• neurotoxin
• tetanus
• nervous system
• motor neurons
• spreading

Tetanus neurotoxin (TeNT) and botulinum neurotoxins (BoNTs) are clostridial neurotoxins, which show high specificity for the nervous system. They impair neurotransmitter release, causing spastic and flaccid paralysis, respectively. Their activity is specific for components of the SNARE complex, which are essential for neurotransmitter release at synaptic terminals. The precise mechanisms by which TeNT and BoNTs carry out their action, and which cell types are preferentially affected are not yet completely understood. The routes of transport and spreading exploited by the neurotoxins could be also hijacked by pathological protein aggregates, as it has been shown for tau in the brain, it has become increasingly important to study and understand such mechanisms. I thus began to study the molecular mechanisms of neurotoxin spreading and cell specificity, by using both in vitro and in vivo approaches.
In the in vivo experiments, I analysed the diffusion of TeNT in the central nervous system after injection of toxin in peripheral muscles. It has always been thought that the toxin acts at the level of inhibitory interneurons at central nervous system, blocking their neurotransmitter release and leading to the characteristic spastic paralysis in the affected muscles. However, there is no evidence showing which neuronal subtypes are actually affected in the central nervous system. I injected TeNT in the whisker pad (WP) musculature of wild type mice and analysed the presence of its cleaved substrate (cleaved VAMP/synaptobrevin) in the central nervous system. I have determined which synaptic terminals in the brain are preferentially targeted by the neurotoxin after retrograde axonal transport from the periphery. For this purpose, I stained brain sections with primary antibodies directed against different synaptic markers to quantify the degree of co-localisation between cleaved VAMP and inhibitory/excitatory markers. I compared the presence of cl.VAMP in cholinergic, GABAergic and glutamatergic synaptic inputs to motor neurons and I found a higher colocalisation in inhibitory terminals. A second part of the in vivo experiments focusses on the toxins activity in the spinal cord, after injection in the hind-limb muscles. As preliminary analysis, I used the HcT fragment to determine the its localisation at central level and its ability to undergo to trans-synaptic transfer. I then started to study the full-length toxin activity in the spinal cord and the molecular mechanisms exploited by these neurotoxins to propagate within the nervous system.
For the in vitro part, I exploited the expertise of the Schiavo’s laboratory at the UCL Queen Square Institute of Neurology in London. I used microfluidic chambers (MCFs) with two or three compartments hosting distinct neuronal types to analyse the trans-synaptic transfer of the binding fragment of tetanus neurotoxin (HcT) labelled with a fluorophore. Different mouse lines were used for the primary neuronal cultures to distinguish different neuronal subtypes and understand which populations are preferentially targeted by the HcT fragment. I was able to successfully grow primary motor neurons in MFCs and to show that the HcT fragment can undergo to trans-synaptic transfer. The next step will be to apply the full- length TeNT and BoNTs to visualise their actual spreading and activity.