In der synaptischen Vesikelmembran adulter Neuronen bildet Synaptobrevin mit Synaptophysin den Synaptophysin-Synaptobrevin-Komplex. Der Komplex wird im Gegensatz zum SNARE-Komplex nicht in embryonalen Membranen gebildet, sondern erst während der neuronalen Entwicklung hochreguliert. Dabei erfährt Synaptophysin wahrscheinlich eine posttranslationale Modifizierung, die durch einen niedermolekularen Faktor bewirkt wird. Der Synaptophysin-Synaptobrevin-Komplex spielt eine entscheidende Rolle innerhalb der Präsynapse bei der Bereitstellung von Synaptobrevin zur Bindung seiner SNARE-Partner an der Plasmamembran. Im Zustand erhöhter exozytotischer Aktivität der Synapse beschleunigt der Synaptophysin-Synaptobrevin-Komplex die Rekrutierung von Synaptobrevin für eine erneute Bildung des SNARE-Komplexes und ermöglicht damit schnelle Exozytose-Endozytose-Zyklen bei erhöhter präsynaptischer Stimulation. Der Synaptophysin-Synaptobrevin-Komplex und der SNARE-Komplex schließen sich gegenseitig aus. Synaptische Membranen sind aus Lipiden und Proteinen aufgebaut, welche miteinander in Wechselwirkungen stehen. Innerhalb der Membranen formieren sich Subdomänen wie Lipid Rafts die durch eine besondere Lipidzusammensetzung stabilisiert werden und mit speziellen Proteinen bevorzugt assoziieren. Durch die spezielle Organisation des Membranaufbaus können die Prozesse der Endozytose und der Exozytose zum Teil reguliert werden. Synaptische Vesikel, die zu den kleinsten Zellorganellen zählen, zeigen einen besonders hohen membranären Cholesterolgehalt. Synaptophysin ist ein integrales Membranprotein synaptischer Vesikel und konnte zusätzlich als spezifisch cholesterolbindendes Protein identifiziert werden. Durch die Assoziation mit cholesterolreichen Nanodomänen der synaptischen Vesikelmembran könnten die Funktionen von Synaptophysin bei der Membranstabilisation und im Synaptophysin-Synaptobrevin-Komplex der synaptischen Vesikelmembran beeinflusst werden. In dieser Arbeit wurde gezeigt, dass nach Cholesterolverminderung der Membranen von CHOp38-Zellen und PC12-Zellen mittels Filipin und Methyl-ß-cyclodextrin Synaptophysin in dem Detergens Triton X-100 unlöslicher wird. Die Cholesterolverminderung der Membranen von Neuronen aus Rattengehirngewebe und Hippokampuskulturen mittels Methyl-ß-cyclodextrin und Lovastatin führte weiterhin zu einer verminderten Bildung des Synaptophysin-Synaptobrevin-Komplexes in der synaptischen Vesikelmembran. Somit scheint die Cholesterolassoziation von Synaptophysin und damit die Organisation der synaptischen Vesikelproteine innerhalb von Membrandomänen entscheidend an der Regulation der Proteininteraktionen im Synaptophysin-Synaptobrevin-Komplex beteiligt zu sein. Zusätzlich trägt Synaptophysin durch seine Cholesterolbindung wahrscheinlich zur Stabilisierung des hohen Krümmungsgrades der Membran der synaptischen Vesikel bei. Die Auswirkungen des verminderten Cholesterolgehaltes auf Synaptophysin und den Synaptophysin-Synaptobrevin-Komplex konnten auch bei homozygoten Mausmutanten für die Niemann-Pick Krankheit nachgewiesen werden. Der Cholesterolgehalt synaptischer Vesikel ist also für die Bildung des Synaptophysin-Synaptobrevin-Komplexes entscheidend und beeinflusst direkt die synaptische Effizienz. / Synaptobrevin interacts with synaptophysin in membranes of adult small synaptic vesicles and forms the synaptophysin/synaptobrevin complex. In contrast to the SNARE complex the synaptophysin/synaptobrevin complex only occurs in adult rat brain but is absent in embryonic brain. Changes in the binding properties of synaptophysin are probably induced by a factor of low molecular weight and correlate with posttranslational modifications of the protein. The synaptophysin/synaptobrevin complex plays an important role within the presynaptic terminal promoting synaptobrevin to bind its SNARE partners at the plasma membrane. In times of increased synaptic activity at the synapse the synaptophysin/synaptobrevin complex accelerates the recruitment of synaptobrevin to form new SNARE complexes and allows for fast exocytotic/endocytotic cycles. The synaptophysin/synaptobrevin complex and the SNARE complex are mutually exclusive. Major constituents of synaptic membranes are lipids and proteins which are subjected to continuous interactions. Within the membrane form