Walter Volknandt

A plethora of presynaptic proteins associated with ATP-storing organelles in cultured astrocytes

Cultured astrocytes can release a variety of messenger substances via receptor‐mediated mechanisms, implicating their potential for regulated exocytosis and the participation of proteins of the SNARE complex. Here we demonstrate the astrocytic expression and organellar association of a large variety of synaptic proteins (synaptobrevin II, synaptotagmin I, synaptophysin, rab3a, synapsin I, SNAP‐25, and syntaxin I) and also of the ubiquitous cellubrevin. As revealed by immunoblotting the expression of synaptic proteins was highest within the first few days after plating. Synaptophysin and SNAP‐25 showed the most significant decline with prolonged culture time. Rab3a and synaptobrevin II were retained at a high level and synaptotagmin I, synapsin I, and syntaxin I at a lower level until 20 DIV. The immunoreaction for cellubrevin was low at the beginning and increased with prolonged culture time. As revealed by light microscopical immunocytochemistry the proteins are expressed by GFAP‐positive astrocytes and associated with organelles of varying size. Immunoelectron microscopical analysis allocates synaptobrevin II and synaptophysin to the membranes of vesicular organelles. Double labeling experiments for pairs of synaptic proteins reveal that individual synaptic proteins can be entirely colocalized or partly reside on different organelles. Subcellular fractionation of astrocyte cultures by sucrose density gradient centrifugation after 2, 6, 13, and 20 DIV showed that the proteins sediment with ATP containing organelles of a broad density range. Our data suggest that messenger substances may be released from cultured astrocytes via receptor‐mediated, Ca2+‐dependent exocytosis. GLIA 26:233–244, 1999.

Immunoisolation of two synaptic vesicle pools from synaptosomes: a proteomics analysis.

The nerve terminal proteome governs neurotransmitter release as well as the structural and functional dynamics of the presynaptic compartment. In order to further define specific presynaptic subproteomes we used subcellular fractionation and a monoclonal antibody against the synaptic vesicle protein SV2 for immunoaffinity purification of two major synaptosome‐derived synaptic vesicle‐containing fractions: one sedimenting at lower and one sedimenting at higher sucrose density. The less dense fraction contains free synaptic vesicles, the denser fraction synaptic vesicles as well as components of the presynaptic membrane compartment. These immunoisolated fractions were analyzed using the cationic benzyldimethyl‐n‐hexadecylammonium chloride (BAC) polyacrylamide gel system in the first and sodium dodecyl sulfate–polyacrylamide gel electrophoresis in the second dimension. Protein spots were subjected to analysis by matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS). We identified 72 proteins in the free vesicle fraction and 81 proteins in the plasma membrane‐containing denser fraction. Synaptic vesicles contain a considerably larger number of protein constituents than previously anticipated. The plasma membrane‐containing fraction contains synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery and numerous other proteins potentially involved in regulating the functional and structural dynamics of the nerve terminal.

Acetylcholine, ATP, and proteoglycan are common to synaptic vesicles isolated from the electric organs of electric eel and electric catfish as well as from rat diaphragm.

Abstract Cholinergic synaptic vesicles were isolated from the electric organs of the electric eel (Electrophorus electricus) and the electric catfish (Malapterurus electricus) as well as from the diaphragm of the rat by density gradient centrifugation followed by column chromatography on Sephacryl‐1000. This was verified by both biochemical and electron microscopic criteria. Differences in size between synaptic vesicles from the various tissue sources were reflected by their elution pattern from the Sephacryl column. Specific activities of acetylcholine (ACh; in nmol/mg of protein) of chromatography‐purified vesicle fractions were 36 (electric eel), 2 (electric catfish), and 1 (rat diaphragm). Synaptic vesicles from all three sources contained ATP in addition to ACh (molar ratios of ACh/ATP, 9–12) as well as binding activity for an antibody raised against Torpedo cholinergic synaptic vesicle proteoglycan. Synaptic vesicles from rat diaphragm contained binding activity for the monoclonal antibody asv 48 raised against a rat brain 65‐kilodalton synaptic vesicle protein. Antibody asv 48 binding was absent from electric eel and electric catfish synaptic vesicles. These antibody binding results, which were obtained by a dot blot assay on isolated vesicles, directly correspond to the immuno‐cytochemical results demonstrating fluorescein isothiocyanate staining in the respective nerve terminals. Our results imply that ACh, ATP, and proteoglycan are common molecular constituents of motor nerve terminal‐derived synaptic vesicles from Torpedo to rat. In addition to ACh, both ATP and proteoglycan may play a specific role in the process of cholinergic signal transmission.

Localization of SNARE proteins and secretory organelle proteins in astrocytes in vitro and in situ.

