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Title: Targeting SNARE proteins and the trafficking of sodium channels in inflammatory pain
Author: Alves Simoes, Marta
ISNI:       0000 0004 7226 4718
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2017
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Dorsal root ganglion (DRG) neurones perceive and discriminate diverse types of sensations. Nociceptors are a subgroup of DRG neurones specialised in translating noxious pain stimuli to the spinal cord and higher brain centres. Following a noxious insult, nociceptors are known to have enhanced excitability and peptide secretion both of which, are likely to be a consequence of increased membrane trafficking and vesicle fusion with the plasma membrane. Vesicle-associated membrane proteins (VAMPs) are vesicular SNARE proteins (v-SNAREs) which complex together with cognate target SNARE proteins (t-SNAREs) found on 'acceptor' compartments. Together they regulate membrane trafficking and vesicle fusion; while much is known about the v- and t-SNAREs involved in the fusion of neurotransmitter and neuropeptide-containing vesicles with the plasma membrane, to date the identity of SNAREs involved in ion channel trafficking or secretion in nociceptors is limited. To explore the role of SNAREs on DRG neurones' secretion and excitability, an in vitro inflammation model was established. An inflammatory soup containing ATP, bradykinin, prostaglandin E2, histamine, noradrenaline, nerve growth factor, and serotonin was added for 22 h. This incubation induced hyperexcitability. In voltage-clamp, sodium currents resistant to tetrodotoxin, Nav1.9 and Nav1.8 currents, were increased. However, a decrease in the expression of Nav1.7 (TTX-sensitive) and Nav1.9 (TTX-resistant) at the plasma membrane was observed, which likely reflects changes in subcellular location of these channels induced by the inflammatory model. This study also identified the expression of seven vesicle membrane-associated proteins in DRG neurones (VAMP1-5, 7 and 8). This expression was found across all soma diameters. VAMP1/2/7 were observed in the neurites. The potential of the botulinum chimaeras, tetbot A and tetbot B, in reducing CGRP (calcitonin gene-related peptide) release and preventing excitability induced by an inflammatory soup was also explored. These chimaeras are designed to target isolectin B4-negative DRG neurones and cleave SNAP25 (t-SNARE) and VAMP1/2/3 (v-SNAREs). Tetbot A cleaved SNAP25 and significantly reduced CGRP release elicited by 60 mM KCl. The interpretation of the electrophysiology results is problematic as the detergent used in the chimaera preparations altered baseline properties of the DRG neurones.
Supervisor: Liz, Seward Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available