specialized environments known as lipid rafts that are stabilized through tightly packed lipids and proteins that associate preferentially with these domains. The characteristic organisation of membrane structures is crucial for regulating the process of endocytosis and exocytosis. Synaptic vesicles are among the smallest cell organelles and are especially enriched in cholesterol. The integral membrane protein synaptophysin in addition was identified as a major specifically cholesterol-binding protein. Lateral association with cholesterol enriched subunits of the synaptic vesicle membrane may contribute to mediate the functions of synaptophysin in stabilising membrane structures and may in part regulate synaptophysin/synaptobrevin complex formation. Here we show that depletion of the cholesterol content of CHOp38 cell and PC12 cell membranes by Filipin and Methyl-ß-cyclodextrin significantly changes the solubility of synaptophysin in non-ionic detergents like Triton X-100. After cholesterol depletion of adult rat brain and primary cultures of mouse hippocampus by Methyl-ß-cyclodextrin and the HMGCoA-reductase inhibitor Lovastatin the synaptophysin/synaptobrevin complex was seen to be downregulated. Thus, the synaptophysin/synaptobrevin interaction critically depends on high cholesterol content of the synaptic vesicle membrane. Thereby, synaptophysin likely contributes to stabilise the high membrane curvature of synaptic vesicles. The effects of cholesterol depletion on functional properties of synaptophysin and the synaptophysin/synaptobrevin complex could also be shown on homozygous littermates of the mouse model of Niemann-Pick type C disease. Our investigation indicates that the cholesterol content of synaptic vesicles appears to be important for the fusion of the synaptophysin/synaptobrevin complex and directly affects synaptic efficiency.

Ziel der vorliegenden Arbeit war es, die Rolle der synaptischen Vesikelproteine Synaptophysin und Synaptobrevin bei Wachstum und Differenzierung der primär dissoziierten hippocampalen Zellkultur 17 Tage alter Mäuseembryonen zu untersuchen. Besonderes Interesse galt dabei dem Fortsatzwachstum und der Synaptogenese. Durch Beobachtung von Zellmorphologie und Zellwachstum wurde gezeigt, daß die Ausbildung charakteristischer Zelltypen ein intrinsischer Prozeß der hippocampalen Neurone ist. Die Synaptogenese wurde durch die Darstellung der entwicklungsabhängigen Verteilung der synaptischen Vesikelproteine Synaptophysin und Synaptobrevin mittels Immunfluoreszenzmarkierung dokumentiert. Durch elektronenmikroskopische Aufnahmen wurde die Differenzierung subzellulärer Strukturen der hippocampalen Zellkultur charakterisiert und die Verteilung von Synaptophysin und Synaptobrevin entwicklungsabhängig dargestellt. Synaptophysin fand sich nicht nur in axonalen Präsynapsen, sondern auch in Dendriten. Synaptobrevin war im Gegensatz zu Synaptophysin nicht in allen synaptischen Vesikeln der Axonterminalen darstellbar. Um die Wirkungen von Synaptophysin und Synaptobrevin auf hippocampale Neurone in der Kultur genauer zu untersuchen, standen zwei Versuchsmodelle zur Verfügung: 1. die Synaptophysin-defiziente Maus und 2. das clostridiale Neurotoxin Tetanustoxin (TeNT), das Synaptobrevin spezifisch spaltet. Anhand der Depletion von Synaptophysin wurde untersucht, ob in vitro das Fehlen des Proteins Konsequenzen in Bezug auf Synapsenbildung und morphologisches Erscheinungsbild der Neurone hat. Es konnten lichtmikroskopisch und auch im elektronenmikroskopischen Bild keine Unterschiede zu Kontrollkulturen festgestellt werden. Durch morphometrische Messungen zeigte sich, daß in der Kultur der Synaptophysin-depletierten Maus nach 2 DIV mit hoher Signifikanz die Dendriten länger als in Kontrollkulturen waren. Dies spricht für regulatorische Funktionen des Proteins bei Exo- und Endozytosevorgängen während des dendritischen Wachstums. Nach Zugabe von Tetanustoxin zur pränatalen Zellkultur konnte gezeigt werden, daß die Inaktivierung von Synaptobrevin durch TeNT in vitro keine Konsequenzen in Bezug auf das morphologische Erscheinungsbild der Neurone, die Synapsenbildung und das Wachstumsverhalten hat. Mit morphometrischen Messungen konnten für TeTx-behandelte Neurone keine hochsignifikanten Unterschiede zu Kontrollkulturen festgestellt werden. Synaptobrevin