Quantitative proteomics analysis of detergent-resistant membranes from chemical synapses: evidence for cholesterol as spatial organizer of synaptic vesicle cycling

Synaptic vesicles (SVs) in the central nervous system upon stimulation undergo rapid calcium-triggered exoendocytic cycling within the nerve terminal that at least in part depends on components of the clathrin- and dynamin-dependent endocytosis machinery. How exocytic SV fusion and endocytic retrieval are temporally and spatially coordinated is still an open question. One possibility is that specialized membrane microdomains characterized by their high content in membrane cholesterol may assist in the spatial coordination of synaptic membrane protein recycling. Quantitative proteomics analysis of detergent-resistant membranes (DRMs) isolated from rat brain synapses or cholesterol-depleted control samples by liquid chromatography-tandem mass spectrometry identified a total of 159 proteins. Among these 122 proteins were classified as cholesterol-dependent DRM or DRM-associated proteins, many of which with proven or hypothesized functions in exoendocytic vesicle cycling including clathrin, the clathrin adaptor complex AP-2, and a variety of SV proteins. In agreement with this, SV membrane and endocytic proteins displayed a partial resistance to extraction with cold Triton X-100 in cultured rat hippocampal neurons where they co-localized with labeled cholera toxin B, a marker for cholesterol-enriched DRMs. Moreover SV proteins formed cholesterol-dependent complexes in CHAPS-extracted synaptic membrane lysates. Our combined data suggest that lipid microdomains may act as spatial coordinators for exoendocytic vesicle cycling at synapses.     

Endocytic proteins: An expanding repertoire of presynaptic functions

From a presynaptic perspective, neuronal communication mainly relies on two interdependent events: The fast Ca²⁺-triggered fusion of neurotransmitter-containing synaptic vesicles (SVs) and their subsequent high-fidelity reformation. To allow rapid neurotransmission, SVs have evolved into fascinating molecular nanomachines equipped with a well-defined set of proteins. However, upon exocytosis, SVs fully collapse into the presynaptic plasma membrane leading to the dispersal of their molecular components. While the canonical function of endocytic proteins at the presynapse was believed to be the retrieval of SV proteins via clathrin-mediated endocytosis, it is now evident that clathrin-independent endocytic mechanisms predominate. We will highlight in how far these mechanisms still rely on the classical endocytic machinery and discuss the emerging functions of endocytic proteins in release site clearance and SV reformation from endosomal-like vacuoles.     

How synapsin I may cluster synaptic vesicles

Synapsin I is the most abundant brain phosphoprotein present in conventional synapses of the CNS. Knockout and rescue experiments have demonstrated that synapsin is essential for clustering of synaptic vesicles (SVs) at active zones and the organization of the reserve pool of SVs. However, in spite of intense efforts it remains largely unknown how exactly synapsin I performs this function. It has been proposed that synapsin I in its dephosphorylated state may tether SVs to actin filaments within the cluster from where SVs are released in response to activity-induced synapsin phosphorylation. Recent studies, however, have failed to detect actin filaments inside the vesicle cluster at resting central synapses. Instead, proteins with established functional roles in SV recycling have been found within this presynaptic compartment. Here we discuss potential alternative mechanisms of synapsin I-dependent SV clustering in the reserve pool.     

The molecular physiology of activity-dependent bulk endocytosis of synaptic vesicles

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.     

Signals involved in targeting membrane proteins to synaptic vesicles

1. Synaptic vesicles (SVs) mediate fast regulated secretion of classical neurotransmitters. In order to perform their task SVs rely on a restrict set of membrane proteins. The mechanisms responsible for targeting these proteins to the SV membrane are still poorly understood.2. Likewise, little is known about the intracellular routes taken by these proteins in their way to SV membrane. Recently, several domains and motifs necessary for correct localization of SV proteins have been identified.3. In this review we summarize the sequence motifs that have been identified in the cytoplasmic domains of SV proteins that are involved in endocytosis and targeting of SVs. We suggest that the vesicular acetylcholine transporter, a protein found predominantly in synaptic vesicles, is perhaps a model protein to understand the pathways and interactions that are used for synaptic vesicle targeting.     

