Botulinum toxin modulation of mouse bladder sensory afferent signalling

Abstract

This thesis is an investigation of the effects of botulinum neurotoxins (BoNTs) on the ability of the sensory nerves innervating the bladder to detect and transduce mechanical and chemical stimuli. The healthy functioning of the bladder is dependent on the sensory limb of the micturition cycle, and hyperactivation may lead to the development of urgency, a symptom associated with overactive bladder (OAB). Currently, the treatment options for patients with OAB are not ideal, however, injections of BoNT serotype A is a well-tolerated and effective third line treatment. The mechanisms behind the BoNT/A dependent reduction in urgency symptoms are not well understood, and it is unclear how a neurotoxin best known for paralysing the neuromuscular junction may affect the ability of sensory nerves to detect stretch during filling.

Using a well-characterised ex vivo mouse electrophysiology preparation where the responses of afferent nerves to bladder filling are recorded directly, a wide variety of BoNT serotypes and recombinant constructs were investigated. In chapter three, studies into the entry mechanisms of BoNT/A into the bladder wall revealed the well-establised double-receptor mechanism was not necessary for internalisation, as the light chain (LC/A) alone significantly inhibited afferent neurotransmission. In chapter four, two other serotypes of BoNT, BoNT/B and BoNT/E were investigated, to understand whether modulation of afferent neurotransmission was a feature specific to BoNT/A. These experiments revealed both BoNTs /B and /E directly inhibited distension-induced firing. In chapter five, the role of SNARE cleavage on BoNT/A induced afferent inhibition was investigated using the catalytically inactive construct BoNT/A (0) unable to cleave SNAP-25. This construct had no effect on hemidiaphragm contractility yet potently inhibited distension induced afferent firing, and was more effective at doing so than catalytically active BoNT/A. These findings are novel and reveal a SNAP-25 independent modulation of sensory signalling.