scheint also sowohl beim Axon- als auch beim Dendritenwachstum keine essentielle Rolle zu spielen. / Goal of this work was to investigate the role of the synaptic vesicle proteins synaptophysin and synaptobrevin during development and differentiation of mouse fetal hippocampal neurons in primary culture. The outgrowth of dendritic and axonal fibers and the mechanisms of synaptogenesis were of special interest. Observation of morphology and development showed that generation of characteristic cell types is an intrinsic process of hippocampal neurons. Differentiation of cellular and subcellular structures in hippocampal cell culture, characteristics of synaptogenesis and the stage-dependent distribution of synaptophysin and synaptobrevin were demonstrated by immunofluorescence and electron microscopy. Synaptophysin was localized in presynaptic axon terminals but also in dendrites. In contrast with synaptophysin immunoreaction for synaptobrevin could not be found in all synaptic vesicles of the presynaptic axon terminal. To investigate the influence of synaptophysin and synaptobrevin on hippocampal neurons in culture two experimental models were used, first a synaptophysin-knock-out-mouse and second the clostridial neurotoxin tetanustoxin (TeNT) which selectively cleaves synaptobrevin. With the depletion of synaptophysin we investigated if there are consequences in development of synapses and morphological features of the neurons in vitro. In both, light and electron microscopy, there was no difference to control cultures. In morphometric measurements there was a significant difference in the lenght of developing dendrites after 2 DIV with longer dendrites in the synaptophysin-knock-out-mouse. Therefore synaptophysin may have a regulatory function for exo-/ endocytosis during dendritic growth. Specific inactivation of synaptobrevin by tetanustoxin had no consequences in morphological features, synaptogenesis and growing of the hippoacampal neurons in vitro. Morphometric measurements showed no significant differences between TeNT and control. Therefore we conclude that synaptobrevin has no essential function during axon growth or dendrite elongation.

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Synaptic transmission requires the fusion of neurotransmitter containing vesicles with the neuron's plasma membrane in a temporally restrictive manner. In Drosophila, this challenge is accomplished in part by the SNARE protein neuronal-Synaptobrevin (n-Syb). The juxtamembrane region of this molecule, linking the cytosolic SNARE motif and transmembrane region, is hypothesized to play a functional role in facilitating membrane fusion. This short, 10 amino acid, segment contains numerous charged residues and one conserved tryptophan residue. Its short rigid structure may be important in transducing force during SNARE complex assembly. Tryptophan residues, common in membrane proteins, are often observed at the membrane-water interface. It was hypothesized that this conserved tryptophan residue was important for anchoring and positioning n-Syb in the membrane. Proteins produced with tryptophan mutated were tested for anchoring and stability in a membrane model using NMR spectroscopy. Experiments testing depth of insertion using exposure to oxygen, a paramagnetic species, and exchange with deuterium demonstrated that tryptophan anchored n-Syb in the membrane. To test a potential functional role for the juxtamembrane region of n-Syb in synaptic transmission, a reverse genetic approach was employed. Wild-type and mutant P-element clones were made using the genomic sequence of n-syb including the endogenous promoter. n-Syb was found to be expressed, integrate and orient correctly in the membrane of Drosophila S2 cells. Transgenic Drosophila, produced via P-element transformation, were also found to produce transgenic protein. Transgenic expression of wild-type n-syb was found to restore an n-syb hypomorphic mutant from severe motor impairment and limited lifespan to wild-type levels. Synaptic transmission was assessed in 3rd instar larval preparations of mutant and wild-type transgenics. Mutation of the tryptophan residue and insertion of a short flexible linker were both found to inhibit synaptic transmission, while insertion of a long flexible linker was not.