Clathrin-mediated endocytosis at synapses

Neurons are communication specialists that convert electrical into chemical signals at specialized cell-cell junctions termed synapses. Arrival of an action potential triggers calcium-regulated exocytosis of neurotransmitter (NT) from small synaptic vesicles (SVs), which then diffuses across the synaptic cleft and binds to postsynaptic receptors to elicit specific changes within the postsynaptic cell. Endocytosis of pre- and postsynaptic membrane proteins including SV components and postsynaptic NT receptors is essential for the proper functioning of the synapse. During the past several years, we have witnessed enormous progress in our understanding of the mechanics of clathrin-mediated endocytosis (CME) and its role in regulating exo-endocytic vesicle cycling at synapses. Here we summarize the molecular machinery used for recognition of synaptic membrane protein cargo and its clathrin-dependent internalization, and describe the inventory of tools that can be used to monitor vesicle cycling at synapses or to inhibit CME in a stage-specific manner.     

AP180 Couples Protein Retrieval to Clathrin‐Mediated Endocytosis of Synaptic Vesicles

How clathrin‐mediated endocytosis (CME) retrieves vesicle proteins into newly formed synaptic vesicles (SVs) remains a major puzzle. Besides its roles in stimulating clathrin‐coated vesicle formation and regulating SV size, the clathrin assembly protein AP180 has been identified as a key player in retrieving SV proteins. The mechanisms by which AP180 recruits SV proteins are not fully understood. Here, we show that following acute inactivation of AP180 in Drosophila, SV recycling is severely impaired at the larval neuromuscular synapse based on analyses of FM 1‐43 uptake and synaptic ultrastructure. More dramatically, AP180 activity is important to maintain the integrity of SV protein complexes at the plasma membrane during endocytosis. These observations suggest that AP180 normally clusters SV proteins together during recycling. Consistent with this notion, SV protein composition and distribution are altered in AP180 mutant flies. Finally, AP180 co‐immunoprecipitates with SV proteins, including the vesicular glutamate transporter and neuronal synaptobrevin. These results reveal a new mode by which AP180 couples protein retrieval to CME of SVs. AP180 is also genetically linked to Alzheimer's disease. Hence, the findings of this study may provide new mechanistic insight into the role of AP180 dysfunction in Alzheimer's disease.      

Proper synaptic vesicle formation and neuronal network activity critically rely on syndapin I

Synaptic transmission relies on effective and accurate compensatory endocytosis. F-BAR proteins may serve as membrane curvature sensors and/or inducers and thereby support membrane remodelling processes; yet, their in vivo functions urgently await disclosure. We demonstrate that the F-BAR protein syndapin I is crucial for proper brain function. Syndapin I knockout (KO) mice suffer from seizures, a phenotype consistent with excessive hippocampal network activity. Loss of syndapin I causes defects in presynaptic membrane trafficking processes, which are especially evident under high-capacity retrieval conditions, accumulation of endocytic intermediates, loss of synaptic vesicle (SV) size control, impaired activity-dependent SV retrieval and defective synaptic activity. Detailed molecular analyses demonstrate that syndapin I plays an important role in the recruitment of all dynamin isoforms, central players in vesicle fission reactions, to the membrane. Consistently, syndapin I KO mice share phenotypes with dynamin I KO mice, whereas their seizure phenotype is very reminiscent of fitful mice expressing a mutant dynamin. Thus, syndapin I acts as pivotal membrane anchoring factor for dynamins during regeneration of SVs.     

Synapsin Condensates Recruit alpha-Synuclein

Neurotransmission relies on the tight spatial and temporal regulation of the synaptic vesicle (SV) cycle. Nerve terminals contain hundreds of SVs that form tight clusters. These clusters represent a distinct liquid phase in which one component of the phase are SVs and the other synapsin 1, a highly abundant synaptic protein. Another major family of disordered proteins at the presynapse includes synucleins, most notably α-synuclein. The precise physiological role of α-synuclein in synaptic physiology remains elusive, albeit its role has been implicated in nearly all steps of the SV cycle. To determine the effect of α-synuclein on the synapsin phase, we employ the reconstitution approach using natively purified SVs from rat brains and the heterologous cell system to generate synapsin condensates. We demonstrate that synapsin condensates recruit α-synuclein, and while enriched into these synapsin condensates, α-synuclein still maintains its high mobility. The presence of SVs enhances the rate of synapsin/α-synuclein condensation, suggesting that SVs act as catalyzers for the formation of synapsin condensates. Notably, at physiological salt and protein concentrations, α-synuclein alone is not able to cluster isolated SVs. Excess of α-synuclein disrupts the kinetics of synapsin/SV condensate formation, indicating that the molar ratio between synapsin and α-synuclein is important in assembling the functional condensates of SVs. Understanding the molecular mechanism of α-synuclein interactions at the nerve terminals is crucial for clarifying the pathogenesis of synucleinopathies, where α-synuclein, synaptic proteins and lipid organelles all accumulate as insoluble intracellular inclusions.     

