Berlin, Freie Univ., Diss., 2001. / Dateiformat: zip, Dateien im PDF-Format. Computerdatei im Fernzugriff.

Berlin, Freie Univ., Diss., 2001. / Dateiformat: zip, Dateien im PDF-Format. Computerdatei im Fernzugriff.

Berlin, Freie Universiẗat, Diss., 2001. / Dateiformat: zip, Dateien im PDF-Format.

Neurotransmission is essential for neuronal communication. At the presynapse, synaptic vesicles (SVs) undergo exocytosis to release neurotransmitter in response to incoming action potentials, and endocytosis to maintain the supply of SVs needed for further rounds of exocytosis. A key event during SV endocytosis is the efficient sorting and localisation of SV proteins at the plasma membrane. This ensures that nascent SVs that are formed have the correct molecular composition to participate in subsequent exocytic events. The sorting of SV proteins at the plasma membrane is usually facilitated by adaptor proteins (e.g. AP-2) which recognise binding motifs present on key SV proteins and facilitate their internalisation during endocytosis. In addition to this, certain SV proteins possess the ability to chaperone each other as part of an endocytic transport complex throughout the SV recycling process. In conjunction with AP-2-facilitated sorting, the transport of complexed SV proteins during endocytosis provides further mechanistic insight into how SVs are generated with consistent high fidelity for functional viability. Using pHluorins as a tool to visualise SV protein trafficking in hippocampal cultures, the relationship between two key SV proteins, synaptic vesicle protein 2A (SV2A) and synaptotagmin I (SYT1), was investigated. SYT1 predominantly acts as the Ca2+ sensor for fast synchronous release at the presynapse, whilst the exact function of SV2A remains unknown to this day. In this study, the ablation of the AP-2 binding site in SV2A (Y46A) resulted in increased SYT1 surface expression and accelerated SYT1 retrieval compared to WT SV2A. No additive defects were observed when a second point mutation (T84A) was introduced to SV2A that disrupts the phosphorylation-dependent interaction between SV2A and SYT1, thus confirming that SYT1 localisation and retrieval is dependent on normal SV2A retrieval by AP-2. The hypothesis that disruption of the SV2A-SYT1 interaction may provide an underlying mechanism for motor onset seizures in epilepsy was also investigated. An epilepsy-related mutation (R383Q) in SV2A also resulted in increased SYT1 surface expression and accelerated SYT1 retrieval mirroring the defects caused by the Y46A mutation. Introduction of Y46A or T84A mutation into SV2A R383Q resulted in no additive defects compared to the single mutant, suggesting that the observed defects in SYT1 localisation and retrieval kinetics in the epilepsy-related mutant may be caused by the ablation of normal SV2A internalisation. GST pulldown assays, mass spectrometry and western blotting data indicate that presence of the mutation disrupts normal binding of the SV2A cytosolic loop with actin, tubulin and certain subunits of V-ATPase. Finally, a link between SV2A-dependent presynaptic dysfunction and epilepsy was examined through studies utilising the anti-epileptic drug, levetiracetam (LEV). SV2A contains a binding site for LEV, suggesting that it may act as a carrier for the drug into the presynapse. Hippocampal neuronal cultures were treated with LEV at various concentrations in the presence of specific patterns of neuronal activity. No observed effects of the drug on synaptophysin, vesicular glutamate transporter 1 (VGLUT1) and SYT1 recycling were observed, suggesting that LEV is unlikely to function as a modulator of excitatory presynaptic activity or by influencing SV2A function. In conclusion, this work demonstrates that SV2A is essential for accurate SYT1 trafficking and a link has been established between defective SV2A internalisation and subsequent downstream effects on SYT1 localisation and retrieval during SV recycling.