Astrocytes are capable of regulated release of messenger molecules. Astrocytes cultured from new born rodent brain express a variety of classical presynaptic proteins. We investigated the question whether the capability to express synaptic proteins in culture was a feature only of immature astrocytes, and whether these proteins were also expressed by astrocytes in situ. Experiments were performed with transgenic mice expressing the enhanced green fluorescent protein under the control of the human glial fibrillary acidic protein promoter. Using double fluorescence and astrocytes cultured from 1 to 16 day-old animals we show that the astrocytic expression of synaptic proteins in culture is invariant of the age of donor animals. Culturing can induce the astrocytic expression of specific synaptic proteins such as SV2, synaptophysin and SNAP-25. Astrocytes in brain sections of 1-16 day-old animals revealed a punctuate immunofluorescence for secretory carrier membrane protein (SCAMP), SNAP-23, synaptobrevin II, and cellubrevin, to a minor extent for SNAP-25 and synaptophysin, and none for SV2. Our results demonstrate that cultured astrocytes express synaptic proteins not present in situ. Nevertheless, astrocytic organelles in situ are equipped with molecules that could be involved in regulated exocytosis of messenger substances.

The hemicholinium-3 sensitive high affinity choline transporter is internalized by clathrin-mediated endocytosis and is present in endosomes and synaptic vesicles.

Synthesis of acetylcholine depends on the plasma membrane uptake of choline by a high affinity choline transporter (CHT1). Choline uptake is regulated by nerve impulses and trafficking of an intracellular pool of CHT1 to the plasma membrane may be important for this regulation. We have generated a hemagglutinin (HA) epitope tagged CHT1 to investigate the organelles involved with intracellular trafficking of this protein. Expression of CHT1‐HA in HEK 293 cells establishes Na+‐dependent, hemicholinium‐3 sensitive high‐affinity choline transport activity. Confocal microscopy reveals that CHT1‐HA is found predominantly in intracellular organelles in three different cell lines. Importantly, CHT1‐HA seems to be continuously cycling between the plasma membrane and endocytic organelles via a constitutive clathrin‐mediated endocytic pathway. In a neuronal cell line, CHT1‐HA colocalizes with the early endocytic marker green fluorescent protein (GFP)‐Rab 5 and with two markers of synaptic‐like vesicles, VAMP‐myc and GFP‐VAChT, suggesting that in cultured cells CHT1 is present mainly in organelles of endocytic origin. Subcellular fractionation and immunoisolation of organelles from rat brain indicate that CHT1 is present in synaptic vesicles. We propose that intracellular CHT1 can be recruited during stimulation to increase choline uptake in nerve terminals.

The synaptic vesicle proteome

Synaptic vesicles are key organelles in neurotransmission. Vesicle integral or membrane‐associated proteins mediate the various functions the organelle fulfills during its life cycle. These include organelle transport, interaction with the nerve terminal cytoskeleton, uptake and storage of low molecular weight constituents, and the regulated interaction with the pre‐synaptic plasma membrane during exo‐ and endocytosis. Within the past two decades, converging work from several laboratories resulted in the molecular and functional characterization of the proteinaceous inventory of the synaptic vesicle compartment. However, up until recently and due to technical difficulties, it was impossible to screen the entire organelle thoroughly. Recent advances in membrane protein identification and mass spectrometry (MS) have dramatically promoted this field. A comparison of different techniques for elucidating the proteinaceous composition of synaptic vesicles revealed numerous overlaps but also remarkable differences in the protein constituents of the synaptic vesicle compartment, indicating that several protein separation techniques in combination with differing MS approaches are required to identify and characterize the synaptic vesicle proteome. This review highlights the power of various gel separation techniques and MS analyses for the characterization of the proteome of highly purified synaptic vesicles. Furthermore, the newly detected protein assignments to synaptic vesicles, especially those proteins which are new to the inventory of the synaptic vesicle proteome, are critically discussed.

The proteome of the presynaptic active zone: from docked synaptic vesicles to adhesion molecules and maxi-channels.

