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.

Synaptophysin (Syp) is a major integral synaptic vesicle (SV) protein; there are 31 copies of Syp per vesicle, which totals up to 10% of the total SV protein content. Despite being the major SV protein, little is known about the interaction partners of Syp and as a result there has been no clear role attributed to it. One key feature of Syp is that its cytoplasmic C-terminus contains 10 pentapeptide repeats, nine of which are initiated by a tyrosine residue. Syp is the major tyrosine phospho-protein on SVs. The kinase thought to phosphorylate Syp in vivo is the ubiquitously expressed non-receptor kinase C-Src. There are two splice variants of C-Src, N1- and N2-Src, which are only expressed in neuronal tissues. Although the 3 Srcs are structurally similar, they differ by a small insert of amino acids into their SH3 domains (the N-Src loop). Examination of the amino acid sequence of the cytosolic C-terminus of Syp revealed a putative type one SH3 domain interaction motif. A screen using SH3 domains of synaptic proteins as bait in GST-pull downs from nerve terminal lysate allowed an inventory of potential interaction partners of Syp to be created. Reciprocal experiments using the C-terminal of Syp as bait confirmed many of these interactions. Single point mutations of the SH3 interaction motif on Syp highlighted that syndapin and C-Src bound to Syp via this motif. These binding mutants were inserted in Syp superecliptic synaptophluorin (SypHy) to determine the functional consequences of these interactions. These mutants did not affect the trafficking of Syp when expressed in cortical neurons derived from Syp knockout mice. However, the SH3 interaction motif was fundamental for the retrieval of VAMP (vesicle associated membrane protein) when expressed in Syp knockout cultures. Importantly, this role is not mediated through a direct interaction with VAMP with the SH3 interaction motif implicating either syndapin, C-Src or both in Syp-dependent VAMP retrieval. The 3 different Srcs had different methods of interaction with Syp, and in vitro protein kinase assays the ability of the three Src splice variants to phosphorylate Syp was assessed. Key differences in both speed and efficiency of Syp phosphorylation was observed for the different Src splice variants. Mutagenesis of either all 9 tyrosine residues, only previously identified sites resulted in changes in Syp interactions in GST-pull down assays from nerve terminal lysates. To investigate the role of Syp phosphorylation in the SV lifecycle, the tyrosine pentapeptide repeats were truncated from the C-terminal of Syp in both a mCerulean tagged Syp and SypHy. The experiments showed that these potential tyrosine phosphorylation sites were not involved in the trafficking of Syp but key in the retrieval of VAMP from the plasma membrane during the SV lifecycle. I have indentified an SH3 interaction motif on the C-terminal of Syp that is critical in forming a complex of proteins that are responsible for the retrieval of VAMP during the SV lifecycle. Further experiments have shown that this key interaction is potentially phosphorylation dependent. My preliminary mass spectrometry analysis has provided a catalogue of proteins that can potentially interact with Syp, identifying proteins that may bind to either the Syp C-terminus SH3 interaction motif or to other regions in a phosphorylation dependent manner. This has provided a list of potential candidate proteins for the VAMP retrieval complex.

