Increased levels of SV2A botulinum neurotoxin receptor in clinical sensory disorders and functional effects of botulinum toxins A and E in cultured human sensory neurons

Yiangos Yiangou1 Uma Anand^1,2, William R. Otto², Marco Sinisi³, Michael Fox³, Rolfe Birch3 Keith A. Foster⁴, Gaurav Mukerji^1,5, Ayesha Akbar^1,6, Sanjiv K. Agarwal⁵, Praveen Anand^1
1Department of Clinical Neuroscience, Imperial College London, Hammersmith Hospital, London; ²Histopathology Laboratory, Cancer Research UK, London Research Institute, London; ³Peripheral Nerve Injury Unit, Royal National Orthopaedic Hospital, Stanmore; ⁴Syntaxin Ltd, Oxford; ⁵Department of Urology; ⁶Department of Gastroenterology, Imperial College London, Hammersmith Hospital, London, United Kingdom

Background: There is increasing evidence that botulinum neurotoxin A may affect sensory nociceptor fibers, but the expression of its receptors in clinical pain states, and its effects in human sensory neurons, are largely unknown.
Methods: We studied synaptic vesicle protein subtype SV2A, a receptor for botulinum neurotoxin A, by immunostaining in a range of clinical tissues, including human dorsal root ganglion sensory neurons, peripheral nerves, the urinary bladder, and the colon. We also determined the effects of botulinum neurotoxins A and E on localization of the capsaicin receptor, TRPV1, and functional sensitivity to capsaicin stimuli in cultured human dorsal root ganglion neurons.
Results: Image analysis showed that SV2A immunoreactive nerve fibers were increased in injured nerves proximal to the injury (P = 0.002), and in painful neuromas (P = 0.0027); the ratio of percentage area SV2A to neurofilaments (a structural marker) was increased proximal to injury (P = 0.0022) and in neuromas (P = 0.0001), indicating increased SV2A levels in injured nerve fibers. In the urinary bladder, SV2A nerve fibers were found in detrusor muscle and associated with blood vessels, with a significant increase in idiopathic detrusor overactivity (P = 0.002) and painful bladder syndrome (P = 0.0087). Colon biopsies showed numerous SV2A-positive nerve fibers, which were increased in quiescent inflammatory bowel disease with abdominal pain (P = 0.023), but not in inflammatory bowel disease without abdominal pain (P = 0.77) or in irritable bowel syndrome (P = 0.13). In vitro studies of botulinum neurotoxin A-treated and botulinum neurotoxin E-treated cultured human sensory neurons showed accumulation of cytoplasmic vesicles, neurite loss, and reduced immunofluorescence for the heat and capsaicin receptor, TRPV1. Functional effects included dose-related inhibition of capsaicin responses on calcium imaging after acute treatment with botulinum neurotoxins A and E.
Conclusion: Differential levels of SV2A protein expression in clinical disorders may identify potential new targets for botulinum neurotoxin therapy. In vitro studies indicate that treatment with botulinum neurotoxins A and E may affect receptor expression and nociceptor function in sensory neurons.

Keywords: SV2A, human, pain, botulinum neurotoxin, neurons