The presynaptic proteome controls neurotransmitter release and the short and long term structural and functional dynamics of the nerve terminal. Using a monoclonal antibody against synaptic vesicle protein 2 we immunopurified a presynaptic compartment containing the active zone with synaptic vesicles docked to the presynaptic plasma membrane as well as elements of the presynaptic cytomatrix. Individual protein bands separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis were subjected to nanoscale‐liquid chromatography electrospray ionization‐tandem mass spectrometry. Combining this method with 2‐dimensional benzyldimethyl‐n‐hexadecylammonium chloride/sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and matrix‐assisted laser desorption ionization time of flight and immunodetection we identified 240 proteins comprising synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery, proteins involved in intracellular signal transduction, a large variety of adhesion molecules and proteins potentially involved in regulating the functional and structural dynamics of the pre‐synapse. Four maxi‐channels, three isoforms of voltage‐dependent anion channels and the tweety homolog 1 were co‐isolated with the docked synaptic vesicles. As revealed by in situ hybridization, tweety homolog 1 reveals a distinct expression pattern in the rodent brain. Our results add novel information to the proteome of the presynaptic active zone and suggest that in particular proteins potentially involved in the short and long term structural modulation of the mature presynaptic compartment deserve further detailed analysis.

Colocalization on the same synaptic vesicles of syntaxin and SNAP-25 with synaptic vesicle proteins: a re-evaluation of functional models required?

Synaptic vesicle docking and calcium dependent exocytosis are thought to require the specific interaction of proteins of the synaptic vesicle membrane (such as VAMP/synaptobrevin and synaptotagmin) and their plasma membrane-located counterparts (such as syntaxin and SNAP-25). When isolating synaptic vesicles by glycerol velocity gradient centrifugation we found cosedimentation of the presumptive presynaptic plasma membrane proteins syntaxin and SNAP-25 with synaptic vesicle membrane proteins. In order to further identify the antibody binding organelles we performed an immunoelectron microscopical analysis of synaptosomal profiles. Syntaxin and SNAP-25 were not only associated with the plasma membrane but to a large extent also with synaptic vesicle profiles. In order to answer the question whether the syntaxin and SNAP-25 containing vesicular compartment would also carry classical synaptic vesicle membrane markers we performed double labeling experiments using poly- and monoclonal antibodies. We found colocalization on the same vesicle not only of SNAP-25 and syntaxin but also of SNAP-25 with the synaptic vesicle membrane proteins SV2 and synaptotagmin and of syntaxin with the vesicular membrane protein synaptophysin. Our results demonstrate that syntaxin and SNAP-25 are colocalized with classical vesicle membrane proteins on the same vesicle and suggest that the functional models for the interaction of presynaptic proteins need to be re-evaluated.

Association of three small GTP-binding proteins with cholinergic synaptic vesicles

Several small (low molecular weight) GTP‐binding proteins are associated with cholinergic synaptic vesicles derived from the electric organ of electric ray. Using GTP overlay techniques and direct micro sequencing we analyzed the association of small GTP‐binding proteins with synaptic vesicles. Both experimental procedures revealed the specific occurrence of multiple small GTP‐binding proteins with this organelle. Moreover, direct amino acid sequence analysis assigned at least three different small GTP‐binding proteins, ora3, o‐ral and o‐rab3, to the vesicular compartment. Furthermore, the data reflect the relative abundance of these three proteins on the vesicle membrane, thereby demonstrating the predominant occurrence of o‐rab3, the only exclusively synaptic vesicle specific small GTP‐binding protein.

Intraneuronal distribution of a synaptic vesicle membrane protein: Antibody binding sites at axonal membrane compartments andtrans-Golgi network and accumulation at nodes of Ranvier

The distribution of a cholinergic synaptic vesicle-specific transmembrane glycoprotein (Buckley and Kelly, 1985, J. Cell Biol. 100, 1284-1294) was investigated in the entire electromotor neuron of Torpedo marmorata using a monoclonal antibody and immunocytochemistry at the light- and electron-microscopical level (immunoperoxidase, colloidal gold). In the nerve, terminal binding of immunogold particles is restricted to synaptic vesicles. In the axon a number of additional membrane compartments like multivesicular bodies, vesiculotubular structures, lamellar bodies and electron-dense granules share the surface located synaptic vesicle-specific transmembrane glycoprotein-epitope. Membranous structures likely to represent the axoplasmic reticulum inside axons and nerve terminals are not labelled. Antibody-binding membrane compartments are accumulated at nodes of Ranvier. In the perikaryon the tubules of the trans-Golgi network as well as multivesicular bodies, lamellar bodies, electron-lucent vesicles, granules with electron-dense core and peroxisomes are labelled. Immunotransfer blots of isolated synaptic vesicles and tissue extracts of electric organ display a 100,000 mol. wt band of broad electrophoretic mobility typical of the synaptic vesicle-specific transmembrane glycoprotein. Extracts of electromotor nerve and electric lobe contain in addition a strong band at 85,000 mol. wt and a few lower molecular weight bands. We suggest that the synaptic vesicle originates directly from the trans-Golgi network. The endoplasmic reticulum is not involved in vesicle formation or retrieval. On retrograde transport the vesicle membrane compartment is likely to fuse with other intra-axonal (endosomal?) organelles.