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.     

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.     

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.     

Synaptic vesicles interchange their membrane proteins with a large surface reservoir during recycling

During recycling of synaptic vesicles (SVs), the retrieval machinery faces the challenge of recapturing SV proteins in a timely and precise manner. The significant dilution factor that would result from equilibration of vesicle proteins with the much larger cell surface would make recapture by diffusional encounter with the endocytic retrieval machinery unlikely. If SV proteins exchanged with counterparts residing at steady state on the cell surface, the dilution problem would be largely avoided. In this scenario, during electrical activity, endocytosis would be driven by the concentration of a pre-existing pool of SVs residing on the axonal or synaptic surface rather than the heavily diluted postfusion vesicular pool. Using both live cell imaging of endogenous synaptotagmin Ia (sytIa) as well as pHluorin-tagged sytIa and VAMP-2, we show here that synaptic vesicle proteins interchange with a large pool on the cell axonal surface whose concentration is approximately 10-fold lower than that in SVs.     

Age-related changes in synaptic vesicle pools of axo-dendritic synapses on hippocampal CA1 pyramidal neurons in mice

The ultrastructure of symmetric (putatively inhibitory) axo-dendritic synapses on the membrane of hippocampal CA1 pyramidal neurons was investigated in young (20-day-old) and adult (1-year-old) mice. It was shown that synapses of adult animals contain, on average, fewer synaptic vesicles (SVs), and resting SVs of the reserve pool are mostly responsible for this difference. At the same time, in the synapses of adult mice SVs are localized closer to active zones, and the readily releasable pool of SVs is larger in these animals than in young mice. The observed changes in the spatial structure of SV pools presumably demonstrate the age-associated adaptation of inhibitory synapses providing the maintenance of adequate functional properties of hippocampal neuronal networks. Neirofiziologiya/Neurophysiology, Vol. 38, Nos. 5/6, pp. 407–411, September–December, 2006.     

Synaptophysin I controls the targeting of VAMP2/synaptobrevin II to synaptic vesicles

Synaptic vesicle (SV) proteins are synthesized at the level of the cell body and transported down the axon in membrane precursors of SVs. To investigate the mechanisms underlying sorting of proteins to SVs, fluorescent chimeras of vesicle-associated membrane protein (VAMP) 2, its highly homologous isoform VAMP1 and synaptotagmin I (SytI) were expressed in hippocampal neurons in culture. Interestingly, the proteins displayed a diffuse component of distribution along the axon. In addition, VAMP2 was found to travel in vesicles that constitutively fuse with the plasma membrane. Coexpression of VAMP2 with synaptophysin I (SypI), a major resident of SVs, restored the correct sorting of VAMP2 to SVs. The effect of SypI on VAMP2 sorting was dose dependent, being reversed by increasing VAMP2 expression levels, and highly specific, because the sorting of the SV proteins VAMP1 and SytI was not affected by SypI. The cytoplasmic domain of VAMP2 was found to be necessary for both the formation of VAMP2-SypI hetero-dimers and for VAMP2 sorting to SVs. These data support a role for SypI in directing the correct sorting of VAMP2 in neurons and demonstrate that a direct interaction between the two proteins is required for SypI in order to exert its effect.     

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.     

Influence of synapsin I on synaptic vesicles: an analysis by force-volume mode of the atomic force microscope and dynamic light scattering

Synaptic vesicles (SVs) are small neuronal organelles that store neurotransmitters and release them by exocytosis into the synaptic cleft for signal transmission between nerve cells. They consist of a highly curved membrane composed of different lipids containing several proteins with specific functions. A family of abundant extrinsic SV proteins, the synapsins, interact with SV proteins and phospholipids and play an important role in the regulation of SV trafficking and stability. We investigated the interactions of one these proteins with the SV membrane using atomic force microscope and dynamic light scattering. We examined SVs isolated from rat forebrain both under native conditions and after depletion of endogenous synapsin I. We used the atomic force microscope in two modes: imaging mode for characterizing the shape and size of SVs, and force-volume mode for characterizing their stiffness. Synapsin-depleted SVs were larger in size and showed a higher tendency to aggregate than native vesicles, although their stiffness was not significantly different. Because synapsins are believed to cross-link SV to each other and to the actin cytoskeleton, we also measured the SV aggregation kinetics induced by synapsin I by dynamic light scattering and atomic force microscopy and found that the addition of synapsin I promotes a rapid aggregation of SVs. The data indicate that synapsin directly affects SV stability and aggregation state and support the physiological role of synapsins in the assembly and regulation of SV pools within nerve terminals.     

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.     

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.     

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.      

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.     