Presynaptic active zones in invertebrates and vertebrates

The regulated release of neurotransmitter occurs via the fusion of synaptic vesicles (SVs) at specialized regions of the presynaptic membrane called active zones (AZs). These regions are defined by a cytoskeletal matrix assembled at AZs (CAZ), which functions to direct SVs toward docking and fusion sites and supports their maturation into the readily releasable pool. In addition, CAZ proteins localize voltage‐gated Ca²⁺ channels at SV release sites, bringing the fusion machinery in close proximity to the calcium source. Proteins of the CAZ therefore ensure that vesicle fusion is temporally and spatially organized, allowing for the precise and reliable release of neurotransmitter. Importantly, AZs are highly dynamic structures, supporting presynaptic remodeling, changes in neurotransmitter release efficacy, and thus presynaptic forms of plasticity. In this review, we discuss recent advances in the study of active zones, highlighting how the CAZ molecularly defines sites of neurotransmitter release, endocytic zones, and the integrity of synapses.     

Glycosylation Is Dispensable for Sorting of Synaptotagmin 1 but Is Critical for Targeting of SV2 and Synaptophysin to Recycling Synaptic Vesicles

Glycosylation is a major form of post-translational modification of synaptic vesicle membrane proteins. For example, the three major synaptic vesicle glycoproteins, synaptotagmin 1, synaptophysin, and SV2, represent ∼30% of the total copy number of vesicle proteins. Previous studies suggested that glycosylation is required for the vesicular targeting of synaptotagmin 1, but the role of glycosylation of synaptophysin and SV2 has not been explored in detail. In this study, we analyzed all glycosylation sites on synaptotagmin 1, synaptophysin, and SV2A via mutagenesis and optical imaging of pHluorin-tagged proteins in cultured neurons from knock-out mice lacking each protein. Surprisingly, these experiments revealed that glycosylation is completely dispensable for the sorting of synaptotagmin 1 to SVs whereas the N-glycans on SV2A are only partially dispensable. In contrast, N-glycan addition is essential for the synaptic localization and function of synaptophysin. Thus, glycosylation plays distinct roles in the trafficking of each of the three major synaptic vesicle glycoproteins.     

Vesicular Glutamate Transporter 1 Orchestrates Recruitment of Other Synaptic Vesicle Cargo Proteins during Synaptic Vesicle Recycling

A long standing question in synaptic physiology is how neurotransmitter-filled vesicles are rebuilt after exocytosis. Among the first steps in this process is the endocytic retrieval of the transmembrane proteins that are enriched in synaptic vesicles (SVs). At least six types of transmembrane proteins must be recovered, but the rules for how this multiple cargo selection is accomplished are poorly understood. Among these SV cargos is the vesicular glutamate transporter (vGlut). We show here that vGlut1 has a strong influence on the kinetics of retrieval of half of the known SV cargos and that specifically impairing the endocytosis of vGlut1 in turn slows down other SV cargos, demonstrating that cargo retrieval is a collective cargo-driven process. Finally, we demonstrate that different cargos can be retrieved in the same synapse with different kinetics, suggesting that additional post-endocytic sorting steps likely occur in the nerve terminal.     

Endophilin functions as a membrane-bending molecule and is delivered to endocytic zones by exocytosis

Two models have been proposed for endophilin function in synaptic vesicle (SV) endocytosis. The scaffolding model proposes that endophilin's SH3 domain recruits essential endocytic proteins, whereas the membrane-bending model proposes that the BAR domain induces positively curved membranes. We show that mutations disrupting the scaffolding function do not impair endocytosis, whereas those disrupting membrane bending cause significant defects. By anchoring endophilin to the plasma membrane, we show that endophilin acts prior to scission to promote endocytosis. Despite acting at the plasma membrane, the majority of endophilin is targeted to the SV pool. Photoactivation studies suggest that the soluble pool of endophilin at synapses is provided by unbinding from the adjacent SV pool and that the unbinding rate is regulated by exocytosis. Thus, endophilin participates in an association-dissociation cycle with SVs that parallels the cycle of exo- and endocytosis. This endophilin cycle may provide a mechanism for functionally coupling endocytosis and exocytosis.     