Die integralen Vesikelmembranproteine Synaptophysin und Synaptobrevin interagieren in adulten Neuronen. Zusätzlich bildet Synaptobrevin mit den Plasmamembranproteinen Syntaxin und synaptosome-associated protein 25kDa (SNAP25) den SNAP-Rezeptor (SNARE)-Proteinkomplex, der Voraussetzung für die Fusion zwischen synaptischen Vesikeln und präsynaptischer Membran ist. Mit Synaptophysin interagierendes Synaptobrevin bindet jedoch nicht an den SNARE-Proteinen. Es wird daher vermutet, dass der Synaptophysin/Synaptobrevin-Komplex eine Art Reservepool für Synaptobrevin bei erhöhter neuronaler Aktivität darstellt und die Verfügbarkeit von Synaptobrevin während der Exozytose reguliert. Mit verschiedenen Ansätzen wurde versucht, den auf dem Vesikel befindlichen Komplex genauer zu charakterisieren und in seiner Funktion näher zu beschreiben. Nach Stimulation mit exozytosevermittelnden Substanzen dissoziierte der Synaptophysin/ Synaptobrevin-Komplex, sowohl unter nativen Bedingungen als auch bei Blockierung des finalen Fusionsereignisses. Dieser Prozess war calciumabhängig, konnte jedoch nicht durch die direkte Wirkung von Calcium ausgelöst werden. Die Untersuchung des Komplexes mit Hilfe von clostridialen Neurotoxinen zeigte, dass Synaptobrevin bevorzugt in Bindung an Synaptophysin und als Dimer gespalten wurde. Die Spaltung des SNARE-Proteins SNAP25 hatte keinen Einfluss auf die Komplexbildung. Die Verringerung des Cholesterolgehaltes der Membran führte zur Abnahme der Interaktion von Synaptophysin und Synaptobrevin, umgekehrt zeigte sich ein Anstieg bei zusätzlicher Cholesterolapplikation. In weiteren Experimenten konnte der C-terminale Teil des Synaptobrevins als für die Bindung zu Synaptophysin entscheidende Abschnitt identifiziert werden. Weiterhin konnte die erfolgreiche Translokation von rekombinanten Konstrukten aus Botulinumtoxin D und einem angekoppelten funktionstüchtigen Protein ins Zytosol gezeigt werden. / The vesicle associated membrane proteins synaptophysin and synaptobrevin interact in ma-ture neurones. Additionally synaptobrevin forms a complex with the plasma membrane pro-teins syntaxin and synaptosome-associated protein 25kDa (SNAP25), better known as the SNAP-Receptor (SNARE) complex, which is a prerequisite for fusion of the presynaptic and vesicle membranes. These two protein complexes however are mutually exclusive. It is as-sumed that the synaptophysin/synaptobrevin complex resembles a reserve pool for synapto-brevin and regulates the availability of synaptobrevin for the fusion process in case of in-creased synaptic activity. Different approaches where chosen to characterize this protein complex and to examine its function in more detail. After excessive stimulation the synaptophysin/synaptobrevin complex dissociates, even when the final fusion process is blocked. This step was dependent on the presence of cal-cium, though it could not be triggered directly by calcium administration. When using clos-tridial neurotoxins, synaptobrevin was preferentially cleaved in its homodimeric form and in the complex with synaptophysin. Cleavage of SNAP25 had no effect on the complex forma-tion. Depletion of cholesterol content decreases the interaction of synaptophysin with synap-tobrevin, while cholesterol treatment increases interaction. Further experiments indicated that synaptophysin binds to the the carboxy-terminal transmembrane part of synaptobrevin. Fur-thermore it could be shown that proteins attached to botulinum toxin can be delivered to the cytosol of neuronal cells, being fully active.

Synaptic transmission requires the fusion of neurotransmitter containing vesicles with the neuron's plasma membrane in a temporally restrictive manner. In Drosophila, this challenge is accomplished in part by the SNARE protein neuronal-Synaptobrevin (n-Syb). The juxtamembrane region of this molecule, linking the cytosolic SNARE motif and transmembrane region, is hypothesized to play a functional role in facilitating membrane fusion. This short, 10 amino acid, segment contains numerous charged residues and one conserved tryptophan residue. Its short rigid structure may be important in transducing force during SNARE complex assembly. Tryptophan residues, common in membrane proteins, are often observed at the membrane-water interface. It was hypothesized that this conserved tryptophan residue was important for anchoring and positioning n-Syb in the membrane. Proteins produced with tryptophan mutated were tested for anchoring and stability in a membrane model using NMR spectroscopy. Experiments testing depth of insertion using exposure to oxygen, a paramagnetic species, and exchange with deuterium demonstrated that tryptophan anchored n-Syb in the membrane. To test a potential functional role for the juxtamembrane region of n-Syb in synaptic transmission, a reverse genetic approach was employed. Wild-type and mutant P-element clones were made using the genomic sequence of n-syb including the endogenous promoter. n-Syb was found to be expressed, integrate and orient correctly in the membrane of Drosophila S2 cells. Transgenic Drosophila, produced via P-element transformation, were also found to produce transgenic protein. Transgenic expression of wild-type n-syb was found to restore an n-syb hypomorphic mutant from severe motor impairment and limited lifespan to wild-type levels. Synaptic transmission was assessed in 3rd instar larval preparations of mutant and wild-type transgenics. Mutation of the tryptophan residue and insertion of a short flexible linker were both found to inhibit synaptic transmission, while insertion of a long flexible linker was not.

