furthermore, blocking synaptic vesicle release increased the frequency that new interactions formed and retracted. In contrast, tracking the movements of all process extensions of singly-labeled oligodendrocytes revealed that synaptic vesicle release does not regulate overall process motility or exploratory behavior. Blocking synaptic vesicle release influenced the density of oligodendrocyte process extensions interacting with reticulospinal and serotonergic axons, but not commissural interneuron or dopaminergic axons. Taken together, these data indicate that alterations to synaptic vesicle release cause changes to oligodendrocyte-axon interactions that are neuron subtype specific.

Excessive sucrose consumption reduces synaptic density and increases cannabinoid receptors in Göttingen minipigs.

Diets high in sucrose and fat are becoming more prevalent the world over, accompanied by a raised prevalence of cardiovascular diseases, cancers, diabetes, obesity, and metabolic syndrome. Clinical studies link unhealthy diets with the development of mental health disorders, particularly depression. Here, we investigate the effects of 12 days of sucrose consumption administered as 2 L of 25% sucrose solution daily for 12 days in Göttingen minipigs on the function of brain receptors involved in reward and motivation, regulating feeding, and pre- and post-synaptic mechanisms. Through quantitative autoradiography of cryostat sections containing limbic brain regions, we investigated the effects of sucrose restricted to a 1-h period each morning, on the specific binding of [3H]raclopride on dopamine D2/3 receptors, [3H]UCB-J at synaptic vesicle glycoprotein 2A (SV2A), [3H]MPEPγ at metabotropic glutamate receptor subtype 5 (mGluR5) and [3H]SR141716A at the cannabinoid receptor 1 (CB1). Compared to control diet animals, the sucrose group showed significantly lower [3H]UCB-J and [3H]MPEPγ binding in the prefrontal cortex. The sucrose-consuming minipigs showed higher hippocampal CB1 binding, but unaltered dopamine D2/3 binding compared to the control group. We found that the sucrose diet reduced the synaptic density marker while increasing CB1 binding in limbic brain structures, which may subserve maladaptive changes in appetite regulation and feeding. Further studies of the effects of diets and lifestyle habits on brain neuroreceptor and synaptic density markers are warranted.

Exploring the Interactions between two Ligands, UCB-J and UCB-F, and Synaptic Vesicle Glycoprotein 2 Isoforms.

In silico modeling was applied to study the efficiency of two ligands, namely, UCB-J and UCB-F, to bind to isoforms of the synaptic vesicle glycoprotein 2 (SV2) that are involved in the regulation of synaptic function in the nerve terminals, with the ultimate goal to understand the selectivity of the interaction between UCB-J and UCB-F to different isoforms of SV2. Docking and large-scale molecular dynamics simulations were carried out to unravel various binding patterns, types of interactions, and binding free energies, covering hydrogen bonding and nonspecific hydrophobic interactions, water bridge, π-π, and cation-π interactions. The overall preference for bonding types of UCB-J and UCB-F with particular residues in the protein pockets can be disclosed in detail. A unique interaction fingerprint, namely, hydrogen bonding with additional cation-π interaction with the pyridine moiety of UCB-J, could be established as an explanation for its high selectivity over the SV2 isoform A (SV2A). Other molecular details, primarily referring to the presence of π-π interactions and hydrogen bonding, could also be analyzed as sources of selectivity of the UCB-F tracer for the three isoforms. The simulations provide atomic details to support future development of new selective tracers targeting synaptic vesicle glycoproteins and their associated diseases.