Mixing and matching during synaptic vesicle endocytosis

The question of how synapses maintain an active recycling pool of synaptic vesicles to support high-frequency synaptic transmission has been a perplexing and often controversial problem. In this issue of Neuron, Fernandez-Alfonso et al. present data indicating that at least two synaptic vesicle proteins, synaptotagmin 1 and VAMP-2, are present in a large pool on the synaptic and axonal plasma membrane and can interchange with recently exocytosed proteins. These findings suggest that a plasma membrane pool of synaptic vesicle proteins provides a reservoir that can facilitate rapid endocytosis.     

Synaptic vesicle proteins and early endosomes in cultured hippocampal neurons: differential effects of Brefeldin A in axon and dendrites

The pathways of synaptic vesicle (SV) biogenesis and recycling are still poorly understood. We have studied the effects of Brefeldin A (BFA) on the distribution of several SV membrane proteins (synaptophysin, synaptotagmin, synaptobrevin, p29, SV2 and rab3A) and on endosomal markers to investigate the relationship between SVs and the membranes with which they interact in cultured hippocampal neurons developing in isolation. In these neurons, SV proteins are detected as punctate immunoreactivity that is concentrated in axons but is also present in perikarya and dendrites. In the same neurons, the transferrin receptor, a well established marker of early endosomes, is selectively concentrated in perikarya and dendrites. In the perikaryal- dendritic region, BFA induced a dramatic tubulation of transferrin receptors as well as a cotubulation of the bulk of synaptophysin. Synaptotagmin, synaptobrevin, p29 and SV2 immunoreactivities retained a primarily punctate distribution. No tubulation of rab3A was observed. In axons, BFA did not produce any obvious alteration of the distribution of SV proteins, nor of peroxidase- or Lucifer yellow- labeled early endosomes. The selective effect of BFA on dendritic membranes suggests the existence of functional differences between the endocytic systems in dendrites and axons. Cotubulation of transferrin receptors and synaptophysin in the perikaryal-dendritic region is consistent with a functional interconnection between the traffic of SV proteins and early endosomes. The heterogeneous effects of BFA on SV proteins in this cell region indicates that SV proteins are differentially sorted upon exit from the TGN and are coassembled into SVs at the cell periphery.     

Genetic and molecular analysis of synaptic vesicle recycling in Drosophila

Following exocytosis, one of the major presynaptic events is replenishing synaptic vesicles (SVs) to ensure the possibility of continuous synaptic transmission. The nerve terminal is thought to recycle SVs through clathrin-mediated endocytosis and by a clathrin-independent pathway called ‘kiss and run’. This review highlights the use of the genetic model organism, the fruit fly (Drosophila melanogaster), in dissecting the molecular mechanisms of clathrin-mediated endocytosis in recycling SVs at neuromuscular junctions (NMJs). Analyses of endocytotic mutants in Drosophila indicate that clathrin-mediated endocytosis may be essential for SV recycling, including a putative fast recycling mechanism uncovered recently. Further, a rather complex picture begins to emerge suggesting that clathrin-mediated endocytosis involves several sequential steps mediated by a large number of proteins. Finally, these studies also reveal that SV proteins may be selectively retrieved into nascent SVs by clathrin accessory proteins and defects in protein retrieval have significant impacts on synaptic transmission. Following the completion of the Drosophila Genome Project and the development of gene targeting and RNAi approaches, genetic studies in Drosophila have become increasingly efficient. Hence, Drosophila is expected to continue to serve as an important model organism for studies of SV recycling.     