The crayfish claw opener neuromuscular junction (NMJ) is a biological model for studying presynaptic neuromodulation by serotonin and synaptic vesicle recycling. Serotonin acts on crayfish axon terminals to increase the release of the neurotransmitter glutamate, but a complete understanding of its mechanisms of action are unknown. In order to sustain enhanced neurotransmission over long periods of time, it was hypothesized that serotonin recruits (activates) a population of previously non-recycling vesicles to become releasable and contribute to neurotransmission. To determine if serotonin activates a distinct population of synaptic vesicles, FM1-43 fluorescence unloading experiments were performed on crayfish excitatory opener axon terminals. These experiments could not resolve a serotonin-activated population of synaptic vesicles, but instead revealed that synaptic vesicles change behaviour in axon terminals independent of serotonin, with vesicles becoming less likely to exocytose and unload FM1-43 dye over time. The change in behaviour was hypothesized to be due to conversion of vesicles from a recycling (releasable) status to a reserve (reluctant to release) status. Synaptic vesicle pool localization was then tested using photoconversion of FM1-43 and transmission electron microscopy techniques. The spatial location of FM1-43-labeled vesicles fixed 2 minutes following 20 Hz stimulation did not reveal retention of vesicles specifically near release sites and the distribution of FM1-43-labeled vesicles was not significantly different between early (2 min) and late (180 min) time points. Terminals fixed 30 seconds following stimulation, however, contained numerous endosome-like structures - the most frequently observed structures resembled large vesicles, which were the equivalent of 2-5 regular vesicle sizes. These results suggest that crayfish axon terminals recycle vast amounts of membrane in response to sustained 20-Hz stimulation and endocytosis appears to occur via multiple routes with the most common being through large vesicle intermediates. / Graduate

博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University

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The development of the central nervous system is dysregulated in neurodevelopmental disorders such as intellectual disability, autism spectrum disorder, and epilepsy. These three disorders have different clinical features, yet there is high comorbidity between them. They can be difficult to study due to their highly complex aetiologies, however there are various monogenic diseases that can cause all of them, including SYNGAP1 haploinsufficiency where the synaptic guanosine triphosphatase (GTPase)-activating protein (SYNGAP) protein levels are highly reduced; Fragile X syndrome where the fragile X mental retardation protein (FMRP) is no longer translated; and DNM1 epileptic encephalopathy where mutations in the Dynamin1 gene alter the protein function. These monogenic conditions are synaptopathies as the proteins affected play important roles in synapse stability and neurotransmission. Because of the high comorbidity between these disorders, it is hypothesised that there may be a common mechanism underlying them. We hypothesise that a deficit in presynaptic vesicle recycling may be part of a common mechanism underlying intellectual disability, autism spectrum disorder, and epilepsy especially in SYNGAP1 haploinsufficiency, Fragile X syndrome, and DNM1 epileptic encephalopathy. Using various fluorescent presynaptic activity reporters including synaptic pHluorins, tetramethylrhodamine dextran and calcium dyes to compare presynaptic activity in in vitro models of these monogenic conditions, we found differences in synaptic vesicle (SV) endocytosis in the genetically altered conditions compared to wildtype controls. We observed various SV endocytosis defects in clathrin-mediated endocytosis (CME) or activity-dependent bulk endocytosis (ADBE) in our models. We observed enhanced CME in SynGAP1 KO mouse hippocampal neurons. This enhanced SV endocytosis was accompanied by decreased SV cargo on the plasma membrane. Rat SynGAP1 KO hippocampal neurons did not display enhanced SV endocytosis, nor did neurons with the GTPase-activating (GAP) domain of SynGAP deleted. This was perhaps due to the altered time course of development between these rodent species. In mouse and rat models of Fragile X syndrome, CME was not altered compared to wildtype controls. However, in a rat model, we observed fewer nerve terminals undergoing ADBE which is the dominant SV endocytosis mode during elevated neuronal activity. De novo epileptic encephalopathy-associated mutations in DNM1 had differential effects on SV recycling through both CME and ADBE. Mouse hippocampal neurons overexpressing Dyn1R237W, Dyn1I289F and Dyn1H396D all showed less CME compared to overexpression of Dyn1WT. Moreover, fewer nerve terminals overexpressing Dyn1H396D were found to undergo ADBE. We also found that a large-conductance potassium (BK) channel opener can accelerate clathrin-mediated endocytosis and thus may be able to rescue the impaired SV endocytosis caused by these mutants. Although there is not yet a common underlying pathway at the presynaptic level between these conditions, SV recycling dysfunction is present across all of these models. Furthermore, we propose an axis of pathophysiology model where optimal SV endocytosis is required for optimised neural performance. We propose that either decreased or increased SV endocytosis can lead to the synaptic dysfunction observed in these models.