Aktive Zonen (AZs) sind hoch spezialisierte, subzelluläre Kompartimente von Neuronen, die der synaptischen Übertragung dienen. Sie enthalten Gerüstproteine wie RIM (Rab3 interacting molecule) sowie elektronendichte Projektionen bestehend aus Bruchpilot bei Drosophila melanogaster oder Bassoon im Säuger, welche Schlüsselkomponenten des Vesikelverkehrs darstellen. Bei der Fliege sind Anzahl und Verteilung von Bruchpilot-Molekülen in AZs relevant für die funktionelle Differenzierung. Ihre Anordnung wird im Abstand von weniger als einem Mikrometer innerhalb einer präsynaptischen Endigung reguliert. Im Rahmen der vorliegenden Arbeit wurden elektrophysiologische Ableitungen und konfokale sowie höchstauflösende, immunhistochemische Bildgebung mit dem dSTORM (direct Stochastic Optical Reconstruction Microscopy) Verfahren an larvalen, neuromuskulären Synapsen von Drosophila durchgeführt. Dabei wurde das genetische Potenzial des Modellorganismus genutzt, um relevante Proteinfunktionen und -interaktionen zu analysieren. RIM als zentrale Komponente Aktiver Zonen ist relevant für synaptische Plastizität. Eine als CORD7 (cone-rod dystrophy type 7) bezeichnete Punktmutation (Arginin zu Histidin) innerhalb der 310 Helix der C2A-Domäne von RIM wurde mit erhöhten kognitiven Fähigkeiten einer Patientengruppe in Verbindung gebracht. Weil die Drosophila C2A-Domäne eine hohe Homologie zur Säugerdomäne aufweist, konnte der Einfluss dieser Mutation auf Struktur und Funktion von Synapsen untersucht werden. Es zeigte sich, dass der Aminosäureaustausch der CORD7-Position und des benachbarten Arginin-Restes die synaptische Organisation und Transmission beeinflussen. In einer Reihe weiterer Experimente wurde das Zusammenspiel von Bruchpilot und Synaptotagmin, dem Calciumsensor der evozierten Transmitterfreisetzung, analysiert. Während AZs ohne Bruchpilot auch ohne Synaptotagmin funktionieren, führt dessen Reduktion zu einer Umverteilung von Bruchpilot-Molekülen innerhalb von AZs und zu dramatischen Änderungen in ihrer Anzahl. Abschließend wurde so ein Beitrag zum Verständnis der molekularen Organisation synaptischer Informationsverarbeitung und Plastizität geleistet, wobei zu klären bleibt, wie die zuverlässige Speicherung von Informationen an AZs erreicht werden kann. / Vesicle cycle in active zones

The microfilament system consists of actin filaments as the major component and is regulated by a number of actin binding proteins. It is juxtaposed to the plasma membrane where it forms a dense cortical weave from where it pervades into the cell interior. This filament system has multiple roles and participates in virtually all motile processes where its dynamic activities depend on receptor mediated signaling leading to constant polymerizations and depolymerizations. These activities are now also known to affect gene regulation. This thesis discusses these dynamic reorganizations of the microfilament system and how components are supplied to support these motile processes. The focus is on profilin/profilin:actin, actin polymerization and the localization of the transcripts of these proteins. The localization of profilin mRNA was examined in relation to the distribution of β-actin mRNA using fluorescent in situ hybridization. It was concluded that both these mRNAs localize to sites of massive actin polymerization called dorsal ruffles albeit the data obtained suggests that this localization must be dependent on distinct mechanisms. Additionally signal transduction and cell motility was studied after depleting the two profilin isoforms 1 and 2. The activity of the transcription factor SRF is known to be coupled to microfilament system dynamics via the cofactor MAL which binds to actin monomers and is released upon receptor mediated actin polymerization. Depletion of profilin was seen to influence SRF dependent signaling, most likely because the lack of profilin enables more MAL to bind actin monomers thereby preventing SRF dependent transcription. Finally, it was also investigated how the synaptic vesicle protein synaptotagmin 1 which is involved in exocytosis, has a role in actin polymerization. This protein has previously been described to cause filopodia formation when ectopically expressed. A polybasic sequence motif was identified as the effector sequence for this activity and it was established that this sequence interacts with anionic lipids. It is also discussed how this sequence could have a role in neurotransmitter release and actin polymerization in the nerve synapse. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Submitted.

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