The t-SNAREs syntaxin 1 and SNAP-25 are present on organelles that participate in synaptic vesicle recycling

Syntaxin 1 and synaptosome-associated protein of 25 kD (SNAP-25) are neuronal plasmalemma proteins that appear to be essential for exocytosis of synaptic vesicles (SVs). Both proteins form a complex with synaptobrevin, an intrinsic membrane protein of SVs. This binding is thought to be responsible for vesicle docking and apparently precedes membrane fusion. According to the current concept, syntaxin 1 and SNAP-25 are members of larger protein families, collectively designated as target-SNAP receptors (t-SNAREs), whose specific localization to subcellular membranes define where transport vesicles bind and fuse. Here we demonstrate that major pools of syntaxin 1 and SNAP-25 recycle with SVs. Both proteins cofractionate with SVs and clathrin-coated vesicles upon subcellular fractionation. Using recombinant proteins as standards for quantitation, we found that syntaxin 1 and SNAP-25 each comprise approximately 3% of the total protein in highly purified SVs. Thus, both proteins are significant components of SVs although less abundant than synaptobrevin (8.7% of the total protein). Immunoisolation of vesicles using synaptophysin and syntaxin specific antibodies revealed that most SVs contain syntaxin 1. The widespread distribution of both syntaxin 1 and SNAP-25 on SVs was further confirmed by immunogold electron microscopy. Botulinum neurotoxin C1, a toxin that blocks exocytosis by proteolyzing syntaxin 1, preferentially cleaves vesicular syntaxin 1. We conclude that t- SNAREs participate in SV recycling in what may be functionally distinct forms.     

Docking and fusion of synaptic vesicles in cell-free model system of exocytosis

The present study involves the testing and characterization of synaptic vesicle (SV) docking and fusion as the steps of exocytosis using two different approaches in vitro.The interaction of SVs was determined by the changing of particles size in suspensions by the method of dynamic light scattering (DLS). Fluorescence assay is represented for studying the mechanism of SV membrane fusion. The sizes of membrane particles were shown to increase in the medium containing cytoplasmic proteins of synaptosomes. Therefore, the cytosolic proteins are suggested to promote the SVs into close proximity where they may become stably bound or docked. The specific effect of synaptosomal cytosolic proteins on the interaction of SVs in the cell-free system was demonstrated. The incubation of SVs with liver cytosol proteins or in the bovine serum albumin solution did not lead to the enlargement of the particles size. The fusion reaction of the SVs membranes occurred within the micromolar range of Ca²⁺ concentrations. Our studies have shown that in vitro process of exocytosis can be divided into Ca²⁺-independent step, termed docking and followed by fusion step that is triggered by Ca²⁺. The role of cytosolic proteins of synaptosomes in docking and fusion of SVs in cell-free system was further confirmed.     

Bulk endocytosis at neuronal synapses

Neurotransmitter-containing synaptic vesicle(SV)fusion with the nerve terminal plasma membrane initiates neurotransmission in response to neuronal excitation.Under mild stimulation,the fused vesicular membrane is retrieved via kiss-and-run and/or clathrin-mediated endocytosis,which is sufficient to maintain recycling of SVs.When neurons are challenged with very high stimulation,the number of fused SVs can be extremely high,resulting in significant plasma membrane addition.Under such conditions,a higher capacity retrieval pathway,bulk endocytosis,is activated to redress this large membrane imbalance.Despite first being described more than 40 years ago,the molecular mechanisms underpinning this important process have yet to be clearly defined.In this review,we highlight the current evidence for bulk endocytosis and its prevalence in various neuronal models,as well as discuss the underlying molecular components.     

Docking and fusion of synaptic vesicles in cell-free model system of exocytosis

The present study involves the testing and characterization of synaptic vesicle (SV) docking and fusion as the steps of exocytosis using two different approaches in vitro.The interaction of SVs was determined by the changing of particles size in suspensions by the method of dynamic light scattering (DLS). Fluorescence assay is represented for studying the mechanism of SV membrane fusion. The sizes of membrane particles were shown to increase in the medium containing cytoplasmic proteins of synaptosomes. Therefore, the cytosolic proteins are suggested to promote the SVs into close proximity where they may become stably bound or docked. The specific effect of synaptosomal cytosolic proteins on the interaction of SVs in the cell-free system was demonstrated. The incubation of SVs with liver cytosol proteins or in the bovine serum albumin solution did not lead to the enlargement of the particles size. The fusion reaction of the SVs membranes occurred within the micromolar range of Ca²⁺ concentrations. Our studies have shown that in vitro process of exocytosis can be divided into Ca²⁺-independent step, termed docking and followed by fusion step that is triggered by Ca²⁺. The role of cytosolic proteins of synaptosomes in docking and fusion of SVs in cell-free system was further confirmed.