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Title: Regulation of exocytosis and peptide release by synaptotagmins in sensory neurons
Author: Mészáros, Judit
ISNI:       0000 0004 8510 7519
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2020
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In response to inflammation and nerve injury, nociceptive DRG (dorsal root ganglion) neurons become hyperexcitable and this hyperexcitability persists in chronic pain. A key feature of chronic pain states is enhanced secretion of neuropeptides (such as CGRP or Substance P) as a result partly of gene expression changes, and partly changes at the level of regulated secretion. To date however no studies have investigated what changes occur at the level of exocytosis and what changes in the secretory machinery are required to support enhanced neuronal transmission. The research described in this thesis addresses this fundamental question and identifies the role of two regulators of vesicle fusion, synaptotagmin 4 and 7 (syt4/7), in enhanced peptide secretion from nociceptors following exposure to neuronal growth factor (NGF). Synaptotagmins are vesicular membrane proteins that are known for their role in coupling excitation and increased calcium concentration to vesicle fusion and release of transmitters. In this thesis four synaptotagmin isoforms are described for the first time in DRG neurons using a combination of western blotting and immunofluorescence: syt2, syt4, syt7 and 11. Syt2 and 7 are low and high affinity Ca2+ sensors respectively that are important regulators of vesicle fusion. Syt4 and 11 are isoforms that are unable to bind calcium, and therefore their role in regulated secretion is unclear. Syt4 is the more well-known isoform of the two, but studies published to date have provided conflicting views, supporting both an inhibitory and potentiating role. Our results have shown that all the above isoforms are expressed in peptide expressing DRG neurons and are present on CGRP containing vesicles. NGF is a well-known inflammatory mediator of the adult nervous system; it enhances peptide secretion in nociceptive DRG neurons that express its receptor TrkA and contributes to hyperalgesia by sensitising TRPV1 channels. Here we show that syt4 is present in the majority of TrkA positive DRG neurons, and that the enhancement in CGRP secretion following NGF exposure is reduced in neurons isolated from Syt4 knockout (KO) mice. In order to dissect further the role of syt4 and 7 in exocytosis in live DRG neurons, we have established a pHluorin-based fusion assay using total internal reflection (TIRF) microscopy. Using this technique, we found that syt4 and syt7 are functionally targeted to an overlapping population of vesicles, where they mediate fusion events with long fusion pore open times and slow endocytosis. To our knowledge this is the first study to address directly the role of Syt4 in peptide secretion from nociceptors. Our results provide novel insight into the molecular mechanisms contributing to pain signalling and in particular the secretion of CGRP, a well-known mediator of pain conditions.
Supervisor: Seward, Elizabeth Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available