Synapses vary widely in the probability of transmitter release. For instance, in response to an action potential the phasic synapses of the crayfish have a 100-1000-fold higher release probability than tonic synapses. The difference in release probability is attributed to differences in the exocytotic machinery such as the degree of “zippering” of the trans-SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) complex. I used physiological and molecular approaches to determine if the zippered state of SNAREs associated with synaptic vesicles and the interaction between the SNARE complex and Complexin influence the probability of release at the synapse. I used three Botulinum neurotoxins which bind and cleave at different sites on VAMP to determine whether these sites were occluded by SNARE interaction (zippering) or open to proteolytic attack. Under low stimulation conditions, the light-chain fragment of botulinum B (BoNT/B-LC) but not BoNT/D-LC or tetanus neurotoxin (TeNT-LC) cleaved VAMP and inhibited evoked release at both phasic and tonic synapses. In addition, a peptide based on the C-terminal half of crayfish VAMP’s SNARE motif (Vc peptide) designed to interfere with SNARE complex zippering at the C-terminal end inhibited release at both synapses. The susceptibility of VAMP to only BoNT/B-LC and interference by the Vc peptide indicated that SNARE complexes at both phasic and tonic synapses were partially zippered only at the N-terminal end with the C-terminal end exposed under resting conditions. I used a peptide containing part of the crayfish Complexin central α-helix domain to interfere with the interaction between Complexin and the SNARE complex. The peptide enhanced phasic evoked release and inhibited tonic evoked release under low stimulation but attenuated release at both synapses under intense stimulation. Therefore, Complexin appeared to exhibit a dual function under low synaptic activity but only promoted release under high synaptic activity. The results showed that the zippered state of the SNARE complex does not determine initial release probability as a similar zippered SNARE complex structure under resting conditions is common to both phasic and tonic synapses. However, Complexin may have a role in influencing the initial release probability of a synapse. Therefore, the interaction between the SNARE complex and Complexin is important for release but other factors contribute more significantly to synaptic strength.

Synapses vary widely in the probability of transmitter release. For instance, in response to an action potential the phasic synapses of the crayfish have a 100-1000-fold higher release probability than tonic synapses. The difference in release probability is attributed to differences in the exocytotic machinery such as the degree of “zippering” of the trans-SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) complex. I used physiological and molecular approaches to determine if the zippered state of SNAREs associated with synaptic vesicles and the interaction between the SNARE complex and Complexin influence the probability of release at the synapse. I used three Botulinum neurotoxins which bind and cleave at different sites on VAMP to determine whether these sites were occluded by SNARE interaction (zippering) or open to proteolytic attack. Under low stimulation conditions, the light-chain fragment of botulinum B (BoNT/B-LC) but not BoNT/D-LC or tetanus neurotoxin (TeNT-LC) cleaved VAMP and inhibited evoked release at both phasic and tonic synapses. In addition, a peptide based on the C-terminal half of crayfish VAMP’s SNARE motif (Vc peptide) designed to interfere with SNARE complex zippering at the C-terminal end inhibited release at both synapses. The susceptibility of VAMP to only BoNT/B-LC and interference by the Vc peptide indicated that SNARE complexes at both phasic and tonic synapses were partially zippered only at the N-terminal end with the C-terminal end exposed under resting conditions. I used a peptide containing part of the crayfish Complexin central α-helix domain to interfere with the interaction between Complexin and the SNARE complex. The peptide enhanced phasic evoked release and inhibited tonic evoked release under low stimulation but attenuated release at both synapses under intense stimulation. Therefore, Complexin appeared to exhibit a dual function under low synaptic activity but only promoted release under high synaptic activity. The results showed that the zippered state of the SNARE complex does not determine initial release probability as a similar zippered SNARE complex structure under resting conditions is common to both phasic and tonic synapses. However, Complexin may have a role in influencing the initial release probability of a synapse. Therefore, the interaction between the SNARE complex and Complexin is important for release but other factors contribute more significantly to synaptic strength.