Pulsed-laser creation and characterization of giant plasma membrane vesicles from cells

Femtosecond-pulsed laser irradiation was found to initiate giant plasma membrane vesicle (GPMV) formation on individual cells. Laser-induced GPMV formation resulted from intracellular cavitation and did not require the addition of chemical stressors to the cellular environment. The viscosity, structure, and contents of laser-induced GPMVs were measured with fluorescence microscopy and single-particle tracking. These GPMVs exhibit the following properties: (1) GPMVs grow fastest immediately after laser irradiation; (2) GPMVs contain barriers to free diffusion of incorporated fluorescent beads; (3) materials from both the cytoplasm and surrounding media flow into the growing GPMVs; (4) the GPMVs are surrounded by phospholipids, including phosphatidylserine; (5) F-actin is incorporated into the vesicles; and (6) caspase activity is not essential for GPMV formation. The effective viscosity of 65 nm polystyrene nanoparticles within GPMVs ranged from 32 to 434 cP. The nanoparticle diffusion was commonly affected by relatively large, macromolecular structures within the bleb.     

Molecular basis of synaptic vesicle cargo recognition by the endocytic sorting adaptor stonin 2

Synaptic transmission depends on clathrin-mediated recycling of synaptic vesicles (SVs). How select SV proteins are targeted for internalization has remained elusive. Stonins are evolutionarily conserved adaptors dedicated to endocytic sorting of the SV protein synaptotagmin. Our data identify the molecular determinants for recognition of synaptotagmin by stonin 2 or its Caenorhabditis elegans orthologue UNC-41B. The interaction involves the direct association of clusters of basic residues on the surface of the cytoplasmic domain of synaptotagmin 1 and a β strand within the μ–homology domain of stonin 2. Mutation of K783, Y784, and E785 to alanine within this stonin 2 β strand results in failure of the mutant stonin protein to associate with synaptotagmin, to accumulate at synapses, and to facilitate synaptotagmin internalization. Synaptotagmin-binding–defective UNC-41B is unable to rescue paralysis in C. elegans stonin mutant animals, suggesting that the mechanism of stonin-mediated SV cargo recognition is conserved from worms to mammals.     

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.     

Serum cholesterol and expression of ApoAI, LXRβ and SREBP2 in vitamin D receptor knock-out mice

Vitamin D insufficiency has been reported to be associated with increased blood cholesterol concentrations. Here we used two strains of VDR knock-out (VDR-KO) mice to study whether a lack of vitamin D action has any effect on cholesterol metabolism. In 129S1 mice, both in male and female VDR-KO mice serum total cholesterol levels were significantly higher than those in wild type (WT) mice (20.7% (P = 0.05) and 22.2% (P = 0.03), respectively). In addition, the serum high-density lipoprotein-bound cholesterol (HDL-C) level was 22% (P = 0.03), respectively higher in male VDR-KO mice than in WT mice. The mRNA expression levels of five cholesterol metabolism related genes in livers of 129S1 mice were studied using quantitative real-time PCR (QRT-PCR): ATP-binding cassette transporter A1 (ABCA1), regulatory element binding protein (SREBP2), apolipoprotein A-I (ApoAI), low-density lipoprotein receptor (LDLR) and liver X receptor beta (LXRβ). In the mutant male mice, the mRNA level of ApoAI and LXRβ were 49.2% (P = 0.005) and 38.8% (P = 0.034) higher than in the WT mice. These changes were not observed in mutant female mice, but the female mutant mice showed 52.5% (P = 0.006) decrease of SREBP2 mRNA expression compared to WT mice. Because the mutant mice were fed with a special rescue diet, we wanted to test whether the increased cholesterol levels in mutant mice were due to the diet. Both the WT and mutant NMRI mice were given the same diet for 3 weeks before the blood sampling. No difference in cholesterol or in HDL-C between WT and mutant mice was found. The results suggest that the food, gender and genetic background have an effect on the cholesterol metabolism. Although VDR seems to regulate some of the genes involved in cholesterol metabolism, its role in the regulation of serum cholesterol seems to be minimal.     

Exo-endocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons

In mature neurons synaptic vesicles (SVs) undergo cycles of exo-endocytosis at synapses. It is currently unknown whether SV exocytosis and recycling occurs also in developing axons prior to synapse formation. To address this question, we have developed an immunocytochemical assay to reveal SV exo-endocytosis in hippocampal neurons developing in culture. In this assay antibodies directed against the lumenal domain of synaptotagmin I (Syt I), an intrinsic membrane protein of SVs, are used to reveal exposure of SV membranes at the cell surface. Addition of antibodies to the culture medium of living neurons for 1 hr at 37 degrees C resulted in their rapid and specific internalization by all neuronal processes and, particularly, by axons. Double immunofluorescence and electron microscopy immunocytochemistry indicated that the antibodies were retained within SVs in cell processes and underwent cycles of exo-endocytosis in parallel with SV membranes. In contrast, another endocytotic marker, wheat germ agglutinin, was rapidly cleared from the processes and transported to the cell body. Antibody-labeled SVs were still present in axons several days after antibody loading and became clustered at presynaptic sites in parallel with synaptogenesis. These results demonstrate that SVs undergo multiple cycles of exo-endocytosis in developing neuronal processes irrespective of the presence of synaptic contacts.     

