Exploring the functional impact of Botulinum neurotoxin type A central action and its contribution to peripheral neuroparalysis

• Applicant: Dr. Sandy Richter
• Subject Area: Molecular and Cellular Neurology and Neuropathology
• Term: from 2021 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 460563594

The present proposal is designed for a two-year Walter Benjamin fellowship. During this time, I aim to explore the central activity of Botulinum neurotoxin serotype A (BoNT/A), which is used as a potent therapeutic agent for the treatment of several human neurological disorders. BoNT/A is a protein neurotoxin that blocks neurotransmitter release at the neuromuscular junction (NMJ) by specifically cleaving SNAP-25, a neuronal SNARE protein essential for synaptic vesicle exocytosis. In addition, a fraction of peripherally injected BoNT/A undergoes retroaxonal transport and acts directly on central synaptic circuits, as indicated by clinical records in which the therapeutic benefit outlasts BoNT/A peripheral blockade. However, the mechanism of BoNT/A central activity and its functional impact on spinal motor pathways are yet unknown. My central hypothesis is that retrogradely transported BoNT/A attenuates the synaptic activity between upper and lower motor neurons in the spinal cord (and brain stem) thereby providing a synergistic contribution to the well-characterized effect at the NMJ. I aim to address this hypothesis as follows. Firstly, I aim to determine the duration of BoNT/A central over peripheral effects by injecting various doses of BoNT/A in hindlimb muscles of adult mice and comparing the level of SNAP-25 cleavage at the NMJ and spinal cord (or brain stem) via western blotting, immunostaining, and confocal imaging. Next, I aim to elucidate the functional impact of BoNT/A central activity on the [cortical motor neuron]-[spinal motor neuron]-[muscle] motor chain by combining in vivo electrophysiology and optogenetics. The idea is that retroaxonally transported BoNT/A undergoes transcytosis into second order neurons through a synaptic contact at the level of the ventral horn in the spinal cord. By stimulating the motor cortex and recording from the muscle, I aim to unveil whether BoNT/A central activity causes a synaptic blockade of (mainly cholinergic) inputs to α-motor neurons, which will reduce their excitability and prevent muscle contraction. Lastly, I aim to explore a novel strategy to enhance the fraction of retroaxonally transported BoNT/A by blocking the synaptic vesicle cycle at the NMJ via pre-treatment with another serotype, namely BoNT/B. Since BoNT/A exploit synaptic vesicle recycling to act at the NMJ, pre-treatment is expected to enhance the fraction of BoNT/A sorted towards the retrograde trafficking route, which I aim to investigate in vitro and in vivo. The rationale of my project is to provide a better understanding on the functional contribution of BoNT/A central action over its well-known peripheral effect, which will be mandatory to improve the toxins current clinical application by addressing additional therapeutic targets in the future.

• DFG Programme: WBP Fellowship
• International Connection: Italy
• Host: Dr. Marco Pirazzini, Ph.D.