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dc.contributor.advisorCousin, Mike
dc.contributor.advisorWiegand, Ulrich
dc.contributor.authorLow, Darryl Weijun
dc.date.accessioned2018-07-16T10:21:32Z
dc.date.available2018-07-16T10:21:32Z
dc.date.issued2018-07-10
dc.identifier.urihttp://hdl.handle.net/1842/31320
dc.description.abstractNeurotransmission 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.en
dc.contributor.sponsorMedical Research Council (MRC)en
dc.language.isoenen
dc.publisherThe University of Edinburghen
dc.subjectSV2Aen
dc.subjectsynaptotagminen
dc.subjectsynaptic vesicleen
dc.titleSynaptic vesicle protein 2A-dependent function and dysfunction at the presynapseen
dc.typeThesis or Dissertationen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD Doctor of Philosophyen


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