Involvement of water channels in synaptic vesicle swelling

Vesicle swelling is critical for secretion; however, the underlying mechanism of synaptic vesicle (SV) swelling is unknown. A G alphai3-phospholipase A2 (PLA2)-mediated involvement of the water channel aquaporin-1 (AQP1) in the regulation of secretory vesicle swelling in the exocrine pancreas has been previously reported. In the present study, the association and involvement of water channels in SV swelling was explored. Results from the study demonstrate that water channels AQP1 and AQP6, and the heterotrimeric Go protein are associated with SVs and participate in their swelling.     

Morphology of egg phosphatidylcholine-cholesterol single-bilayer vesicles,studied by freeze-etch electron microscopy

Summary Homogeneous, small, single-bilayer vesicles were prepared from egg phosphatidylcholine with various concentrations of cholesterol by ultrasonic dispersion in 0.1m KCl, 0.01m Tris, pH 8.0, buffer, followed by gel chromatography. The shape and size distributions of the fractionated vesicles were investigated for preparations with cholesterol compositions from 0 to 50 moles/100 moles, using freeze-etch electron microscopy. The size distribution was estimated from the shadow width of vesicles which were exposed by etching and the vesicle shape was checked by comparing the images obtained by tilting the replicas. The widths of the vesicle diameter distributions were relatively broad, corresponding to standard deviations in the range 60–90 Å, but showing no systematic variation with cholesterol composition. In all cases it was found that 70% of the vesicle diameters lay within 150 Å of the modal value. The apparent vesicle diameters remained constant for cholesterol compositions up to 20 moles/100 moles (modal diameter=330 ± 20 Å, mean diameter = 350 ± 3 Å), but there was a sharp net increase in diameter at 30 moles cholesterol/100 moles reaching a model diameter of 430 ± 20 Å (mean diameter = 430 ± 3 Å) at 50 moles cholesterol/100 moles. Using the tilted microscope stage it was found that all vesicles were spherical at all cholesterol compositions studied, including those above 30 moles cholesterol/100 moles. The molecular mechanism by which cholesterol controls the vesicle size is discussed in terms of the asymmetric distribution of cholesterol across the vesicle bilayer.     

Cytoplasmic viscosity near the cell plasma membrane: measurement by evanescent field frequency-domain microfluorimetry.

The purpose of this study was to determine whether the unique physical milieu just beneath the cell plasma membrane influences the rheology of fluid-phase cytoplasm. Cytoplasmic viscosity was evaluated from the picosecond rotation of the small fluorophore 2',7'-bis-(2-carboxyethyl)-5-carboxyfluorescein (BCECF) by parallel-acquisition Fourier transform microfluorimetry (Fushimi and Verkman, 1991). Information about viscosity within < 200 nm of cell plasma membranes was obtained by selective excitation of fluorophores in an evanescent field created by total internal reflection (TIR) of impulse-modulated s-plane-polarized laser illumination (488 nm) at a glass-aqueous interface. Measurements of fluorescence lifetime and time-resolved anisotropy were carried out in solutions containing fluorescein or BCECF at known viscosities, and monolayers of BCECF-labeled Swiss 3T3 fibroblasts and Madin-Darby canine kidney (MDCK) cells. Specific concerns associated with time-resolved fluorescence measurements in the evanescent field were examined theoretically and/or experimentally, including variations in lifetime due to fluorophore proximity to the interface, and the use of the s and p polarized excitation. In fluorescein solutions excited with s-plane polarized light, there was a 5-10% decrease in fluorescein lifetime with TIR compared to trans (subcritical) illumination, but no change in rotational correlation time (approximately 98 ps/cP). Intracellular BCECF had a single lifetime of 3.7 +/- 0.1 ns near the cell plasma membrane. Apparent fluid-phase viscosity near the cell plasma membrane was 1.1 +/- 0.2 cP (fibroblast) and 1.0 +/- 0.2 cP (MDCK), not significantly different from the viscosity measured in bulk cytoplasm far from the plasma membrane. The results establish the methodology for time-resolved microfluorimetric measurement of polarization in the evanescent field and demonstrate that the cell plasma membrane has little effect on the fluid-phase viscosity of adjacent cytoplasm.